Zinc prevents Salmonella enterica serovar Typhimurium-induced loss of intestinal mucosal barrier function in broiler chickens

Abstract

The study was carried out to evaluate the beneficial effects of supplemental zinc (Zn) on the intestinal mucosal barrier function in Salmonella enterica serovar Typhimurium-challenged broiler chickens in a 42-day experiment. A total of 336 1-day-old male Arbor Acres broiler chicks were assigned to eight treatment groups. A 4×2 factorial arrangement of treatments was used in a completely randomized experimental design to study the effects of levels of supplemental Zn (0, 40, 80 and 120 mg/kg diet), pathogen challenge (with or without S. Typhimurium challenge), and their interactions. S. Typhimurium infection caused reduction of growth performance (P<0.05) and intestinal injury, as determined by reduced (P<0.05) villus height/crypt depth ratio and sucrase activity in the ileum, increased (P<0.05) plasma endotoxin levels, and reduced (P<0.05) claudin-1, occludin and mucin-2 mRNA expression in the ileum at day 21. Zn pre-treatment tended to improve body weight gain (P=0.072) in the starter period, to increase the activity of ileal sucrase (P=0.077), to reduce plasma endotoxin levels (P=0.080), and to significantly increase (P<0.05) the villus height/crypt depth ratio and mRNA levels of occludin and claudin-1 in the ileum at day 21. The results indicated that dietary Zn supplementation appeared to alleviate the loss of intestinal mucosal barrier function induced by S. Typhimurium challenge and the partial mechanism might be related to the increased expression of occludin and claudin-1 in broiler chickens.

Introduction

Food-borne diseases in humans caused by members of Salmonella remain a major public health concern worldwide and are commonly associated with the consumption of broiler chicken meat contaminated with salmonellas (Rabsch et al., 2001; Zhang et al., 2003; Liljebjelke et al., 2005). Aside from its importance as a food-borne pathogen, Salmonella causes systemic or subclinical disease characterized by reduced performance, intestinal inflammation, high mortality, and cross-contamination in chicks (Suzuki, 1994; Fasina et al., 2008). In broiler chicken meat, the predominant reported serovar is Salmonella enterica serovar Typhimurium. There is therefore increasing interest in preventing intestinal infection of chickens with salmonellas. Attempts to reduce levels of salmonellas in the intestine of broiler chickens, in part, requires understanding of the interactions between salmonellas and the intestinal mucosal barriers.

The intestinal mucosal barriers, which represent the first line of defence, are composed primarily of epithelial cell membranes and a mucus gel layer covering the epithelium (Turner, 2006). Bacterial enteropathogens must traverse the mucus layer in order to approach and adhere to the mucosal epithelium cells (Hecht, 1999; Opitz et al., 2007). The epithelial cells form a continuous intact physical barrier with interspersing tight junctions (TJs). This epithelial barrier is one of the most important components of the intestinal mucosal barrier against macromolecular transmission (Ballard et al., 1995). The disruption of TJs results in increased permeability to luminal antigens and bacteria, and further deteriorates mucosal barrier function. TJs can be altered by several bacterial pathogens through modification of TJ proteins such as occludin, claudin, and zonula occludens (ZO)-1 (Berkes et al., 2003; Sousa et al., 2005; Landau, 2006). Infection of polarized epithelial cell monolayers by S. Typhimurium disrupted TJ structure and function, and subsequently perturbed the epithelial barrier (Jepson et al., 1995, 2000; Tafazoli et al., 2003; Boyle et al., 2006). Similarly, in vitro experiments infecting T84 intestinal epithelial cells with S. Typhimurium caused a decrease in both ZO-1 expression and in the amount of phosphorylated occludin, redistribution of epithelial TJ proteins claudin-1 and ZO-2, facilitation of bacterial translocation, and loss of barrier function (Koehler et al., 2007). However, the direct interaction of salmonella on component proteins of the TJs has not been investigated in vivo for broilers.

Zinc (Zn) was shown to have an important function in resisting diseases, improving wound healing and maintaining epithelial tissue integrity (Vallee & Falchuk, 1993). The importance of Zn to intestinal development and function has been demonstrated in many studies, such as increased intestinal crypt-cell production, reduced duration of mitosis (Duff & Ettarh, 2002), and improved epithelial cell restitution (Cario et al., 2000), and maintaining the structure and function of the intestinal barrier (Rodriguez et al., 1996; Sturniolo et al., 2001; Lambert et al., 2004). Finamore et al. (2008) reported that Zn deprivation induced a decrease of transepithelial electrical resistance and alterations to tight and adherens junctions. Added Zn in the diet reduced gut lesion scores (Rapp et al., 2004) and prevented reduction in absorption of carotenoids and pigmentation in Eimeria tenella-infected chickens (Zhao et al., 2006). Furthermore, Zn supplementation reduced intestinal permeability and increased expression of occludin and ZO-1 in mammals (Roy et al., 1992; Zhang & Guo, 2009). However, a few broiler chicken studies have demonstrated the beneficial effects of supplemental Zn on the intestinal mucosal barriers of Salmonella-infected chickens. Therefore, it would be of interest to examine whether supplementation of Zn modulates the expression of TJ proteins.

The purpose of the present experiment was to evaluate the beneficial effects of supplemental Zn on the intestinal mucosal barrier function and expression of TJ proteins in S. Typhimurium-challenged broiler chickens. More specifically, effects on intestinal histomorphology, plasma endotoxin, and gene expression of mucin-2 (MUC2) and TJ proteins (mainly occludin and claudin-1) were assessed.

Materials and Methods

Birds, diets and experimental design

The study consisted of a completely randomized experimental design with a 4×2 factorial arrangement of treatments, four levels of supplemental Zn (0, 40, 80 and 120 mg/kg diet) and with or without S. Typhimurium challenge. Three hundred and thirty-six 1-day-old male Arbor Acres broiler chickens were obtained from a commercial hatchery and randomly divided into eight treatments with seven replicates of six birds each. The experimental period lasted 42 days. Chickens were raised in a temperature-controlled room with 24 hours of constant light. The temperature of the room was 35 to 33°C for the first 3 days, and then it declined by 3°C weekly until 22 to 24°C. All birds were provided with feed and water ad libitum throughout the grow-out period. The basal diets were corn-soybean meal based, and formulated to meet or exceed the Feeding Standard of Chicken (China, 2004) requirements. The dietary phases consisted of starter (0 to 21 days) and grower (22 to 42 days) as shown in Table 1. Experimental procedures were approved and conducted under the guidelines of the China Agricultural University Animal Care and Use Committee.

Table 1.  Composition and nutrient levels of the basal diets.

	Composition (g/kg)
Ingredient	Days 1 to 21	Days 22 to 42
Maize	585.15	560.1
Soybean meal	355	360
Soybean oil	19	43
Dicalcium phosphate	19.7	19.8
Limestone	11.3	8.4
Sodium chloride	3.5	3
Vitamin and mineral premix^a	2.2	2.2
Choline chloride (50%)	1.6	2
d,l-Methionine (98%)	1.36	1.2
l-Lysine (99%)	0.99	0
Ethoxyquinoline (33%)	0.2	0.3
Calculated nutrient levels
Metabolizable energy (MJ/kg)	12.04	12.67
Crude protein (g/kg)	205.0	199.7
Calcium (g/kg)	10.0	9.0
Available phosphorus (g/kg)	4.5	4.5
Lysine (g/kg)	115	10.6
Methionine (g/kg)	4.5	4.6
Zinc (mg/kg)^b	28.72	27.65
^aMineral and vitamin premix supplied (per kilogram of final diet): copper, 8 mg (CuSO4·5H2O); iron, 80 mg (FeSO4); manganese, 100 mg (MnSO4·H2O); selenium, 0.15 mg (Na2SeO3); iodine, 0.35 mg (KI); vitamin A, 9500 IU (retinylacetate); cholecalciferol, 62.5 µg; vitamin K3, 2.65 mg; thiamin, 2 mg; riboflavin, 6 mg; vitamin B12, 0.025 mg; vitamin E, 30 IU (α-tocopherol acetate); biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; niacin, 50 mg.
^bAnalysed.

Salmonella enterica serovar Typhimurium challenge

A chicken S. Typhimurium field strain isolated from a clinical case of salmonellosis was obtained from the China Veterinary Culture Collection Center (Beijing, China). The organism was cultured in lactose broth (CM 228, Land Bridge Technology Ltd, Beijing, China) for 18 h at 40°C. To determine the number of colony-forming units (CFUs), the inoculum was diluted and plated on xylose–lysine–deoxycholate agar (Beijing Aoboxing Bio-tech Co., Ltd, Beijing, China) for 24 h at 37°C.

At 7 days of age, all birds in challenged groups were orally gavaged with the actively growing culture of Salmonella (0.5×10⁸ CFU/ml, 1.0 ml/bird), and the non-challenged group chicks were mock-challenged with 1 ml sterile buffered peptone water. The infection protocol was based on results obtained from previous trials in our laboratory, and these showed consistent and uniform salmonella colonization in chicks.

Performance parameters

Chicks and feed were weighed by pen (replicate) at day of hatch, 21 days and 42 days. Feed intake (FI) was measured, and the body weight gain (BWG) and feed conversion ratio were calculated for each period and cumulatively for the grow-out.

Recovery of salmonellas

To confirm the presence of infection, four birds per treatment were killed at 5 days and 14 days post challenge to determine the numbers of salmonellas in the caecum. The caecal contents were collected aseptically into pre-weighed 15-ml sterile plastic tubes, weighed, diluted in peptone water to an initial 10⁻¹ dilution, and homogenized. All homogenates were serially diluted in phosphate-buffered saline to appropriate levels for isolation on xylose–lysine–deoxycholate agar plates. Plates were incubated for 24 h at 37°C and then presumptive S. Typhimurium colonies were counted. Negative samples were placed in tetrathionate broth (1:10), incubated at 37°C for 24 h, and streaked on xylose–lysine–deoxycholate agar. Total concentration of S. Typhimurium in each caecum was expressed as log₁₀ CFU/ml.

Sample collection

On day 21, one bird per replicate (seven replicates/treatment) was randomly selected and blood was collected aseptically from the wing vein into heparinized vacutainers. Plasma was obtained by centrifuging at 2000×g for 15 min at 4°C and stored at −20°C for plasma endotoxin assay. The birds were killed by intracardial administration of sodium pentobarbital (30 mg/kg body weight) and jugular exsanguination on the morning of day 21. The small intestine was then removed and gently flushed with ice-cold saline. Samples (~4 cm) were taken from the midway between Meckel's diverticulum and the ileo-caeca junction (ileum). Samples for mRNA determination were frozen in liquid nitrogen.

Intestinal histomorphology

All small intestinal segments were immediately fixed in 4% paraformaldehyde and then embedded in paraffin. Consecutive sections (5 µm) were stained with haematoxylin and eosin and observed for histomorphology examination. The villus height (from the tip of the villus to the crypt opening) and crypt depth (from the base of the crypt to the level of the crypt opening) were measured from 15 randomly selected villi and associated crypts on one section per chicken at 40× magnification. The villus height to crypt depth ratio was then calculated from these measurements by dividing the villous height with the crypt depth. All examinations and measurements were performed with an Olympus optical microscope using ProgRes CapturePro software, version 2.7 (Jenoptik, Jena, Germany).

Plasma endotoxin assay

Plasma endotoxin was measured as an indicator of intestinal permeability. The endotoxin level in the plasma samples was measured using a quantitative chromogen limulus amoebocyte lysate test (Pyrochrome® Chromogenic Endotoxin Testing; Associates of Cape Cod Inc., East Falmouth Massachusetts, USA). Briefly, 0.1 ml plasma was incubated with 0.1 ml limulus amebocyte lysate at 37°C for 45 min. After several subsequent reactions, the samples were read spectrophotometrically at 405 nm. The plasma endotoxin levels were calculated against a standard curve of control standard endotoxin (Escherichia coli O113:H10) concentrations. All glassware, solutions, and surgical instruments used in the experiment were autoclaved at 121°C for 15 min.

Sucrase activity

Ileal sucrase activity was measured as an additional marker of mucosal integrity. Ileal sucrase activity was assayed colorimetrically according to Lamb-Rosteski et al. (2008) using sucrose as a substrate and expressed as units/g protein. The concentration of protein in the sample was determined using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, Illinois, USA).

RNA isolation and quantitative real-time polymerase chain reaction

Total RNA was isolated using Trizol reagent (Invitrogen Life Technologies, Carlsbad, California, USA) according to the manufacturer's instructions. The concentration and purity of total RNA was estimated by measuring its optical density at 260 and 280 nm. One microgram of total RNA was reverse transcribed by a reverse transcription kit (Invitrogen Life Technologies) according to the manufacturer's instructions. All of the cDNA preparations were stored frozen at −30°C until further use. A quantitative real-time polymerase chain reaction (PCR) assay was performed with the 7500 fluorescence detection system (Applied Biosystems, Foster City, California, USA) according to optimized PCR protocols using the SYBR-Green PCR kit (Applied Biosystems). The gene-specific primers for claudin-1, occludin, MUC2 and β-actin were as follows: forward 5′-CATACTCCTGGGTCTGGTTGGT-3′, reverse 5′-GACAGCCATCCGCATCTTCT-3′ for claudin-1 (GenBank accession number AY750897.1); forward 5′-ACGGCAGCACCTACCTCAA-3′, reverse 5′-GGGCGAAGAAGCAGATGAG-3′ for occludin (GenBank accession number: D21837.1); forward 5′-TTCATGATGCCTGCTCTTGTG-3′, reverse 5′-CCTGAGCCTTGGTACATTCTTGT-3′ for MUC2 (GenBank accession number XM_421035); forward 5′-ACTCTGGTGATGGTGTTAC-3′, reverse 5′-GGCTGTGATCTCCTTCTG-3′ for β-actin (GenBank accession number NM_205518). The following PCR conditions were employed: an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 30 sec, the annealing and extension temperature at 60°C for 1 min, and a final extension step of 72°C for 10 min. To confirm amplification specificity, the PCR products from each primer pair were subjected to a melting curve analysis and subsequent agarose gel electrophoresis. Gene expression was quantified using the comparative threshold cycle method and the data were expressed as the relative value to the control group.

Statistical analysis

All analyses were performed with SPSS 16.0 software (SPSS Inc., Chicago, Illinois, USA). The data were analysed by two-way analysis of variance. Differences were considered significant at P<0.05, and a tendency was defined as P<0.1. All values are expressed as treatment means with their pooled standard error of the mean.

Results

Establishment of Salmonella infection

The Salmonella concentration in the caecal contents of challenged group chicks was 6.55±1.33 log₁₀ CFU on day 5 post challenge and 4.14±1.27 log₁₀ CFU on day 14 post challenge, as opposed to nil values for the non-challenged group chicks. This confirms that Salmonella infection was established.

Growth performance

There were no significant interactions (P>0.05) between Zn and Salmonella challenge observed in any feeding period (Table 2). The main effects of Zn on growth performance were only observed for FI during days 1 to 21. The BWG showed a tendency (P=0.072) for an improvement in the starter period when Zn was added to the diets. Birds fed Zn at 120 mg/kg diet had significantly higher (P<0.05) BWG and FI than those fed diets without Zn supplementation during days 1 to 21. There was a significant reduction (P<0.01) in BWG of birds in the challenged treatment compared with that of birds in the non-challenged treatment. Infection with S. Typhimurium significantly decreased (P<0.05) FI of chicks in comparison with uninfected birds during days 1 to 21, and tended to decrease (P=0.056) FI of chicks during days 1 to 42. No differences were observed for the feed conversion ratio in any feeding period (P>0.05).

Table 2.  Growth performance of broiler chickens treated with dietary supplemental zinc and challenged with S. Typhimurium^a.

		Days 1 to 21			Days 1 to 42
Supplemental Zn level (mg/kg)	S. Typhimurium	BWG (g)	FI (g)	FCR	BWG (g)	FI (g)	FCR
	−	522	753	1.44	1881	3303	1.76
0	+	484	716	1.48	1849	3226	1.72
	−	570	833	1.47	2054	3438	1.66
40	+	510	747	1.47	1864	3241	1.75
	−	537	751	1.40	1926	3300	1.72
80	+	491	715	1.46	1840	3194	1.71
	−	537	788	1.47	1901	3317	1.73
120	+	488	711	1.46	1818	3205	1.76
SEM		6.22	8.38	0.097	19.56	30.84	0.016
Source of variation (P value)
Zn		0.093	0.02	0.506	0.208	0.725	0.784
Challenged		<0.001	<0.001	0.269	0.011	0.056	0.539
Zn×challenged		0.892	0.442	0.572	0.484	0.916	0.578
^aEach value represents the mean values of seven pens of six birds each (n=7). FCR, feed conversion ratio; SEM, standard error of the mean. −, without S. Typhimurium challenge; +, with S. Typhimurium challenge.

Intestinal morphology

S. Typhimurium challenge resulted in decreased (P<0.05) villus height and villus height/crypt depth ratio in the ileum at day 21, but Zn supplementation significantly increased (P<0.05) the villus height and villus height/crypt depth ratio in the ileum (Table 3). Birds fed Zn at 80 or 120 mg/kg diet had significantly higher (P<0.05) villus height and villus height/crypt depth ratio than those fed diets without Zn supplementation. Neither S. Typhimurium challenge nor Zn addition affected crypt depth in the ileum (P>0.05). There were no significant interactions of Zn by Salmonella challenge observed for intestinal morphology in any feeding period (P>0.05).

Table 3.  Plasma levels of endotoxin, histomorphological parameters and sucrase activity in the ileum of broiler chickens at 21 days of age treated with dietary supplemental Zn and challenged with S. Typhimurium^a.

Supplemental Zn level (mg/kg)	S. Typhimurium	Plasma endotoxin (EU/ml)	Villus height (µm)	Crypt depth (µm)	Villus height/crypt depth ratio	Sucrase activity (units/g protein)
	−	0.1166	441	136	3.27	15.04
0	+	0.2633	400	133	3.02	13.66
	−	0.1013	451	132	3.44	16.48
40	+	0.2400	413	136	3.08	13.97
	−	0.1038	477	130	3.77	17.20
80	+	0.2310	460	133	3.48	14.11
	−	0.0853	509	139	3.76	18.89
120	+	0.2130	474	135	3.48	15.33
SEM		0.01124	7.42	3.18	0.074	0.421
Source of variation (P value)
Zn		0.080	0.001	0.822	0.035	0.077
Challenged		<0.001	0.011	0.733	0.041	0.001
Zn×challenged		0.902	0.898	0.999	0.993	0.746
^aEach value represents the mean values of seven pens of six birds each (n=7). SEM, standard error of the mean. −, without S. Typhimurium challenge; +, with S. Typhimurium challenge.

Plasma endotoxin levels and ileal sucrase activity

Infection with S. Typhimurium significantly increased (P<0.05) plasma endotoxin levels of chicks at day 21, and significantly decreased (P<0.05) the activity of ileal sucrase (Table 3). Zn supplementation had the tendency to reduce plasma endotoxin levels (P=0.080), but tended (P=0.077) to increase the activity of ileal sucrase when compared with the control group. Birds fed Zn at 120 mg/kg diet had significantly lower (P<0.05) plasma endotoxin levels than those fed diets without Zn supplementation.

Claudin-1, occludin and MUC2 mRNA expression

Infection with S. Typhimurium significantly decreased (P<0.05) claudin-1, occludin and MUC2 mRNA expression in the ileum of chicks at day 21 (Table 4). Adding Zn to the diet of chicks significantly enhanced (P<0.05) mRNA levels of occludin and claudin-1 in ileal mucosa. Birds fed Zn at 80 or 120 mg/kg diet significantly enhanced (P<0.05) mRNA levels of occludin and claudin-1 in ileal mucosa when compared with other groups. Inclusion of Zn in the diet of chicks did not affect the mRNA levels of MUC2 (P > 0.05). There were no significant interaction effects on claudin-1, occludin and MUC2 mRNA expression in the ileum between dietary Zn and S. Typhimurium challenge.

Table 4.  Relative expression of claudin-1, occludin and MUC2 in the ileum of broiler chickens at 21 days of age treated with dietary supplemental Zn and challenged with S. Typhimurium^a.

Supplemental Zn level (mg/kg)	S. Typhimurium	Claudin-1	Occludin	Mucin-2
	−	1.06	1.02	1.02
0	+	0.68	0.55	0.77
	−	1.39	1.27	1.34
40	+	0.58	0.70	1.06
	−	2.16	1.76	1.23
80	+	0.92	1.06	1.08
	−	1.84	1.58	1.64
120	+	1.19	1.27	1.31
SEM		0.116	0.082	0.086
Source of variation (P value)
Zn		0.008	<0.001	0.132
Challenged		<0.001	<0.001	0.048
Zn×challenged		0.774	0.527	0.975
^aEach value represents the mean values of seven pens of six birds each (n=7). SEM, standard error of the mean.−, without S. Typhimurium challenge;+, with S. Typhimurium challenge.

Discussion

Infection with salmonellas leads to polydypsia, diarrhoea, and severe reduction in BWG. The effect of Salmonella infection on performance was reported previously (Nakamura et al., 2002; Vandeplas et al., 2009) and was confirmed in this study. In the present study, reduction in BWG occurred after oral challenge, whereas dietary Zn tended to alleviate the reduced BWG resulting from the S. Typhimurium infection. Regarding the effects of Zn supplementation on Salmonella infection, only a few reports in the literature can found. Hegazy & Adachi (2000) reported a significant improvement in growth performance, represented by relative body gain and feed efficiency, for the Zn-enriched diets fed to the Salmonella-challenged group. These data suggest that the Zn might exert a protection role in controlling Salmonella infection. However, the actual mechanisms underlying the protective effects of Zn are still not fully understood.

The intestinal mucosal barrier is essential in maintaining health and preventing tissue injury and several diseases, and in ensuring adequate provision of dietary nutrients to the whole body. Regarding its great importance, the present study focused on the protective role of Zn for intestinal mucosal barrier function.

The villous height/crypt ratio and the activities of sucrase are the most widely used as a marker of mucosal integrity and intestinal function (Clarke et al., 2006; Lamb-Rosteski et al., 2008). A reduction of both the villous/crypt height ratio and the activity of the ileal sucrase were observed after S. Typhimurium challenge in the present study, and this may indicate an impairment of mucosal barrier functional capacity by infection with S. Typhimurium. In support of this view, it was observed that the plasma endotoxin levels, as an indicator of intestinal permeability, were increased in the challenged birds. Endotoxin is produced by Gram-negative bacteria including S. enterica (Loppnow et al., 1986). Previous studies indicated that S. Typhimurium increased intestinal permeability and impaired the intestinal mucosal barrier of chicken (Turnbull & Snoeyenbos, 1974; Van Leeuwen et al., 2004; Fasina et al., 2010). An in vitro study conducted by Kops (1996) found that infection of intestinal epithelial cell line by S. Typhimurium increased permeability of the monolayer to mannitol, leading to bacteria consistently transmigrating in larger numbers. S. Typhimurium infection could cause bacterial translocation to the liver, spleen and blood, leading to endotoxin translocation to the blood circulation. Therefore, the enhanced villus height/crypt depth ratio and the tendency to reduce plasma endotoxin levels supported the idea of a tightening of intestinal permeability in chicks supplemented with Zn. This was consistent with the report of Roselli et al. (2003) that Zn might prevent the increase in TJ permeability induced by pathogens. This was also consistent with our previous results in mammals (Zhang & Guo, 2009).

Bacterial enteropathogens breach the mucus layer, and then they proceed to adhere to, or penetrate into, the underlying mucosal epithelium cells (Hecht, 1999; Gaudier et al., 2004; Opitz et al., 2007). Both the mucus layer and the underlying epithelium cell are components of the intestinal mucosal barriers. The mucus layer is the first line of defence between the vulnerable mucosa and the intestinal lumen. Any qualitative or quantitative changes in the mucin secretion and/or the expression patterns may affect the efficiency of the protective barrier. Mucin production is affected by both numbers of terminally differentiated goblet cells and mucin gene expression (Theodoropoulos & Carraway, 2007). In the intestine, the major mucin gene is MUC2, which codes for the main secreted mucin and is confined to goblet cells (Van Klinken et al., 1995). In the present study, the thickness of the mucus layer was indirectly evaluated by determining the expression of MUC2 genes in intestinal contents. S. Typhimurium challenge decreased the expression of MUC2, but Zn did not affect the expression of MUC2 in the ileum, suggesting that mucin synthesis might be inhibited by S. Typhimurium challenge but not by Zn. However, Fasina et al. (2010) showed that S. Typhimurium infection increased goblet cell density in the intestine of broiler chicks. Further investigation is needed into the thickness of the mucus layer and the amount of mucin produced in intestinal contents or faeces.

Another essential component of intestinal mucosal barriers was primarily regulated by a well-organized system of the epithelial junctional complex termed TJs (Gumbiner, 1993; Kucharzik et al., 2001). TJs create a paracellular permeability barrier and act as a fence preventing macromolecular transmission (Ballard et al., 1995). This is comprised of several unique proteins, including the transmembrane proteins occludin, junctional adhesion molecule, members of the claudin family, and linker proteins such as ZO-1, ZO-2 and ZO-3. Especially the members of the claudin family are key determinants in the regulation of epithelial barrier function in the intestine (Bucker et al., 2010). Occludin and claudin-1 are two of the most important components in the regulation of epithelial barrier function in the intestine (Fanning et al., 1998). TJs are primarily involved in the regulation of paracellular permeability. Disruption of the TJs will lead to an increase in intestinal permeability. To better clarify the molecular mechanism of Zn preventing S. Typhimurium-induced loss of epithelial barrier function, we detected the changes in the expression of occludin and claudin-1 at the mRNA level. The results of our work indicated that supplementation of Zn up-regulated occludin and claudin-1 mRNA expression in the ileum. These results suggested that regulation of occludin and claudin-1 expression by Zn may be involved in ameliorating increased intestinal permeability induced by S. Typhimurium challenge.

Invasion of S. Typhimurium in chickens produces a strong inflammatory response, leading to the severe systemic disease (Kaiser et al., 2000), and this might be associated with harmful effects to the integrity of the intestinal epithelium (Desmidt et al., 1998). The impact of S. Typhimurium on the TJ proteins in broilers was examined in this study for the first time. The present work indicated that S. Typhimurium challenge decreased claudin-1 and occludin mRNA expression in the ileum. This finding is supported by in vitro studies (Boyle et al., 2006; Koehler et al., 2007; Bucker et al., 2010). The reduction of claudin-1 and occludin expression might imply that less claudin-1 and occludin was present at TJs during S. Typhimurium infection in our study. Down-regulation of TJ proteins will result in enhancement of paracellular permeability and disruption of the intestinal barrier, thereby allowing the diffusion of macromolecules such as antigens, bacterial toxins (endotoxin) and pathogens from the intestinal lumen into the blood circulation (Parlesak et al., 2000; Kucharzik et al., 2001).

Zn has been shown to enhance intestinal mucosal repair and possess gastrohepatic protective effects (Cho, 1989; Roy et al., 1992; Sturniolo et al., 2002). Increasing evidence suggests that Zn plays an important role in maintaining epithelial barrier integrity and function of the gastrointestinal tract (Semrad, 1999; Roselli et al., 2003). Dietary supplementation of Zn has been shown to reduce intestinal permeability under a variety of conditions such as disease, malnutrition, stress and enteral pathogen challenge (Roy et al., 1992; Altaf et al., 2002; Sturniolo et al., 2002; Roselli et al., 2003; Tran et al., 2003; Zhang & Guo, 2009). Furthermore, electron microscopy studies showed that the number of perfused TJ complexes in experimental colitis were increased by 50% with Zn supplementation (Sturniolo et al., 2002), and our previous results indicated that Zn supplementation increased the expression of TJ proteins in mammals (Zhang & Guo, 2009). Thus, our result is consistent with these opinions. The present experiments did not evaluate the amount of changes, the distribution or the phosphorylation/dephosphorylation of TJ proteins in the intestine. There is still insufficient knowledge of the mechanisms of the protective effects of Zn against Salmonella.

In conclusion, dietary Zn supplementation appears to alleviate the loss of intestinal mucosal barrier function induced by S. Typhimurium challenge, and the partial mechanism might be related to the increased expression of occludin and claudin-1 in broiler chickens. These results provide new information on the critical role played by dietary Zn in controlling Salmonella infection.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 30901031), the Beijing Natural Science Foundation (No. 6102015), the Chinese Universities Scientific Fund (No. 2012QJ106), and the System for Poultry Production Technology, Beijing Innovation Research Team of Modern Agriculture.