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Dietary sodium selenite affects host intestinal and systemic immune response and disease susceptibility to necrotic enteritis in commercial broilers ab

bc

b

d

S. Z. Xu , S. H. Lee , H. S. Lillehoj & D. Bravo a

Department of Preventive Veterinary Medicine, College of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao, China b

Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD, USA c

National Academy of Agricultural Science, Rural Development Administration, WanjuGun, Jeollabuk-do, Korea

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d

InVivo ANH, Saint-Nolff, France Accepted author version posted online: 11 Nov 2014.Published online: 06 Jan 2015.

To cite this article: S. Z. Xu, S. H. Lee, H. S. Lillehoj & D. Bravo (2015): Dietary sodium selenite affects host intestinal and systemic immune response and disease susceptibility to necrotic enteritis in commercial broilers, British Poultry Science, DOI: 10.1080/00071668.2014.984160 To link to this article: http://dx.doi.org/10.1080/00071668.2014.984160

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British Poultry Science, 2015 http://dx.doi.org/10.1080/00071668.2014.984160

Dietary sodium selenite affects host intestinal and systemic immune response and disease susceptibility to necrotic enteritis in commercial broilers S. Z. XU1,2, S. H. LEE2,3, H. S. LILLEHOJ2,

AND

D. BRAVO4

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Department of Preventive Veterinary Medicine, College of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao, China, 2Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD, USA, 3National Academy of Agricultural Science, Rural Development Administration, Wanju-Gun, Jeollabuk-do, Korea, and 4InVivo ANH, Saint-Nolff, France

Abstract 1. This study was to evaluate the effects of supplementary dietary selenium (Se) given as sodium selenite on host immune response against necrotic enteritis (NE) in commercial broiler chickens. 2. Chicks were fed from hatching on a non-supplemented diet or diets supplemented with different levels of Se (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, broiler chickens were orally infected with Eimeria maxima at 14 d of age and then with Clostridium perfringens 4 d later using our previously established NE disease model. 3. NE-associated clinical signs and host protective immunity were determined by body weight changes, intestinal lesion scores, and serum antibodies against α-toxin and necrotic enteritis B (NetB) toxin. The effects of dietary Se on the gene expression of pro-inflammatory cytokines e.g., interleukin (IL)-1β, IL-6, IL-8LITAF (lipopolysaccharide-induced TNFα-factor), tumour necrosis factor (TNF) SF15, and inducible nitric oxide synthase (iNOS), glutathione peroxidase 7 (GPx7), and avian β-defensins (AvBD) 6, 8, and 13 (following NE infection) were analysed in the intestine and spleen. 4. The results showed that dietary supplementation of newly hatched broiler chicks with 0.25 Se mg/kg from hatch significantly reduced NE-induced gut lesions compared with infected birds given a nonsupplemented diet. The levels of serum antibody against the NetB toxin in the chicks fed with 0.25 and 0.50 mg/kg Se were significantly higher than the non-supplemented control group. The transcripts for IL-1β, IL-6, IL-8, iNOS, LITAF, and GPx7, as well as AvBD6, 8, and 13 were increased in the intestine and spleen of Se-supplemented groups, whereas transcript for TNFSF15 was decreased in the intestine. 5. It was concluded that dietary supplementation with optimum levels of Se exerted beneficial effects on host immune response to NE and reduced negative consequence of NE-induced immunopathology.

INTRODUCTION Necrotic enteritis (NE), caused by Clostridium perfringens, is among the most severe diseases for the poultry industry worldwide (Timbermont et al., 2011; Lee et al., 2013). It was estimated that NE inflicts a global economic loss of over $2 billion annually largely due to medical treatments and impaired growth performance in commercial poultry production (Van Immerseel et al., 2009). NE is difficult to reproduce experimentally using infection with C. perfringens alone because it usually manifests itself when a

variety of predisposing factors are present in the field, especially the serious enteric damage caused by coccidiosis or immunosuppressive factors including viruses and toxins (Williams et al., 2003). The experimental model of NE using co-infection with Eimeria maxima (E. maxima) and C. perfringens has been successfully established (Lee et al., 2011b). With the growing concerns over the emergence of drug-resistant bacteria and drug residues in the food chain, the traditional use of antibiotics to control NE is being reconsidered, even banned (Mot et al., 2013). Therefore, alternative strategies

Correspondence to: Hyun S. Lillehoj, Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service-United States Department of Agriculture, Building 1043, BARC-East, Beltsville, MD 20705, USA. E-mail: [email protected] Accepted for publication 1 October 2014. S. Z. Xu and S. H. Lee contributed equally to this manuscript.

© 2015 British Poultry Science Ltd

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S.Z. XU ET AL.

are urgently needed to mitigate the use of antibiotics and reduce the economic impact of NE. Vaccination offers an attractive approach to control avian NE, but there is currently no effective NE vaccine commercially available for broiler chickens (Lillehoj et al., 2007). The use of feed additives including prebiotics, probiotics, organic acids, essential oils, and phytonutrients can only marginally decrease the incidence of NE in broilers (Geier et al., 2010; Lillehoj and Lee, 2012; Lee et al., 2013). Selenium (Se) plays important roles in immune function, health, and productivity as an essential trace element (Kiremidjian-Schumacher and Stotzky, 1987). Earlier studies have shown that chickens fed on Se-deficient diets exhibited lesions in lymphoid organs and impaired immune function (Peng et al., 2011; Zhang et al., 2012), and Se supplementation exerted favourable effects on host immune responses including T cell responses, antibody production, and intracellular killing of microbes (Swain et al., 2000). Moreover, it has been reported that Se had an anti-cryptosporidial effect and inhibited Cryptosporidium parvum infection mainly by reducing oxidative stress (Huang and Yang, 2002). Enhancement of immune responses in chickens to coccidiosis was observed when diets were supplemented with Se (Colnago et al., 1984). However, the mechanisms exerted by dietary Se in NE infection remain unknown. The present study was conducted to evaluate the effects of dietary Se given as sodium selenite on host protective immunity and to explore the possible immune mechanisms which are influenced by Se by measuring the transcripts of pro-inflammatory cytokines and avian β-defensins (AvBD) in broilers afflicted with NE.

MATERIALS AND METHODS

d 1 and 18. To facilitate the development of NE, birds were switched to a standard grower diet (Table 1) containing 24.0% crude protein between d 18 and 24. All standard feeds include 0.1 mg Se/kg diet. Broilers were randomly distributed into the following 5 groups (15 chickens/group): (1) uninfected control (fed with basal diet and uninfected with E. maxima and C. perfringens); (2) control (fed with basal diet and infected with E. maxima and C. perfringens); (3) 0.25 mg Se/kg diet (fed with basal diet + 0.25 mg Se/kg diet and infected with E. maxima and C. perfringens); (4) 0.50 mg Se/kg diet (fed with basal diets + 0.50 mg Se/kg diet and infected with E. maxima and C. perfringens); and (5) 1.00 mg Se/kg diet (fed with basal diets + 1.00 mg Se/kg diet and infected with E. maxima and C. perfringens). Se was used in the form of sodium selenite. Feed and water were provided ad libitum throughout the experiment. All protocols were approved by the Beltsville Animal Care and Use Committee (BACUC) of the Beltsville Agricultural Research Center. Reproduction of experimental NE NE was induced using an earlier described disease model using E. maxima and C. perfringens (Jang et al., 2012). Briefly, chicks were housed in brooder pens in an Eimeria-free facility for 14 d post hatch and transferred into large hanging cages (3 birds/cage) at a separate location where they were infected and kept until the end of the experimental period. On d 14, chicks were orally infected with E. maxima Beltsville strain 41A (1.0 × 104 sporulated oocysts/bird), followed by an oral inoculation with C. perfringens strain Del-1 (1.0 × 109 CFU/bird) on d 18.

Measurement of body weights and lesion scores

Experimental animals and diets One-d-old male broiler chicks (Ross 308, Longenecker’s Hatchery, Elizabethtown, PA) housed in Petersime starter brooder units were fed with an antibiotic-free certified organic starter diet (Table 1) containing 17.0% crude protein between

Body weights were measured between 0 and 6 d post-infection with E. maxima. Gut lesion scores were evaluated at 2 d post-infection with C. perfringens on a scale of 0 (none) to 4 (high) in a blinded fashion by three independent observers (Prescott, 1979).

Table 1. Composition of the basal diets 1

Ingredients , g/kg Crude protein Carbohydrate Selenium-free mineral and vitamin mixture Fat Fibre Selenium2 (mg/kg) 1

Starter diet

Grower diet

170.0 610.0 150.0

240.0 540.0 150.0

47.0 24.0 0.10

Data were from USDA/FeedMill, Beltsville, MD. The Se concentration was calculated.

2

47.0 24.0 0.10

Measurement of serum antibody levels against Clostridium toxins Blood samples (4 randomly collected birds/ group) were taken by cardiac puncture at 6 d post-infection with C. perfringens immediately following euthanasia. Sera were prepared by lowspeed centrifugation and used to measure antibodies against necrotic enteritis B (NetB) toxin and α-toxin by ELISA as described by Lee et al. (2011c). Recombinant C. perfringens NetB toxin

DIETARY SELENIUM AND DISEASE

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and α-toxin were expressed in E. coli (Lee et al., 2011a). Briefly, 96-well microtitre plates were coated overnight with 1.0 μg/well of purified recombinant α-toxin or NetB toxin proteins. The plates were washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked with PBS containing 1% BSA for 1 h at room temperature. Diluted serum samples (1:20) were added (100 μl/well), incubated with gentle shaking for 2 h at room temperature, and washed with PBS-T. Bound antibodies were detected with peroxidase-conjugated rabbit anti-chicken IgG and 3,3ʹ,5,5ʹ-tetramethylbenzidine substrate (Sigma, St. Louis, MO). The optical density at 450 nm (OD450) was determined with an automated microplate reader (Bio-Rad, Richmond, CA). All samples were analysed in quadruplicate. Quantification of transcript levels of proinflammatory cytokine, GPx7, and AvBD Changes in cytokine, glutathione peroxidase 7 (GPx7), and AvBD levels in NE-afflicted chickens were measured in the spleen and intestine as described (Hong et al., 2006; Kim et al., 2008). At 2 d post-infection with C. perfringens, the spleen and jejunum tissues located proximal to the Meckel’s diverticulum were freshly collected from 4 chicks per group. The jejunums were cut longitudinally and washed three times with ice-cold Hank’s balanced salt solution (HBSS) containing 100 U/ml of penicillin and 100 μg/ml of streptomycin (Sigma, St. Louis, MO). The mucosal layer was carefully scraped away using a surgical scalpel (Nunc, Thermo Fisher Scientific Inc., Roskilde, Table 2. RNA target GAPDH IL-1β IL-6 IL-8 LITAF TNFSF15 iNOS GPx7 AvBD6 AvBD8 AvBD13

3

Denmark). The tissue was washed several times with HBSS containing 0.5 mM EDTA and 5% foetal calf serum (FCS) and incubated for 20 min at 37°C with constant swirling. Cells released into the supernatant were pooled, passed through nylon wool (Robbins Scientific, Sunnyvale, CA) to remove dead cells and cell aggregates and washed twice with HBSS. Intraepithelial lymphocytes (IEL) were purified on a discontinuous Percoll density gradient by centrifugation at 600 × g for 25 min at 24°C. Total RNA from intestine and spleen was extracted using TRIzol (Invitrogen, Carlsbad, CA). Five micrograms of total RNA were treated with 1.0 U of DNase I in 1.0 μl of 10 × reaction buffer (Sigma, St. Louis, MO) for 15 min at room temperature, 1.0 μl of stop solution was added to inactivate DNase I, and the mixture was heated at 70ºC for 10 min. RNA was reverse-transcribed using the StrataScript firststrand synthesis system (Stratagene, La Jolla, CA) according to the manufacturer’s recommendations. Quantitative RT-PCR oligonucleotide primers for chicken pro-inflammatory cytokines e.g., interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor (TNF) SF15, lipopolysaccharide-induced TNFα-factor (LITAF) and inducible nitric oxide synthase (iNOS), glutathione peroxidase (GPx7), avian β-defensins (AvBD6, 8, and 13), and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) as internal control are listed in Table 2. Amplification and detection were carried out using equivalent amounts of total RNA with the Mx3000P system and Brilliant SYBR Green QPCR master mix (Stratagene, La Jolla, CA) as previously described. Standard curves were generated using log10 diluted standard RNA. The levels of individual transcripts

Oligonucleotide primers used in real-time PCR

Primer sequences

PCR product size (bp)

F: 5ʹ-GGTGGTGCTAAGCGTGTTAT-3ʹ R: 5ʹ-ACCTCTGTCATCTCTCCACA-3ʹ F: 5ʹ-TGGGCATCAAGGGCTACA-3ʹ R: 5ʹ-TCGGGTTGGTTGGTGATG-3ʹ F: 5ʹ-CAAGGTGACGGAGGAGGAC-3ʹ R: 5ʹ-TGGCGAGGAGGGATTTCT-3ʹ F: 5ʹ-GGCTTGCTAGGGGAAATGA-3ʹ R: 5ʹ-AGCTGACTCTGACTAGGAAACTGT-3ʹ F: 5ʹ-TGTGTATGTGCAGCAACCCGTAGT-3ʹ R: 5ʹ-GGCATTGCAATTTGGACAGAAGT-3ʹ F: 5ʹ-CCTGAGTATTCCAGCAACGCA-3ʹ R: 5ʹ-ATCCACCAGCTTGATGTCACTAAC-3ʹ F: 5ʹ-TGGGTGGAAGCCGAAATA-3ʹ R: 5ʹ-GTACCAGCCGTTGAAAGGAC-3ʹ F: 5′-TCACCACCTTCAGAATGCAG-3ʹ R: 5′-TCCCAACTGGGAAATTCTTG-3ʹ F: 5ʹ-ATCCTTTACCTGCTGCTGTCTGT-3ʹ R: 5ʹ-GAGGCCATTTGGTAGTTGC-3ʹ F: 5ʹ-TGTGGCTGTTGTGTTTTGT-3ʹ R: 5ʹ-CTGCTTAGCTGGTCTGAGG-3ʹ F: 5ʹ-CATCGTTGTCATTCTCCTCCTC-3ʹ R: 5ʹ-GGTGGAGAACCTGCAGCAGCG-3ʹ

264

NM_204305.1

244

NM_204524.1

254

NM_204628.1

200

NM_205498.1

229

AY765397

292

NM_01024578

241

U46504

223

NM_001163245.1

250

NM_001001193

267

NM_001001781

163

NM_001001780

F, forward; R, reverse.

Accession no.

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S.Z. XU ET AL.

were then normalised to those of GAPDH analysed by the Q-gene program (Muller et al., 2002). Each sample was performed in triplicate. To normalise RNA levels between samples within an experiment, the logarithmic-scaled threshold cycle (Ct) values were transformed to linear units of normalised expression prior to calculating means and SEM for the references and individual targets, followed by the determination of mean normalised expression (MNE) using the Q-gene program.

body weight gain among three Se-supplemented treatment groups, as well as between the treatment and non-treatment groups (Figure 1a). However, NE-induced intestinal lesion scores in the group fed with 0.25 Se mg/kg showed significant reduction (P < 0.05) at 2 d post-infection with C. perfringens compared with the infected control group, but comparison of scores among three Se-supplemented groups revealed no significant differences (Figure 1b).

Effect of Se dietary supplementation on anti-toxin antibody levels

Data expressed as the mean ± SD or mean ± SEM were subjected to one-way ANOVA using SPSS software (SPSS 15.0 for Windows, Chicago, IL), and compared by the Duncan’s multiple range test. Differences between means were considered statistically significant at P < 0.05.

The levels of serum antibody to α-toxin were undetected in the infected groups (Figure 2a). The levels of serum antibody to NetB toxin in the groups fed 0.25 and 0.50 Se mg/kg were significantly (P < 0.05) higher than the group supplemented with 1.00 Se mg/kg and the nonsupplemented group (Figure 2b). In general the serum antibody levels against α-toxin and NetB toxin were linearly increased with the increasing levels of Se from 0.25 to 0.50 mg/kg, with the birds provided with 0.50 mg Se/kg showing the highest antibody levels.

RESULTS Effect of dietary Se supplementation on body weights and intestinal lesion scores During the period from 0 to 6 d post-infection with E. maxima, there were no significant differences on

Body weight gain/bird(g)

(a) 350

a

300

b

Control

0.25

250

b

b

b

200 150 100 50 0 Uninfected

0.50 1.00 Se (mg/kg) Infected with E. maxima /C. perfringens

(b) 4.0 a 3.0 Lesion score

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Statistical analysis

ab

ab

b 2.0

1.0

0.0 Control

0.25

0.50

1.00

Se (mg/kg)

Figure 1. Effects of dietary Se on body weights and intestinal lesions. Chickens were fed with a non-supplemented diet or diets supplemented with 0.25, 0.50 and 1.00 Se mg/kg from 1 d post hatch and uninfected or orally co-infected with 1.0 × 104 sporulated oocysts of E. maxima at 14 d and 1.0 × 109 CFU C. perfringens at 18 d to induce NE. (a) Body weights were calculated between 0 and 6 d post-infection with E. maxima. (b) Intestinal lesion scores were assessed at 2 d post-infection with C. perfringens on a scale of 0 (none) to 4 (high). Each bar represents the mean ± SD values (n = 12). Bars not sharing the same letters are significantly different according to the Duncan’s multiple range test (P < 0.05).

DIETARY SELENIUM AND DISEASE

Antibody to α-toxin (OD,450 nm)

(a)

5

1.00 0.80 0.60 NS 0.40 0.20 0.00 Control

0.25

0.50

1.00

Se (mg/kg) (b)

1.40 a

Antibody to NetB (OD,450 nm)

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1.20 1.00

a b

b

0.80 0.60 0.40 0.20 0.00 Control

0.25

0.50 Se (mg/kg)

1.00

Figure 2. Effects of dietary Se on the levels of antibody against C. perfringens toxin. Chickens were fed with a non-supplemented diet or diets supplemented with 0.25, 0.50, and 1.00 Se mg/kg from 1 d post hatch and uninfected or orally co-infected with 1.0 × 104 sporulated oocysts of E. maxima at 14 d and 1.0 × 109 CFU C. perfringens at 18 d to induce NE. Levels of antibody against α-toxin (a) and NetB toxin (b) were measured by ELISA at 6 d post-infection with C. perfringens. Each bar represents the mean ± SD values (n = 4). Bars not sharing the same letters are significantly different according to the Duncan’s multiple range test (P < 0.05). NS: not significant.

Effect of Se dietary supplementation on proinflammatory cytokine and GPx7 transcripts

Effect of Se dietary supplementation on transcript levels of AvBD

As shown in Figure 3, dietary Se supplementation enhanced (P < 0.05) the transcript levels of proinflammatory cytokines, IL-1β, IL-6, IL-8, LITAF, TNFSF15, iNOS, and GPx7 in the intestine and spleen compared with non-supplemented control at 6 d following E. maxima infection except for TNFSF15 in the intestine and in the spleen in the 1.00 mg Se/kg group as well as intestinal IL-1β in the 0.25 and 0.50 mg Se/kg groups and LITAF in the 0.25 Se mg/kg group. In the intestine, the highest levels of IL-1β, IL-6, TNFSF15, and GPx7 transcripts in the 1.00 mg Se/kg group and IL-8, iNOS and LITAF in the 0.50 mg Se/kg group were observed compared with the other Se-supplemented groups. In the spleen, the transcript levels of IL-1β, iNOS, and TNFSF15 in the 0.25 mg Se/kg group and IL-6, IL-8, LITAF, and GPx7 in the 1.00 mg Se/kg group were highest among Se-supplemented groups, and the transcript levels of the pro-inflammatory cytokines were increased or decreased gradually along with the increase of Se-supplementation in a dosedependent manner.

Analysis of the AvBD transcript levels revealed that chickens fed with Se-supplemented diets exhibited significantly (P < 0.05) increased expression of AvBD6, 8, and 13 in the intestine and spleen compared with non-supplemented control, with the exception of intestinal AvBD8 in the 0.25 and 1.00 mg Se/kg groups and splenic AvBD13 in the 0.25 mg Se/kg group. The mRNA levels of AvBD6 and AvBD13 were highest in the intestine of the 0.25 mg Se/kg group and in the spleen of the 1.00 mg Se/kg group, and the level of AvBD8 was highest both in the intestine and spleen of the 0.50 mg Se/kg group compared to the control (Figure 4).

DISCUSSION This study was to evaluate the effect of feeding different levels of dietary Se given as sodium selenite on immune responses to NE using an established disease model that depends on co-infection with

6

S.Z. XU ET AL. Intestine

Spleen

3.0E–02

Normalised mRNA (IL-1β/GAPDH)

a 2.5E–02 2.0E–02

b

1.5E–02 1.0E–02

b

b

5.0E–03 0.0E+00 Control

0.25

0.50

1.0E–02 9.0E–03 8.0E–03 7.0E–03 6.0E–03 5.0E–03 4.0E–03 3.0E–03 2.0E–03 1.0E–03 0.0E+00

a

b

c

Control

1.00

0.25

Normalised mRNA (IL-6/GAPDH)

3.0E–01

a

6.0E–01

2.5E–01

5.0E–01

2.0E–01

4.0E–01

1.5E–01

b b

3.0E–01

a

b

1.0E–01

5.0E–02 c

0.0E+00 0.25

0.50

Control

1.00

0.25

Se (mg/kg) 3.5E+00

Normalised mRNA (IL-8/GAPDH)

a

a

1.0E–01

Control

6.0E–01 b

5.0E–01

b

2.0E+00

b

4.0E–01

1.5E+00

3.0E–01

1.0E+00

c

2.0E–01 1.0E–01

c

0.0E+00 Control

0.25

0.50

Control

1.00

0.25

0.50

Normalised mRNA (LITAF/GAPDH)

4.0E+00

1.80E+00 1.60E+00 1.40E+00 1.20E+00 1.00E+00 8.00E–01 6.00E–01 4.00E–01 2.00E–01 0.00E+00

a

3.5E+00

a

3.0E+00

b

2.5E+00 b bc

c

2.0E+00 1.5E+00

c

1.0E+00

d

5.0E–01 0.0E+00

Control

0.25

0.50

Control

1.00

0.25

9.0E–05 8.0E–05 7.0E–05 6.0E–05 5.0E–05 4.0E–05 3.0E–05 2.0E–05 1.0E–05 0.0E+00

0.50

1.00

Se (mg/kg)

Se (mg/kg)

Normalised mRNA (TNFSF15/GAPDH)

1.00

Se (mg/kg)

Se (mg/kg)

4.0E–04

a

a

3.5E–04 3.0E–04 b

a

2.5E–04 b

b

2.0E–04 1.5E–04

b

1.0E–04 5.0E–05

b

0.0E+00

Control

0.25

0.50 Se (mg/kg)

a

4.0E–02 3.5E–02

1.0E–02 9.0E–03 8.0E–03 7.0E–03 6.0E–03 5.0E–03 4.0E–03 3.0E–03 2.0E–03 1.0E–03 0.0E+00

a

2.5E–02 2.0E–02 1.5E–02 b

0.0E+00 Control

0.25

0.50

0.25

0.50

1.00

Se (mg/kg)

3.0E–02

1.0E–02 5.0E–03

Control

1.00

a

4.5E–02

Normalised mRNA (iNOS/GAPDH)

1.00

a

7.0E–01 b

2.5E+00

5.0E–01

0.50 Se (mg/kg)

8.0E–01

a

3.0E+00

0.0E+00

a ab

Control

1.00

b

c

0.25

Se (mg/kg)

0.50

1.00

Se (mg/kg) 4.5E–05

2.5E–06 a

Normalised mRNA (GPx7/GAPDH)

1.00

2.0E–01

0.0E+00

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0.50

Se (mg/kg)

Se (mg/kg) 7.0E–01

a

2.0E–06

a

4.0E–05 3.5E–05 3.0E–05

1.5E–06

2.5E–05

ab

2.0E–05

1.0E–06 5.0E–07

1.5E–05 1.0E–05

0.0E+00

0.0E+00

b

b

b

5.0E–06 Control

0.25

0.50

Se (mg/kg)

1.00

b

c Control

0.25

0.50

1.00

Se (mg/kg)

Figure 3. Effects of dietary Se on the transcript levels of pro-inflammatory cytokines and GPx7 in intestine and spleen. Chickens were fed with a non-supplemented diet or diets supplemented with 0.25, 0.50, and 1.00 Se mg/kg from 1 d post hatch and uninfected or orally coinfected with 1.0 × 104 sporulated oocysts of E. maxima at 14 d and 1.0 × 109 CFU C. perfringens at 18 d to induce NE, and the levels of IL-1β, IL-6, IL-8, LITAF, TNFSF15, iNOS, and GPx7 transcript in the intestine and spleen were quantified by real-time quantitative RT-PCR at 2 d post-infection with C. perfringens, and normalised to GAPDH transcript levels. Each bar represents the mean ± SEM values (n = 4). Bars not sharing the same letters are significantly different (P < 0.05) according to the Duncan’s multiple range test.

DIETARY SELENIUM AND DISEASE

7

Intestine

AvBD6

Normalised mRNA (Defensin-6/GAPDH)

Spleen

ab

6.0E–01 4.0E–01 3.0E–01

1.4E+00 1.2E+00

b

5.0E–01

a

1.6E+00

a

7.0E–01

b

1.0E+00

b

8.0E–01

c

6.0E–01

2.0E–01

c

4.0E–01

1.0E–01

2.0E–01

0.0E+00

0.0E+00 Control

0.25

0.50

Control

1.00

0.25

AvBD8

Normalised mRNA (Defensin-8/GAPDH)

a

7.0E–05

a 2.0E–04

6.0E–05 5.0E–05

1.5E–04

4.0E–05 2.0E–05

b

1.0E–04 b

b

b

b

5.0E–05

1.0E–05

c 0.0E+00

0.0E+00 Control

0.25

0.50

Control

1.00

0.25

Se (mg/kg)

AvBD13

Normalised mRNA (Defensin-13/GAPDH)

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2.5E–03

a a

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8.0E–05

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a

bc

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0.50

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Figure 4. Effects of dietary Se on the transcript levels of AvBD6, 8, and 13 in spleen and intestine. Chickens were fed with a nonsupplemented diet or diets supplemented with 0.25, 0.50, and 1.00 Se mg/kg from 1 d post hatch and uninfected or orally co-infected with 1.0 × 104 sporulated oocysts of E. maxima at 14 d and 1.0 × 109 CFU C. perfringens at 18 d, and the transcript levels of AvBD6, 8, and 13 in the intestine and spleen were quantified by real-time quantitative RT-PCR at 2 d post-infection with C. perfringens, and normalised to GAPDH transcript levels. Each bar represents the mean ± SEM values (n = 4). Bars not sharing the same letters are significantly different (P < 0.05) according to the Duncan’s multiple range test.

E. maxima and C. perfringens (Lee et al., 2011b). The main finding from this study is that dietary supplementation of young chickens with 0.25 Se mg/kg significantly decreased gut NE lesions, increased the serum antibody levels against NetB toxin, and upregulated the transcripts of IL-1β, IL-6, IL-8, LITAF, TNFSF15, iNOS and AvBD6, 8, and 13 in the spleen and intestine (except TNFSF15 and AvBD8) following NE induction compared with chickens on nonsupplemented diet. These results indicate that dietary supplementation with optimum levels of Se enhanced the humoral and cellular immune responses and reduced negative consequences of NE-caused gut pathology. The dietary supplementation with 0.25 to 1.00 mg Se/kg in the diet had no effect on the body weights in NE-afflicted young broilers. Previous studies also demonstrated that dietary Se does not alter the growth performance of non-challenged broilers (Bou et al., 2005; Payne and Southern, 2005; Ahmad et al., 2012; Briens et al., 2013). The serum antibodies against C. perfringens αtoxin and NetB toxin play an important role in protection against NE based on our observation that apparently chicks with higher resistance have significantly higher anti-toxin antibody levels compared to those birds with clinical signs of NE (Lee et al., 2012). Therefore increasing resistance of

chicks to NE may be associated with enhanced serum anti-toxin antibody levels. Our study showed that optimum level of Se supplementation between 0.25 and 0.50 mg/kg increased the serum antibodies against α-toxin and NetB toxin. This is consistent with the earlier reports that supplementary Se at 0.20 mg/kg increased serum antibody titres after immunisation against Newcastle disease virus vaccine (Singh et al., 2006), and IgM level in broilers supplemented with 0.3 to 1.0 mg/kg of nanosized Se was increased (Cai et al., 2012). As expected, the high levels of circulating antibodies against α-toxin and NetB toxin coincided with the low levels of αtoxin and NetB toxin in each Se-supplemented group due to their neutralisation (Unpublished data). Pro-inflammatory cytokines regulate host immunity against multiple pathogens through immune cell differentiation, proliferation, apoptosis and NO production. Our present study found that dietary Se enhanced the production of proinflammatory cytokines in the gut and spleen after NE induction, as evidenced by the increased transcripts of the IL-1β, IL-6, IL-8, iNOS, and LITAF. In fact, transient induction of pro-inflammatory cytokines by chicken gut microflora resulted in an activation and normalisation of the innate immune system in the gut and increased resistance to

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S. enteritidis infection (Crhanova et al., 2011). It is possible that these enhanced immune mediators or early activation of certain innate immune response during the early phase of infection process (dependent on the optimum supplementation of Se immediately after hatching) help in providing the enhanced protective effect. Unknown factors that may be relevant in host-pathogen interaction in NE include the relative kinetics of expression of the individual pro-inflammatory cytokines, their sites of production in the gut, and the relative activities of these immune mediators during NE infection. Interestingly, in NE-afflicted chickens fed on a Sesupplemented diet, the expression of TNFSF15 was significantly increased in the spleen and decreased in the intestine, which may indicate that the TNFSF15 regulated by dietary Se may work differently in different organs to maintain homeostasis in chicks exposed to NE. GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum containing a cysteine redox centre. Unlike other members of the GPx family, GPx7 catalyses a peroxidatic cycle using only one cysteine residue through a mechanism in which reduced glutathione and protein disulphide isomerase serve as alternative substrates (Bosello-Travain et al., 2013). In an earlier study, GPx levels in blood are lower in Se-deficient mice or chicks compared with animals with normal Se levels (Wang et al., 2009). In this study, Se supplementation increased the expression of GPx in both the intestine and spleen, and GPx expression is dependent on Se status. AvBD peptides are considered as one of the key components of innate immune system in avian species, and 14 AvBD genes (AvBD1–14) have been identified in the leukocytes, epithelial cells, or EST of chicken genome (Michailidis et al., 2012). Studies have shown that AvBD6 exhibits strong bactericidal and fungicidal activity against food-borne pathogens such as Campylobacter jejuni, Salmonella enterica serovar Typhimurium, C. perfringens, and Escherichia coli (Van Dijk et al., 2007). Our study showed that the levels of AvBD6, 8, and 13 were significantly increased in the Se-supplemented groups compared with the non-supplemented control group, except for the intestinal AvBD8 in the 0.25 and 1.0 Se mg/kg groups and splenic AvBD13 in the 0.25 Se mg/kg group. Consistent with studies that human defensins had a parasiticidal role against Trypanosoma cruzi, Cryptosporidium parvum, and Toxoplasma gondii (Madison et al., 2007; Tanaka et al., 2010; Carryn et al., 2012), the AvBD6, 8, and 13 may have bactericidal and coccidiocidal properties that lead to enhanced immune protection against NE when chicks are fed with an optimum level of dietary Se. Many studies have demonstrated mutual upregulation of human defensins and pro-inflammatory cytokines in various tissues (Pivarcsi et al., 2005). Enhanced expression of defensin genes is

usually associated with heightened innate immune response following the infection with pathogens (Menendez and Brett Finlay, 2007). Our earlier study indicated that not only pro-inflammatory cytokines e.g., IL-1β, IL-6, IL-17 F, and TNFSF15, but also AvBD6, 8, and 13 were all highly induced in broilers following E. maxima and C. perfringens co-infection (Hong et al., 2012). This study also demonstrated that dietary Se given as sodium selenite up-regulated the expressions of AvBD and pro-inflammatory cytokines in broiler chickens in NE model. In conclusion, this study showed that dietary supplementation of newly hatched broiler chicks with sodium selenite enhanced protective immunity against experimental NE and up-regulated the proinflammatory cytokines, glutathione peroxidase and AvBD expression. Since these are important players in the protective host response to pathogens, the exact mechanisms by which dietary Se interacts with the host immune system to enhance responses to NE need to be elucidated.

ACKNOWLEDGEMENTS The authors are grateful to Margie Nichols, Stacy O’Donnell, SeungIk Jang, Misun Jeong, Duk Kyung Kim, and SeungKyoo Lee (USDA-ARS, Beltsville, MD) for their assistance.

FUNDING This study was partially supported by a formal Trust agreement between ARS-USDA and Pancosma S.A., the project “Investigation of the functional activity and the development of functional foods of Allium hookeri (PJ010490)” of Rural Development Administration, Korea, and the International Cooperation Program for Excellent Lecturers of 2011 by Shandong Provincial Education Department, P.R. China.

REFERENCES AHMAD, H., TIAN, J., WANG, J., KHAN, M.A., WANG, Y., ZHANG, L. & WANG, T. (2012) Effects of dietary sodium selenite and selenium yeast on antioxidant enzyme activities and oxidative stability of chicken breast meat. Journal of Agricultural and Food Chemistry, 60: 7111–7120. doi:10.1021/jf3017207 BOSELLO-TRAVAIN, V., CONRAD, M., COZZA, G., NEGRO, A., QUARTESAN, S., ROSSETTO, M., ROVERI, A., TOPPO, S., URSINI, F., ZACCARIN, M. & MAIORINO, M. (2013) Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7. Biochimica Et Biophysica Acta, 1830: 3846–3857. doi:10.1016/j.bbagen.2013.02.017 BOU, R., GUARDIOLA, F., BARROETA, A.C. & CODONY, R. (2005) Effect of dietary fat sources and zinc and selenium supplements on the composition and consumer acceptability of chicken meat. Poultry Science, 84: 1129–1140. doi:10.1093/ps/84.7.1129 BRIENS, M., MERCIER, Y., ROUFFINEAU, F., VACCHINA, V. & GERAERT, P.-A. (2013) Comparative study of a new organic selenium source v. seleno-yeast and mineral selenium sources on muscle selenium enrichment and selenium digestibility in

Downloaded by [New York University] at 13:30 11 January 2015

DIETARY SELENIUM AND DISEASE broiler chickens. British Journal of Nutrition, 110: 617–624. doi:10.1017/S0007114512005545 CAI, S.J., WU, C.X., GONG, L.M., SONG, T., WU, H. & ZHANG, L.Y. (2012) Effects of nano-selenium on performance, meat quality, immune function, oxidation resistance, and tissue selenium content in broilers. Poultry Science, 91: 2532–2539. doi:10.3382/ps.2012-02160 CARRYN, S., SCHAEFER, D.A., IMBODEN, M., HOMAN, E.J., BREMEL, R.D. & RIGGS, M.W. (2012) Phospholipases and cationic peptides inhibit Cryptosporidium parvum sporozoite infectivity by parasiticidal and non-parasiticidal mechanisms. Journal of Parasitology, 98: 199–204. doi:10.1645/GE-2822.1 COLNAGO, G.L., JENSEN, L.S. & LONG, P.L. (1984) Effect of selenium and vitamin E on the development of immunity to coccidiosis in chickens. Poultry Science, 63: 1136–1143. doi:10.3382/ps.0631136 CRHANOVA, M., HRADECKA, H., FALDYNOVA, M., MATULOVA, M., HAVLICKOVA, H., SISAK, F. & RYCHLIK, I. (2011) Immune response of chicken gut to natural colonization by gut microflora and to Salmonella enterica serovar enteritidis infection. Infection and Immunity, 79: 2755–2763. doi:10.1128/IAI.01375-10 GEIER, M.S., MIKKELSEN, L.L., TOROK, V.A., ALLISON, G.E., OLNOOD, C.G., BOULIANNE, M., HUGHES, R.J. & CHOCT, M. (2010) Comparison of alternatives to in-feed antimicrobials for the prevention of clinical necrotic enteritis. Journal of Applied Microbiology, 109: 1329–1338. doi:10.1111/j.13652672.2010.04758.x HONG, Y.H., LILLEHOJ, H.S., LILLEHOJ, E.P. & LEE, S.H. (2006) Changes in immune-related gene expression and intestinal lymphocyte subpopulations following Eimeria maxima infection of chickens. Veterinary Immunology and Immunopathology, 114: 259–272. doi:10.1016/j.vetimm.2006.08.006 HONG, Y.H., SONG, W., LEE, S.H. & LILLEHOJ, H.S. (2012) Differential gene expression profiles of β-defensins in the crop, intestine, and spleen using a necrotic enteritis model in 2 commercial broiler chicken lines. Poultry Science, 91: 1081–1088. doi:10.3382/ps.2011-01948 HUANG, K. & YANG, S. (2002) Inhibitory effect of selenium on Cryptosporidium parvum infection in vitro and in vivo. Biological Trace Element Research, 90: 261–272. doi:10.1385/ BTER:90:1-3:261 JANG, S.I., LILLEHOJ, H.S., LEE, S.-H., LEE, K.W., LILLEHOJ, E.P., HONG, Y.H., AN, D.-J., JEONG, W., CHUN, J.-E., BERTRAND, F., DUPUIS, L., DEVILLE, S. & AROUS, J.B. (2012) Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide™ ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens. Vaccine, 30: 5401–5406. doi:10.1016/j.vaccine.2012.06.007 KIM, D.K., LILLEHOJ, H.S., HONG, Y.H., PARK, D.W., LAMONT, S.J., HAN, J.Y. & LILLEHOJ, E.P. (2008) Immune-related gene expression in two B-complex disparate genetically inbred Fayoumi chicken lines following Eimeria maxima infection. Poultry Science, 87: 433–443. doi:10.3382/ps.2007-00383 KIREMIDJIAN-SCHUMACHER, L. & STOTZKY, G. (1987) Selenium and immune responses. Environmental Research, 42: 277–303. doi:10.1016/S0013-9351(87)80194-9 LEE, K., LILLEHOJ, H.S., LI, G., PARK, M.-S., JANG, S.I., JEONG, W., JEOUNG, H.-Y., AN, D.-J. & LILLEHOJ, E.P. (2011a) Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate: ferredoxin oxidoreductase (PFO) of C. perfringens. Research in Veterinary Science, 91: e80–86. doi:10.1016/j.rvsc.2011.01.017 LEE, K.W., LILLEHOJ, H.S., JEONG, W., JEOUNG, H.Y. & AN, D.J. (2011b) Avian necrotic enteritis: experimental models, host immunity, pathogenesis, risk factors, and vaccine development. Poultry Science, 90: 1381–1390. doi:10.3382/ps.2010-01319 LEE, K.W., LILLEHOJ, H.S., PARK, M.S., JANG, S.I., RITTER, G.D., HONG, Y.H., JEONG, W., JEOUNG, H.Y., AN, D.J. & LILLEHOJ, E.P. (2012) Clostridium perfringens α-Toxin and NetB toxin

9

antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens. Avian Diseases, 56: 230–233. doi:10.1637/9847070711-ResNote.1 LEE, S.H., LILLEHOJ, H.S., JANG, S.I., LEE, K.W., BRAVO, D. & LILLEHOJ, E.P. (2011c) Effects of dietary supplementation with phytonutrients on vaccine-stimulated immunity against infection with Eimeria tenella. Veterinary Parasitology, 181: 97–105. doi:10.1016/j.vetpar.2011.05.003 LEE, S.H., LILLEHOJ, H.S., JANG, S.I., LILLEHOJ, E.P., MIN, W. & BRAVO, D.M. (2013) Dietary supplementation of young broiler chickens with capsicum and turmeric oleoresins increases resistance to necrotic enteritis. British Journal of Nutrition, 110: 840–847. doi:10.1017/S0007114512006083 LILLEHOJ, H.S., KIM, C.H., KEELER, C.L. & ZHANG, S. (2007) Immunogenomic approaches to study host immunity to enteric pathogens. Poultry Science, 86: 1491–1500. doi:10.1093/ps/ 86.7.1491 LILLEHOJ, H.S. & LEE, K.W. (2012) Immune modulation of innate immunity as alternatives-to-antibiotics strategies to mitigate the use of drugs in poultry production. Poultry Science, 91: 1286–1291. doi:10.3382/ps.2012-02374 MADISON, M.N., KLESHCHENKO, Y.Y., NDE, P.N., SIMMONS, K.J., LIMA, M.F. & VILLALTA, F. (2007) Human defensin α-1 causes trypanosoma cruzi membrane pore formation and induces DNA fragmentation, which leads to trypanosome destruction. Infection and Immunity, 75: 4780–4791. doi:10.1128/IAI.00557-07 MENENDEZ, A. & BRETT FINLAY, B. (2007) Defensins in the immunology of bacterial infections. Current Opinion in Immunology, 19: 385–391. doi:10.1016/j.coi.2007.06.008 MICHAILIDIS, G., AVDI, M. & ARGIRIOU, A. (2012) Transcriptional profiling of antimicrobial peptides avian β-defensins in the chicken ovary during sexual maturation and in response to Salmonella enteritidis infection. Research in Veterinary Science, 92: 60–65. doi:10.1016/j.rvsc.2010.10.010 MOT, D., TIMBERMONT, L., DELEZIE, E., HAESEBROUCK, F., DUCATELLE, R. & VAN IMMERSEEL, F. (2013) Day-of-hatch vaccination is not protective against necrotic enteritis in broiler chickens. Avian Pathology, 42: 179–184. doi:10.1080/ 03079457.2013.778955 MULLER, P.Y., JANOVJAK, H., MISEREZ, A.R. & DOBBIE, Z. (2002) Processing of gene expression data generated by quantitative real-time RT-PCR. BioTechniques, 32: 1372–1374, 1376, 1378–1379. PAYNE, R.L. & SOUTHERN, L.L. (2005) Comparison of inorganic and organic selenium sources for broilers. Poultry Science, 84: 898–902. doi:10.1093/ps/84.6.898 PENG, X., CUI, H.-M., DENG, J., ZUO, Z. & CUI, W. (2011) Low dietary selenium induce increased apoptotic thymic cells and alter peripheral blood T cell subsets in chicken. Biological Trace Element Research, 142: 167–173. doi:10.1007/ s12011-010-8756-4 PIVARCSI, A., NAGY, I., KORECK, A., KIS, K., KENDERESSY-SZABO, A., SZELL, M., DOBOZY, A. & KEMENY, L. (2005) Microbial compounds induce the expression of pro-inflammatory cytokines, chemokines and human β-defensin-2 in vaginal epithelial cells. Microbes and Infection, 7: 1117–1127. doi:10.1016/j.micinf.2005.03.016 PRESCOTT, J.F. (1979) The prevention of experimentally induced necrotic enteritis in chickens by avoparcin. Avian Diseases, 23: 1072–1074. doi:10.2307/1589625 SINGH, H., SODHI, S. & KAUR, R. (2006) Effects of dietary supplements of selenium, vitamin E or combinations of the two on antibody responses of broilers. British Poultry Science, 47: 714–719. doi:10.1080/00071660601040079 SWAIN, B.K., JOHRI, T.S. & MAJUMDAR, S. (2000) Effect of supplementation of vitamin E, selenium and their different combinations on the performance and immune response of broilers. British Poultry Science, 41: 287–292. doi:10.1080/ 713654938

10

S.Z. XU ET AL.

Downloaded by [New York University] at 13:30 11 January 2015

TANAKA, T., RAHMAN, M.M., BATTUR, B., BOLDBAATAR, D., LIAO, M., UMEMIYA-SHIRAFUJI, R., XUAN, X. & FUJISAKI, K. (2010) Parasiticidal activity of human α-defensin-5 against Toxoplasma gondii. In Vitro Cellular & Developmental Biology Animal, 46: 560–565. doi:10.1007/s11626-009-9271-9 TIMBERMONT, L., HAESEBROUCK, F., DUCATELLE, R. & VAN IMMERSEEL, F. (2011) Necrotic enteritis in broilers: an updated review on the pathogenesis. Avian Pathology, 40: 341–347. doi:10.1080/03079457.2011.590967 VAN DIJK, A., VELDHUIZEN, E.J., KALKHOVE, S.I., TJEERDSMA-VAN BOKHOVEN, J.L., ROMIJN, R.A. & HAAGSMAN, H.P. (2007) The β-defensin gallinacin-6 is expressed in the chicken digestive tract and has antimicrobial activity against foodborne pathogens. Antimicrobial Agents and Chemotherapy, 51: 912–922. doi:10.1128/AAC.00568-06 VAN IMMERSEEL, F., ROOD, J.I., MOORE, R.J. & TITBALL, R.W. (2009) Rethinking our understanding of the pathogenesis

of necrotic enteritis in chickens. Trends in Microbiology, 17: 32–36. doi:10.1016/j.tim.2008.09.005 WANG, C., WANG, H., LUO, J., HU, Y., WEI, L., DUAN, M. & HE, H. (2009) Selenium deficiency impairs host innate immune response and induces susceptibility to Listeria monocytogenes infection. BMC Immunology, 10: 55. doi:10.1186/14712172-10-55 WILLIAMS, R.B., MARSHALL, R.N., LA RAGIONE, R.M. & CATCHPOLE, J. (2003) A new method for the experimental production of necrotic enteritis and its use for studies on the relationships between necrotic enteritis, coccidiosis and anticoccidial vaccination of chickens. Parasitology Research, 90: 19–26. ZHANG, Z.-W., WANG, Q.-H., ZHANG, J.-L., LI, S., WANG, X.-L. & XU, S.-W. (2012) Effects of oxidative stress on immunosuppression induced by selenium deficiency in chickens. Biological Trace Element Research, 149: 352–361.I. doi:10.1007/s12011-012-9439-0

Dietary sodium selenite affects host intestinal and systemic immune response and disease susceptibility to necrotic enteritis in commercial broilers.

1. This study was to evaluate the effects of supplementary dietary selenium (Se) given as sodium selenite on host immune response against necrotic ent...
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