Research Note: Effects of organic zinc on broiler intestinal permeability and integrity in Clostridium perfringens-challenged condition.

This study evaluates the effects of a zinc-amino acid complex on broiler's intestinal permeability and integrity challenged with Clostridium perfringens. A total of 180 Arbor Acres 1-day-old male broilers were assigned to 6 treatments in a completely randomized 2 × 3 factorial design. The experiment investigated the comparative effects of inorganic and organic zinc supplements, that is (ZnSO4 treatment: 80 mg zinc/kg from ZnSO4; iso-dose replacement group. (ISO) treatment:40 mg zinc/kg from ZnSO4 plus 40 mg zinc/kg from a zinc-amino acid complex; and organic treatment: 40 mg zinc/kg from a zinc-amino acid complex) on C. perfringens-challenged broilers. C. perfringens, on the one hand, compromised intestinal mucosal barrier function by increasing the intestinal permeability of fluorescein isothiocyanate-labeled dextran (P < 0.05) and plasma endotoxin level, on the other hand, decreased both the transepithelial electrical resistance and the relative expression of occludin levels in the ileum at day 21 (P < 0.05). However, zinc supplement alleviates C. perfringens-induced pathologic changes in the intestinal permeability. ISO treatment, in particular, enhanced the villus height-to-crypt depth ratio and transepithelial electrical resistance and also reduced plasma endotoxin levels. In addition, zinc supplement relatively enhanced the expression of occludin levels in the ileum compared with the C. perfringens-challenged group. ISO treatment had the highest relative expression of occludin levels in the ileum. Thereby, the results indicated that partial replacement of ZnSO4 with a zinc-amino acid complex in the broiler diet could promote intestinal mucosal barrier function during C. perfringens infection via increased expression of occludin in the ileum.

Introduction

The intestinal mucosa serves as a link between the external environment and the intestinal immune system, thus functioning as an immune barrier. The integrity of mucosal barrier functions is critical to maximizing nutrient absorption and preventing the development of inflammatory diseases in the intestine (Groschwitz and Hogan, 2009; Bischoff et al., 2014). Clostridium perfringens is present in the broilers' intestine under normal physiological conditions, but its pathogenic consequence on the intestine is of particular interest to researchers. C. perfringens can cause animal cell necrosis, tissue edema, and necrotizing enteritis.

Zinc, an essential trace element for animals, can relieve intestinal inflammation by regulating the permeability of the intestine barrier layers. Inorganic zinc is widely used in animal production owing to its low cost. However, organic zinc potentially has a higher bioavailability level. In comparison, organic zinc poses to be more effective either in its complete or partial replacement for inorganic zinc by promoting intestinal health and tissue morphology—including the intestinal villi height, the goblet cells count, and the villus height–to–crypt depth ratio. Nevertheless, the underlying mechanism of the organic zinc effect to improve intestinal integrity is undefined at the moment (Hu et al., 2013; Bortoluzzi et al., 2019). Thus, the purpose of this study was to evaluate the effect and possible mechanisms of a zinc–amino acid complex on intestinal permeability and integrity of broilers during a C. perfringens challenge.

Materials and methods

Birds, Experimental Design, and Diets

This experiment was carried out with the approval by the Chinese Agricultural University Laboratory Animal Welfare and Experimental Ethical Committee. The study adopted a completely randomized design with a 2 × 3 factorial pattern. Herein, the factors include 3 patterns of Zn supplements (ZnSO4 treatment: 80 mg zinc/kg from ZnSO4; iso-dose replacement group.(ISO) treatment: 40 mg zinc/kg from ZnSO4 and 40 mg zinc/kg from a zinc–amino acid complex; and organic treatment: 40 mg zinc/kg from a zinc–amino acid complex) with or without C. perfringens challenge. A total of 180 1-day-old male Arbor Acres broilers were obtained from a commercial hatchery and randomly divided into 6 treatments with 30 birds each. The birds were raised for 21 d in a temperature-controlled insulator with a continuous 24-h light supply. All birds had ad libitum access to feed and water throughout the experiment period. A corn–soybean basal diet was formulated to meet the NRC requirements for broilers. A zinc–amino acid complex containing zinc lysine and zinc glutamic acid was purchased from Zinpro (Wuxi) Additives Biotechnology Co., Ltd.

C. perfringens Challenge

Field strain type A chicken C. perfringens isolated from a clinical case of necrotic enteritis was obtained from the China Veterinary Culture Collection Center. The bacteria were cultured anaerobically on tryptose-sulfite-cycloserine agar for 8 h at 37°C, and then aseptically inoculated into cooked meat medium and incubated anaerobically overnight at 37°C. All the challenged birds were orally inoculated with 0.5 mL (1.0 × 108 cfu/mL) suspension on day 7, and on day 14–20, the birds were further orally inoculated with 1.0 mL daily. Nonchallenged birds were orally inoculated with the same volume of buffer solution during the same periods.

Sample Collection

On day 14 and day 21, 8 birds were selected from each treatment, and the blood was collected aseptically from the wing vein into heparinized vacutainers, then plasma was obtained by centrifuging blood at 2,000 × g for 15 min at 4°C and stored at −20°C for plasma endotoxin assay. In addition, the ileum was collected for histologic examination, intestinal permeability, and tight junction protein mRNA expression measurement. Samples for mRNA determination were frozen in liquid nitrogen immediately and stored at−80°C.

Sample Analysis

Plasma endotoxin assay: Plasma endotoxin was detected using Pyrochrome Chromogenic Endotoxin Testing (Associates of Cape Cod. Inc., East Falmouth, MA), and the endotoxin standard (Escherichia coli O113: H10) was used as a standard curve.

Small intestinal histomorphology: The small intestine segment at the same position was dissected, immediately fixed in 4% paraformaldehyde, and then embedded in paraffin sectioned (5 μm). The samples were stained and observed with hematoxylin and eosin and an Olympus microscope (CX23, Tokyo, Japan) consecutively. The stained samples were analyzed using ProgRes CapturePro software (Jenoptik, Jena, Germany) to measure the intestinal villi height (from the tip of the villi to the crypt opening) and the crypt depth (from the base of the crypt to the level of the crypt opening). A total of 15 villi and corresponding crypts were randomly selected for each tissue to calculate the villi height–to–the crypt depth ratio.

Intestinal permeability: Ileal segments of each bird were mounted into chambers (Physiological Instruments, San Diego, CA) for the determination of intestinal integrity. Briefly, intestinal samples were placed into chambers, connected to dual-channel current and voltage electrodes. Both the mucosal and serosal sides of the tissue were bathed in Krebs-Henseleit bicar-bonate and provided with a constant O2–CO2 mixture. Individual segments were voltage-clamped at 0 mv, and transepithelial electrical resistance (TER) was determined. After stabilization, intestinal segments were tested for permeability to the macromolecule fluorescein isothiocyanate–labeled dextran (Sigma, St. Louis, MO).

Tight junction protein mRNA expression: Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA), and cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Tokyo, Japan) as per the manufacturer's instructions. Gene expression was detected by the ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA). The claudin-1, occludin, mucin 2, and β-actin gene-specific primer sequences are as follows: claudin-1: forward 5′-CATACTCCTGGGTCTGGTTGGT-3′, reverse 5′-GACAGCCATCCGCATCTTCT-3′ (GenBank accession number: AY750897.1); occludin: forward 5′-ACGGCAGCACCTACCTCAA-3′, reverse 5′-GGGCGAAGAAGCAGATGAG-3′ (GenBank accession number: D21837.1); mucin 2: forward 5′-TTCATGATGCCTGCTCTTGTG-3′, reverse 5′-CCTGAGCCTTGGTACATTCTTGT-3′ (GenBank accession number: NM_001318434); and β-actin: forward 5′-ACTCTGGTGATGGTGTTAC-3′, reverse 5′-GGCTGTGATCTCCTTCTG-3′ (GenBank accession number: NM_205518). The relative gene expression level was calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2000).

Statistical Analyses

All data were analyzed using SPSS 25.0 software (IBM, Armonk, NY). The treatment effects, C. perfringens challenge, and their interactions were analyzed using repeated-measures 2-way ANOVA. The C. perfringens challenge was added to the model as a repeated factor. For zinc levels, it was analyzed using the software's GLM procedure. Comparisons of means for each significant effect were analyzed by Duncan's test using the least-squares mean statement. Final data are presented as means and are considered statistically significant at P-value < 0.05.

Results and discussion

C. perfringens is an important zoonotic pathogen that is widely distributed in nature. Previous studies have shown that oral administration of C. perfringens can successfully infect and establish an intestinal injury model (Liu et al., 2012). In this study, we observed that C. perfringens caused pathologic changes in the intestines of broilers—such as visible thinning of the intestinal wall, congestion, and a large number of bleeding points. The pathologic changes could be because of the C. perfringens proliferation in the small intestine as well as the secretion of exotoxin that results in intestinal damages. C. perfringens (a gram-positive bacterium), on the other hand, can promote the proliferation of several gram-negative bacteria in the ileum (such as E. coli), resulting in the translocation of endotoxin to the blood and increasing the level of endotoxin in the blood (Liu et al., 2012). In agreement, our findings revealed that C. perfringens infection in 21-day-old broilers contributed to elevated plasma endotoxin and impaired intestinal permeability. On the contrary, dietary zinc supplement reduced the plasma endotoxin. Compared with the inorganic zinc treatment, partial replacement of inorganic zinc by organic zinc in the diet (ISO treatment) had the lowest plasma endotoxin level (Table 1). Likewise, dietary zinc treatments significantly affect (P < 0.05) the ileum villus height and villus height/crypt depth ratio of 14-day-old broilers. The ISO treatment group had the highest (P < 0.05) ileum villus height values and villus height/crypt depth ratio than the ZnSO4 group (Table 1). However, both C. perfringens challenge and Zn addition did not have any significant effects (P > 0.05) on ileum villus height, crypt depth, and villus height/crypt depth ratio of 21-day-old broilers (data not shown). Endotoxin is a good bacterial marker for the measurement of intestinal permeability and assessment of epithelial integrity. It has been previously used to evaluate the intestinal barrier impairment and permeability gradient in vivo (Bischoff et al., 2014), whereas the villus height/crypt depth ratio could be used to assess the integrity of the intestinal mucosa. Altogether, this study showed that organic zinc alleviates the intestinal tract damage caused by C. perfringens better than inorganic zinc.

Table 1

Effects of dietary zinc supplements on ileum histomorphologic parameters, plasma endotoxin, and intestinal permeability of broilers challenged with Clostridium perfringens.1

Treatments	C. perfringens	21-d FITC-Dextran permeability (μg/mL)	21-d TER (Ω/cm2)	21-d plasma endotoxin	14-d villus height (μm)	14-d villus height/crypt depth ratio
ZnSO42	−	4.73	114.97	0.0581	277.1	2.60
	+	5.94	81.91	0.1016	326.0	3.27
ISO2	−	4.76	136.62	0.0413	435.8	4.00
	+	5.84	93.07	0.0773	344.4	3.51
Organic2	−	4.05	94.80	0.0544	333.0	3.34
	+	5.96	76.31	0.1050	300.1	3.21
SEM		0.279	12.476	0.00526	13.62	0.096
Zn treatment	ZnSO4	5.29	98.44	0.0798	301.6b	2.96b
	ISO	5.34	114.85	0.0593	376.5a	3.74a
	Organic	5.00	85.56	0.0797	316.6ab	3.27ab
Challenge	−	4.50b	115.46a	0.0513b	348.6	3.31
	+	5.91a	83.77b	0.0946a	323.5	3.33
P-value
Zn treatment		0.604	0.074	0.080	0.024	0.005
Challenge		0.033	0.003	<0.001	0.361	0.928
Zn × Challenge		0.795	0.605	0.776	0.128	0.056

a,bThe same index but the difference is significant (P < 0.05).

Abbreviations: −, without C. perfringens challenge; +, with C. perfringens challenge; FITC-Dextran, fluorescein isothiocyanate–labeled dextran; TER, transepithelial electrical resistance.

Each value represents the mean of 8 replicate (n = 8).

ZnSO4 treatment: 80 mg zinc/kg from ZnSO4; ISO treatment:40 mg zinc/kg plus ZnSO4 plus 40 mg zinc/kg from a zinc–amino acid complex; Organic treatment: 40 mg zinc/kg from a zinc–amino acid complex.

Impairment of the epithelial layers permeability, microbial dysfunction, as well as the immune defense system could potentially compromise the intestinal integrity. As a result, the level of harmful compounds will increase in the mucosa because of epithelial cell wall damage. In this study, our results confirmed that C. perfringens infection significantly increased (P < 0.05) intestinal fluorescein isothiocyanate–labeled dextran permeability and decreased (P < 0.05) TER on day 21 (Table 1); thus, C. perfringens could undermine intestinal permeability and integrity. Zinc has been shown to protect against Salmonella enterica in broiler chickens and enhanced occludin and zonula occludens proteins-1 expressions in weaning piglets in our previous studies (Zhang and Guo, 2009; Zhang et al., 2012). In accordance with the previous data, dietary zinc treatment increased ileal TER on day 21 (P = 0.074) and the ileal TER in ISO treatment was the highest (Table 1) in this study. The results are consistent with the plasma endotoxin and intestinal villus height/crypt depth ratio. Therefore, partial replacement of inorganic zinc by organic zinc deduced to reduce the increased intestinal damage caused by C. perfringens.

The intestinal barrier comprises the unicellular layers of the intestinal epithelium that allows selective absorption of nutrients and water while avoiding the entry of pathogenic materials. The epithelium maintains its selective barrier functions via complex protein networks that form 3 adhesive complexes, including desmosomes, adhesion junctions, and tight junctions (Groschwitz and Hogan, 2009). These complexes help to regulate epithelial cell permeability in the intestine by expressions of cadherin, claudin, occluding, and junctional adhesion molecule proteins (McCarthy et al., 1996; Sonoda et al., 1999). The results in this study showed that zinc alleviated the intestinal epithelial damage caused by the C. perfringens infection. To further elucidate the mechanism involved in the role zinc played to promote intestinal integrity, we investigated the relative expression of occludin, claudin-1, and mucin-2 in the ileum samples. The results showed no significant difference (P > 0.05) between C. perfringens challenge and Zn treatment in the relative expression of claudin-1 and mucin-2 genes (data not shown). However, C. perfringens infection significantly decreased (P < 0.05) the expression of occludin genes, whereas, zinc treatments upregulated the relative expression of occludin in the ileum both at day 14 (P = 0.065) and day 21 (P = 0.056) with the highest expression recorded from ISO treatment (Table 2). In conclusion, we infer that a dietary zinc–amino acid complex could potentially alleviate C. perfringens–induced intestinal mucosal damage by promoting intestinal integrity and upregulating expression of occludin in the ileum.

Table 2

Effects of dietary zinc supplements on relative occludin expressions in the ileum of broilers challenged with Clostridium perfringens.1

Treatments	C. perfringens	14-d occludin	21-d occludin
ZnSO42	−	1.00	1.00
	+	0.75	0.56
ISO2	−	2.02	1.61
	+	1.54	0.83
Organic2	−	1.50	1.19
	+	1.11	0.59
SEM		0.156	0.089
Zn source	ZnSO4	0.88	0.78
	ISO	1.78	1.22
	Organic	1.30	0.89
Challenge	−	1.51	1.27a
	+	1.13	0.66b
P-value
Zn		0.065	0.056
Challenge		0.228	<0.001
Zn × Challenge		0.953	0.671

Abbreviations: −, without C. perfringens challenge; +, with C. perfringens challenge.

Each value represents the mean of 8 replicate (n = 8).

ZnSO4 treatment: 80 mg zinc/kg from ZnSO4; ISO treatment:40 mg zinc/kg plus ZnSO4 plus 40 mg zinc/kg from a zinc–amino acid complex; Organic treatment: 40 mg zinc/kg from a zinc–amino acid complex.