Effects of chicken intestinal antimicrobial peptides on humoral ...

Archives of Animal Nutrition October 2006; 60(5): 427 – 435

Effects of chicken intestinal antimicrobial peptides on humoral immunity of chickens and antibody titres after vaccination with infectious bursal disease virus vaccine in chicken

YANG YURONG1, JIANG YIBAO2, SHE RUIPING1, YIN QINGQIANG2, PENG KAISONG1, BAO HUIHUI1, WANG DECHENG1, LIU TIANLONG1, & ZHOU XUEMEI1 1

College of Veterinary Medicine, China Agricultural University, Beijing, and 2Henan Agricultural University, Zhengzhou, PR China

(Received 7 February 2006; accepted 2 June 2006)

Abstract Sixty chickens were randomly divided into two groups (30 chickens in each group) to determine the effect of oral administration of chicken intestinal antimicrobial peptides (CIAMP) on the humoral immune response. Chickens of both groups were fed the same diet. In the treatment group chickens received drinking water supplemented with CIAMP (1 mg/ml) right after hatching. Samples of blood, bursa of Fabricus, spleen and intestine were taken at day 1, 4, 7, 10 and 17 of experiment. CIAMP supplementation enhanced the content of IgG and IgM in serum from day 4 – 10 and day 10 – 17, respectively, (p 5 0.05), IgM-forming cells in bursa of Fabricus and spleen at the age of 7 days (p 5 0.05) and IgG-forming cells in bursa of Fabricus at the age of 4 days (p 5 0.05). In addition, CIAMP enhanced the IgA-forming cells in caecal tonsils diffuse area at day 4 (p 5 0.05). Furthermore, CIAMP enhanced the antibody response to infectious bursal disease virus vaccine (IBDV) in chickens 21 days following IBDV vaccine administration (p 5 0.05). These results suggested that CIAMP could modulate the humoral immune response of chickens and increased the antibody titres of infectious bursal disease virus vaccine.

Keywords: Antimicrobial peptides, chicken, humoral immunity, immune organ, intestine

1. Introduction Antimicrobial peptides are polypeptides of fewer than 100 amino acids and are found in all species, ranging from plants and insects to animals (Ganz, 2003; Meyerholz & Ackermann, 2005). Antimicrobial proteins are stored in the granules of neutrophils and are produced by leucocytes and epithelial cells lining the environmental interface of the gastrointestinal, urogenital tract, tracheobronchial tree and skin (Oppenheim et al., 2003; Hancock & Correspondence: She Ruiping, College of Veterinary Medicine, China Agricultural University, Beijing 100094, P. R. China. Tel: þ86 10 62733060. Fax: þ86 10 62733321. E-mail: [email protected], [email protected] ISSN 1745-039X print/ISSN 1477-2817 online ª 2006 Taylor & Francis DOI: 10.1080/17450390600884484

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Scott, 2000). Over 880 such peptides have been discovered in plant, insects and animals (http://www.bbcm.units.it/*tossi/pag5.htm). Thirteen chicken antimicrobial peptides have been isolated from bone marrow, tongue, trachea, bursa of Fabricus, brain, kidney, testicle, ovary, male and female reproductive tract, liver and the urogenital tract, colon, rectum and lung. One antimicrobial peptide which is expressed in chicken intestinal epithelial cells is b-defensins Gal-13 (Xiao et al., 2004). Defensins have the capacity to kill and/or inactivate a particular spectrum of bacteria, fungi or some enveloped virus in vitro (Boman, 1995; Ganz & Lehrer, 1998). Recently, some of the mammalian antimicrobial peptides have been shown to have a second major function of rapidly chemo-attracting and activating host cells to engage in innate host defense and/or adaptive immune responses (Chertov et al., 1996; Yang et al., 2000). The use of antibiotics to promote growth has caused harmful residues in the food chain and promoted the spreading of resistance genes (Witte, 1997). Natural compounds have been considered to replace antibiotics, antimicrobial peptides may be used as feed additives to provide an alternative to antibiotics in animal feed. However, there are few studies of the effects of defensins on avian humoral immunity. The objective of this study was to evaluate the effect of chicken intestinal antimicrobial peptides (CIAMP) on humoral immunity of chicken and to investigate the potential use of antimicrobial peptides in modulation of the immune response for animal health.

2. Materials and methods 2.1. Preparation of defensins CIAMP were isolated from chicken intestinal mucus as previous described (Wang & She, 2003; Ghosh et al., 2002). Chicken colon and rectum were washed with sterile saline (0.9% sodium chloride). The chorion and fat were removed and mucosa was isolated from the intestine. The mucosa was homogenized by a muller in ice-cold aqueous 5% acetic acid (1:10 w/v) containing protease inhibitor phenyl methyl sulfonyl fluoride (PMSF) (1 mmol/l). Extracts were placed in a boiling water bath for 10 min, and then cooled rapidly. The pellet was discarded after centrifugation at 6440 g for 30 min at 48C. The clarified extracts were sonicated for 30 sec on ice and stirred overnight at 48C. Ice-cold 5% acetic acid (1:1 v/v) was added in the presence of PMSF and extracted overnight at 48C. The extract was centrifuged at 6440 g for 30 min at 48C. The potential of hydrogen (pH) of the clarified extract was adjusted to 6.0 with sodium hydroxide. The precipitates were removed by centrifugation (6440 g for 30 min at 48C) and the supernatant was loaded onto 106300 mm Sephadex G-100 column and eluted by 0.2 mol sodium acetate buffer per litre. CIAMP elution was analysed by agarose diffusion assay (Lehrer et al., 1991) and Pasteurella cuniculicida was used as test organisms. The fractions of interest were purified with Tricine-PAGE (Schagger & von Jagow, 1987) and subjected to NH2-terminal sequence determination by Edman degradation at the Institute of Process Engineering Chinese Academy of Sciences. The results of amino acid analysis corresponded with the amino acid composition of Gal-13 by one N-terminal residue. Interested fractions were collected and vacuum frozen dry before being stored at 08C for future use. 2.2. Animals and experimental treatments A total of 60 one-day-old healthy chickens (Lohmann Brown) were used in this experiment. These chickens were free of Newcastle disease virus and infectious bursal disease virus. They

Antimicrobial peptides and immunity of chickens

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were randomly assigned to two groups, 30 chickens for each group. Both groups received the same diet. In the CIAMP group, chickens received CIAMP at the final concentration of 1 mg/ml in drinking water every day just after hatching. In the control group, chickens received drinking water free of CIAMP. Each group of chickens was housed in an individual cage. None of the chickens were vaccinated, and feed and water were given ad libitum. 2.3. Sampling Five chickens from each group were randomly sampled at day 1, 4, 7, 10 and 17. Chickens were weighted and sacrificed. Blood samples were collected with heparinized tubes and also blood clotting tubes. Heparinized blood samples were processed immediately for lymphocyte proliferation assays. Serum samples were stored at 7208C until analysis. Spleen, bursa of Fabricus, duodenum, jejunum and caecal tonsils were collected and fixed in 10% (v/v) neutral buffered formalin and stored at 48C for further analysis. Chickens of both groups received infectious bursal disease virus vaccine (medium virulence live vaccine, Beijing Academy of Agriculture and Forestry Science, PR China ) via intranasal (50 ml) and eye drop (50 ml) routes at the age of 20 days. Serum samples were collected for analysis at day 14, 21, 28 and 35 after vaccination. 2.4. Immunological assays 2.4.1. Measurement of the relative content of IgG and IgM in sera. Indirect ELISA test (Yang et al., 2005): ELISA plates, coated with 150 ml serum (diluted 100-fold) per well in sodium carbonate buffer (pH 9.6), were incubated overnight at 48C. Then, plates were washed three times with phosphate buffer saline (PBS). The serum of rabbit anti-chicken IgG (produced by the College of Veterinary Medicine, China Agricultural University, PR China) or rabbit antichicken IgM (Produced by the College of Veterinary Medicine, China Agricultural University, PR China) was diluted 100-fold in PBS, and then 150 ml of diluted serum was placed in each well respectively, and incubated at 378C for 2 h. After washing three times, 150 ml of horseradish peroxidase (HRP) goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc. West Grove, USA), diluted 5000-fold in PBS, was added, then the ELISA-plate was incubated at 378C for 2 h. After that, the plate was washed three times with PBS before 100 ml of o-phenylenediamine (OPD)-hydrogen peroxide were added to each well, then the plate was kept at 378C without light. Twenty minutes later, the reaction was stopped by adding 50 ml H2SO4 (1 mol/l) to each well. The optical density (OD) values were measured at 490 nm by ELISA plate reader (Thermo Electron Corporation, Waltham, USA). Each sample was assayed in triplicate. 2.4.2. Measurement of B lymphocyte proliferation in peripheral blood. The effect of CIAMP on the proliferation of lymphocytes was examined in vitro. Fifty microlitre lipopolysaccharides (LPS) at a concentration of 40 mg/ml diluted with RPMI 1640 (L-glutamine 300 mg/l, NaHCO3 2000 mg/l, 4-(2-hydroxyethyl)-1-piperazineethanesufonic acid (HEPES) 25 mM, Phenol Red 5 mg/l, penicillin 200 IU/m1, streptomycin 200 mg/m1) were placed in 96-well plate. Then 50 ml lymphocytes (1.0 107/ml) were prepared from peripheral blood, which was obtained from both groups. Chickens were placed in 96-well plate in RPMI 1640 supplemented with 5% foetal bovine serum (FBS) per well. After incubation (at 378C, 5% CO2) in a humidified atmosphere for 48 h, 10 ml of 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (final concentration 5 mg/ml) was added to each well. The plate was incubated for 4 h in the humidified atmosphere, and 100 ml of 10% SDS in

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0.01 mol/l HCl solution were added into each well. The plate was kept overnight in the incubator. Absorbance was recorded at 570 nm by ELISA plate reader after the purple formazan crystals was solubilized. Each plate had RPMI 1640 as blank, and each sample had three repetitions. 2.4.3. Measurement of antibody forming cells in immune organ and intestine. In order to count antibody forming cells present in the immune tissue, indirect immunohistochemistry staining on tissue sections was performed as follows: tissue samples of spleen, bursa of Fabricus, duodenum, jejunum and caecal tonsils of chicken, preserved in formalin, were dehydrated and embedded in paraffin using routine methods. Serial paraffin sections (5 mm) of intestine were obtained and kept at 378C for more than 12 h. The sections were immersed in three consecutive washings in xylol for 5 min to remove paraffin, and then hydrated with five consecutive lots of alcohol 100%, 100%, 95%, 80%, 70% and PBS respectively. Afterwards, sections were incubated for 30 min and blocked with 0.3% peroxide-methanol at room temperature. All the following steps were carried out in a moist chamber: (i) Incubation with blocking buffer containing 20% normal goat serum in PBS (0.01 mol/l, pH 7.4) at 378C for 30 min; (ii) Discarding the goat serum and incubating the sections overnight at 48C with the serum of rabbit anti-chicken IgG or rabbit antichicken IgM (College of Veterinary Medicine, China Agricultural University, PR China) or mouse anti-chicken IgA monoclonal antibody (Southern Biotechnology, Birmingham, USA) diluted in PBS; (iii) Rinsing (365 min) in PBS-T (0.01 M PBS, pH 7.4, KH2PO4 0.02%, N2HPO4 0.29%, KCl 0.02%, NaCl 0.8%, BSA 0.05%, Tween-20 0.05%, TritonX-100 0.0015%); (iv) Incubation with HRP anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc. West Grove, USA) or HRP anti-mouse IgG antibody (Zymed Laboratories, Inc. San Diego, USA) diluted in PBS at 378C for 2 h; (v) Rinsing (365 min) in PBS-T; (vi) Incubation with 3,3diaminobenzidin (DAB), kept at 378C without light; (vii) Rinsing (365 min) in PBS-T. (viii) Haematoxylin stained; (ix) Dehydration, clearing and mounting with neutral gums. The number of positive cells in spleen red pulp, bursa of Fabricus, lamina propria of intestine were counted under the light Olympus microscope magnified 200 times. The results were expressed as the number of cells per mm2. 2.4.4. Measurement of antibodies against the infectious bursal disease virus in the sera of chicken. Indirect ELISA test was used to measure antibodies of infectious bursal disease virus in the sera of chicken. ELISA plates were coated with 150 ml of infectious bursal disease virus (China Institute of Veterinary Drug Control, Beijing, PR China) in sodium carbonate buffer (pH 9.6) each well and were incubated overnight at 48C. Then, the plates were washed three times with PBS. The sera (sera of chicken after injected with infectious bursal disease virus vaccine, collected at 14, 21, 28 and 35 days after vaccination) were diluted 100-fold in PBS, and then 150 ml diluted serum was placed in each well, and incubated at 378C for 2 h. After washing three times, 150 ml HRP rabbit anti-chicken IgG antibody (Military Medicine Academy Sciences of Liberation Army, Beijing, PR China), diluted 800-fold in PBS, was added, then the plates were incubated at 378C for 2 h. After being washed, 100 ml OPDhydrogen peroxide was added to each well and incubated for 20 min at 378C without light. The reaction was stopped by adding 50 ml H2SO4 (1 mol/l) to each well. The OD values were recorded at 490 nm by an ELISA plate reader. Each sample was assayed in triplicate. 2.5. Statistical analysis Experimental data were analysed by one-way ANOVA of SAS (Copyright (c) 1999 – 2001 by SAS Institute Inc., Cary, NC, USA.) statistical program. The results were expressed as means

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and standard errors. Differences due to diet treatment were considered significant as p 5 0.05. 3. Results Growth performance of control and CIAMP group were similar (Table I). Compared with the control, CIAMP supplementation (1 mg/ml) promoted the proliferation of LPS-stimulated peripheral blood lymphocytes of chickens at the age of 4 – 7 days. However, no significant differences were observed between groups (Table II). Compared with the control, the content of IgG in serum of CIAMP treated-chickens was enhanced at the age of 4 – 10 days (p 5 0.05). The content of IgM had the same response as IgG at the age of 4 (p 5 0.01), 10 and 17 days (p 5 0.05) (Table II). Furthermore, CIAMP in drinking water increased the number of IgG and IgM-forming cells in red pulp of the spleen at the age of 4 – 10 days, but only IgM-forming cells at the age of 7 days differed significantly (Table III). CIAMP addition also enhanced the number of IgG and IgM-forming cells in bursa of Fabricus of chickens at the age of 4 – 17 days. IgG- and IgM-forming cells differed significantly (p 5 0.05) at the age of 4 and 7 days, respectively (Table III). Compared with the control, CIAMP could enhance the number of IgG- and IgM-forming cells in caecal tonsil diffusion of chickens at the age of 4 – 10 days, but there were no significant differences between groups (Table III).

Table I. Effect of supplementation of chicken intestinal antimicrobial peptides (CIAMP) on body weights [g] of chickens (Means + standard error, n ¼ 5). Age of chickens [d]

Control group CIAMP group

1

4

7

10

17

42.4 + 0.72 42.4 + 0.72

52.8 + 3.74 51.6 + 3.97

64.4 + 0.18 66.7 + 2.68

84.7 + 5.28 82.4 + 3.46

137.2 + 4.82 132.1 + 12.47

Table II. Effect of supplementation of chicken intestinal antimicrobial peptides (CIAMP) on proliferation of B lymphocyte from peripheral blood and content of immunoglobulin in serum of chickens (Means + standard error, n ¼ 5). Age of chickens [d] 1

4

B Lymphocyte proliferation [OD at 570 nm] Control group 0.380 + 0.071 0.330 + 0.093 CIAMP group 0.380 + 0.071 0.415 + 0.096

7

10

17

0.605 + 0.155 0.622 + 0.268

0.730 + 0.088 0.765 + 0.086

0.792 + 0.109 0.711 + 0.050

IgG in serum [OD at 490 nm] Control group 1.43 + 0.133 CIAMP group 1.43 + 0.133

1.44 + 0.050 1.58 + 0.058*

1.43 + 0.166 1.51 + 0.065*

1.46 + 0.074 1.57 + 0.176*

1.44 + 0.094 1.26 + 0.073

IgM in serum [OD at 490 nm] Control group 1.06 + 0.040 CIAMP group 1.06 + 0.040

1.02 + 0.041 1.68 + 0.088**

1.69 + 0.058 1.66 + 0.026

1.71 + 0.069 1.78 + 0.047*

1.69 + 0.042 1.74 + 0.068*

Asterisks indicate significant differences between control and CIAMP group: *p 5 0.05, **p 5 0.01.

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Table III. Effect of chicken intestinal antimicrobial peptides (CIAMP) on the number of antibody forming cells in different tissues of chickens (Means + standard error, n ¼ 5). Age of chickens [d] 1 3

4

7

10

17

2

Spleen red pulp [10 cells/mm ] IgG forming cells Control group 6.16 + 0.40 CIAMP group 5.96 + 0.67 IgM forming cells Control group 6.34 + 0.04 CIAMP group 6.36 + 1.04

6.18 + 0.65 6.46 + 0.72

6.04 + 0.63 6.38 + 0.56

6.26 + 0.49 6.70 + 0.52

6.64 + 1.53 7.30 + 1.18

6.58 + 0.05 6.86 + 0.80

6.48 + 1.05 7.78 + 0.66*

7.06 + 1.19 7.74 + 1.57

8.02 + 1.56 7.60 + 1.48

Bursa of Fabricus [103 cells/mm2] IgG forming cells Control group 5.48 + 1.21 CIAMP group 5.64 + 0.86

5.76 + 0.87 6.74 + 0.35*

5.70 + 0.57 6.22 + 0.53

6.10 + 0.42 6.56 + 0.48

6.40 + 1.16 6.98 + 0.79

5.44 + 0.67 5.74 + 0.91

5.46 + 0.51 6.12 + 0.33*

6.00 + 0.35 6.46 + 0.41

6.12 + 0.94 6.50 + 0.48

1.88 + 0.51 2.02 + 0.19

2.06 + 0.38 2.42 + 0.29

2.04 + 0.17 2.20 + 0.31

2.14 + 0.26 2.10 + 0.16

1.96 + 0.30 2.22 + 0.24

2.06 + 0.15 2.22 + 0.23

2.26 + 0.26 2.50 + 0.37

2.38 + 0.46 2.30 + 0.32

267.6 + 67.18 308.8 + 14.24

422.2 + 53.38 464.8 + 84.34

442.2 + 51.37 500.8 + 93.07

465.0 + 69.18 475.0 + 71.85

182.0 + 15.89 173.0 + 48.58

188.2 + 14.57 213.0 + 45.39

228.2 + 47.35 217.6 + 20.94

220.0 + 35.36 208.4 + 21.48

137.2 + 29.09 195.4 + 39.75*

180.0 + 35.78 203.2 + 38.56

188.6 + 21.05 190.4 + 20.60

208.6 + 42.58 204.2 + 12.64

IgM forming cells Control group CIAMP group

4.92 + 1.09 5.00 + 0.87

Caecal tonsil diffuse area [103 cells/mm2] IgG forming cells Control group 1.46 + 0.50 CIAMP group 1.46 + 0.59 IgM forming cells Control group 1.84 + 0.40 CIAMP group 1.76 + 0.40 IgA-forming cells in lamina propria of Duodenum [cells/mm2] Control group 187.2 + 59.60 CIAMP group 181.0 + 78.35 Jejunum [cells/mm2] Control group 61.4 + 19.58 CIAMP group 90.6 + 33.74 Caecal tonsils [cells/mm2] Control group 65.2 + 10.08 CIAMP group 69.2 + 15.71

Asterisks indicate significant differences between control and CIAMP group: *p 5 0.05.

The number of IgA-forming cells in duodenum and jejunum of CIAMP treated chickens was not significantly different to the control, but the number of IgA-forming cells in caecal tonsils were increased at day 4 in CIAMP group compared with the control (p 5 0.05) (Table III). CIAMP enhanced the level of antibodies against infectious bursal disease virus in sera of chickens from 14 – 35 days after the chickens were injected with infectious bursal disease virus vaccine, but only at day 21 after the vaccination was it significantly increased (p 5 0.05) (Table IV). 4. Discussion Humoral immunity plays an important role in the host defence against pathogens. Many studies concentrated on the role of defensins in innate immunity, such as killing a broad spectrum of microbes. In addition to their antimicrobial activity, defensins have been reported

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Table IV. Effect of chicken intestinal antimicrobial peptides (CIAMP) on antibodies against infectious bursal disease virus in sera of chickens [OD at 490 nm]# (Means + standard error, n ¼ 5). Days after injection with infectious bursal disease virus vaccine

Control group CIAMP group

14

21

28

35

0.552 + 0.049 0.590 + 0.016

0.735 + 0.032 0.811 + 0.033*

0.777 + 0.028 0.844 + 0.041

1.098 + 0.037 1.207 + 0.088

#

Chickens of both groups were injected with infectious bursal disease virus vaccine at the age of 20 days, and sera samples were collected for analysis at 14, 21, 28 and 35 days after vaccination; Asterisk indicates a significant difference between control and CIAMP group: *p 5 0.05.

to induce the acquired host immunity (Oppenheim et al., 2003). Antimicrobial peptides may play an important role in modulation of immune response. The aim of this study was to assess the effects of antimicrobial peptides on humoral immunity of chickens. There have been reports that defensin induced cytotoxicity (Okrent et al., 1990; Lichtenstein et al., 1988). The injection of 1 mg of human defensins into murine skin did not cause any effects indicative of toxicity or tissue damage such as swelling, redness and ulcer formation (Chertov et al., 1996). Our observations indicate that the concentration of CIAMP used in this study was not toxic for chickens. Furthermore, histological analysis showed that CIAMP did not damage the cell structure of the host. This result also demonstrated that CIAMP could be effective by oral administration. The results of our study showed that feeding with CIAMP could enhance systemic humoral immune response of chickens. Compared with the control group, we found that CIAMP supplementation enhanced the content of IgG and IgM in serum, IgM-forming cells in bursa of Fabricus and spleen and IgG-forming cells in bursa of Fabricus at different times after supplementation significantly. An increase in systemic humoral immune response has also been reported in mammalians. Human neutrophil peptide (HNP) defensins can promote acquired systemic immune response, intranasal delivery of HNP defensins plus ovalbumin (OVA) enhanced OVA-specific serum IgG antibody responses and intraperitoneal administration of 1 mg HNP defensins promoted antigen-specific Ig production (Lillard et al., 1999). HNP defensins act as potent adjuvants by producing lymphokines, which promote cellular immunity and antigen-specific Ig production (Tani et al., 2000; Brogden et al., 2003). The results of our study showed that CIAMP addition slightly enhanced the intestinal mucosal humoral immune response at day 4 – 10, but this difference was not significant. In addition, immunohistochemistry did not reveal any statistically significant change in the level of IgM- and IgG-forming cells present in intestine. CIAMP only enhanced the caecal tonsils diffuse area IgA-forming cells at day 4. Several investigators have measured the effect of defensins on mucosal antibody responses. Intranasal immunization with HNPs did not induce Ag-specific IgA Ab responses and IgM anti-OVA Abs in nasal wash or saliva, vaginal washes or faecal samples. HNPs with OVA slightly enhanced OVA-specific IgG titres in vaginal wash or faecal extracts compared with mice receiving OVA alone (Lillard et al., 1999). Intranasally immunized Human b-defensins (HBD) induced low OVA-specific IgG responses in saliva, nasal wash and faecal extracts (Brogden et al., 2003). Antimicrobial proteins are produced by epithelial cells or leucocytes lining the environmental interface of the gastrointestinal tract and protect the epithelium from invading microorganisms through their bactericidal activity. It is interesting that these innate peptides initiated significant systemic immune response, but not mucosal immune response after oral delivery. Defensins may cause subtle changes in epithelial cells; they may be absorbed rapidly

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by the mucosa and thus gain access to peripheral lymphoid tissues or dendritic cells to increase systemic immune responses (Yang et al., 1999, 2002). Additionally, the regulatory effects of defensins on co-stimulatory molecule expression by lymphocytes could also induce differential host immunity. Previous studies have shown that HNP-alpha defensins enhanced IL-2R and CD28 expression in splenic T cells more dramatically than that of Peyer’s patchderived T lymphocytes, which suggests a tissue-specific effect by defensins (Lillard et al., 1999). According to our results, CIAMP could greatly enhance systemic humoral immune responses, and slightly enhanced mucosal humoral immune responses, which may be the reason of tissue specific effect of defensins. This experiment showed that CIAMP enhanced the antibodies against infectious bursal disease virus in sera of chickens at 21 days after receiving infectious bursal disease vaccine. Fritz et al. (2004) also reported that artificial antimicrobial peptides could induce high levels of antigen-specific antibodies after co-injection of antimicrobial peptides with a commercially available influenza vaccine in vivo (Fritz et al., 2004). Immuno-potentiating properties of defensins as candidates are interesting for vaccine design. In addition to direct eradication of microorganisms, defensins may be used as a signal to modulate or amplify adaptive immune responses. Defensins serve as ‘alarm’ signals in mobilizing the immune system and to activate innate and adaptive immune systems (Oppenheim & Yang, 2005). Based on our results, CIAMP are capable of promoting systemic humoral immune responses of chickens at an early age. The increased content of immunoglobulins in serum and antibody-forming cells in bursa of Fabricus strengthens the viability of chicken. However, the costs of CIAMP isolation from the intestine are currently high, therefore, methods should be developed to produce CIAMP cheaper and more effectively. Acknowledgements The authors thank National Natural Science Foundation of China (Grant No. 30471301) for financial support.

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