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Poult Sci 2008. 87:250-254. doi:10.3382/ps.2007-00353
© 2008 Poultry Science Association
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IMMUNOLOGY, HEALTH, AND DISEASE

Effects of Rabbit Sacculus Rotundus Antimicrobial Peptides on the Intestinal Mucosal Immunity in Chickens

T. Liu*, R. She*,1, K. Wang{dagger}, H. Bao*, Y. Zhang*, D. Luo*, Y. Hu*, Y. Ding*, D. Wang* and K. Peng{ddagger}

* College Of Veterinary Medicine, China Agricultural University, Beijing, 100094;{dagger} Shandong Province Laboratory Animal Center, Jinan, 250002, P.R. China; and{ddagger} College Of Veterinary Medicine, Anhui Agricultural University, Hefei, 230036, P.R. China

1 Corresponding author: sheruiping{at}126.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ninety chickens were randomly divided into 2 groups (45 chickens in each group) to determine the effect of oral administration of rabbit sacculus rotundus antimicrobial peptides (RSRP) on the intestinal mucosal immune responses in chicken. On d 7, 14, 21, and 28, the animals received 0.1 mg of RSRP dissolved in 0.5 mL of physiological saline. The control groups received the same dose of physiological salt solution on the same day. The results showed that RSRP increased the villus height of the duodenum (P < 0.01) and jejunum (P < 0.05) at the ages of 28, 42, and 56 d. The numbers of intestinal intraepithelial lymphocytes in different parts of intestine of the RSRP group were increased significantly more than that of the control (P < 0.01 or P < 0.05) at the ages of 28, 42, and 56 d. The RSRP increased the area of IgA-secreting cells of each fragment of intestine at all 3 time points. These results indicated that the presence of RSRP affected and considerably modified the structure of the intestine and mucosal immune parameters in healthy chickens when compared with controls.

Key Words: antimicrobial peptide • mucosal immune • intestine • chicken


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antimicrobial peptides derived from natural sources have received increased attention over the past decade; over 400 antimicrobial peptides have been identified to date in plants, insects, and animals (Hancock and Diamond, 2000). In mammalian species, defensin is one important kind of antimicrobial peptides, and more than 50 defensins have been identified that are either stored in the granules of neutrophils, macrophages, and Paneth cells, or are generated by keratinocytes and mucosal epithelial cells of the respiratory, digestive, urinary, and reproductive systems (Boman, 1995; Hoffmann et al., 1999). Members of the defensin family exhibit a similar protein sequence and are distinguished by a conserved cysteine motif. Defensins show antimicrobial activities against gram-negative and gram-positive bacterial strains and fungi, as well as some parasites and enveloped viruses (Bulet et al., 1999). Recently, some of the mammalian antimicrobial peptides have been shown to have a second major function of rapidly chemoattracting and activating host cells to engage in innate host defense or adaptive immune responses, or both (Broekaert et al., 1995; Boman, 1998; Ganz and Lehrer, 1998). Eighteen rabbit antimicrobial peptides have been isolated from bone marrow, kidney, lung, and peritoneal neutrophils.

Antibiotics have had an enormous impact on treatment of infectious diseases and the success of invasive medical procedures, such as surgery and chemotherapy. However, the rise in antibiotic resistance threatens to reverse some of these gains (Hancock, 2001). One reason for this development is the paucity of truly novel antibiotics since the introduction of quinolones in the early 1960s. Thus, it is important to consider new classes of antibiotics. Natural compounds have been considered to replace antibiotics; antimicrobial peptides may be used as feed additives provide an alternative to antibiotics in animal feed (Yang et al., 2006). However, previous research has failed to consider the effects of antimicrobial peptides on animal intestinal mucosal immunity. The purpose of this paper is to evaluate the effect of rabbit sacculus rotundus peptide (RSRP) on intestinal local immunity of the chicken and to investigate the potential use of antimicrobial peptides in modulation of the immune response for animal health.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of RSRP
The RSRP was isolated from rabbit sacculus rotundus as previous described (Wang and She, 2003). Briefly, rabbit sacculus rotundus were washed with sterile saline (0.9% sodium chloride). The fat was removed and the mucosa of sacculus rotundus was isolated. The mucosa tissue was broken into small pieces with a mortar and pestle in ice-cold aqueous 5% acetic acid (1:10 wt/vol). Extracts were placed in boiling water bath for 10 min and then cooled rapidly. The pellet was discarded after centrifugation at 6,440 x g for 30 min at 4°C. Ice-cold 5% acetic acid (1:1 vol/vol) was added and extracted overnight at 4°C. The extract was centrifuged at 6,440 x g for 30 min at 4°C. The clarified extract was adjusted to pH 7.0 with sodium hydroxide. The precipitates were removed by centrifugation (6,440 x g for 30 min at 4°C), and the supernatant was loaded onto a 10 x 300 mm Sephadex G-100 column [Amersham Pharmacia Biotech (China) Ltd., Hong Kong, China] and eluted with 0.2 mol sodium acetate buffers per liter. The fractions of interest were enriched by bag filter (MWCO3,000, Microdialysis Chambers) and Macrogol-20000 (Guangzhou Shengda Chemical Co., Guangzhou, China) and applied to a 10 x 300 mm DEAE-52 column (Amersham Pharmacia Bio-tech) equilibrated in 0.01 mol dibasic sodium phosphate buffer, pH 7.0, at a flow rate of 10 mL/h. The column was washed with 5 column volumes of the same buffer and eluted stepwise using 0 to 1 M sodium chloride, pH 7.0. Fractions were followed by absorbance at 280 nm and bacteriolytic assay. The fractions having antibacterial activity were pooled and concentrated. Then fractions of interest were purified with Tricine-PAGE (Schagger and von Jagow, 1987) and subjected to NH2-terminal sequence determination by Edman degradation at Beijing SBS Genetech Co. Ltd. (Beijing, China). The result of amino acid analysis corresponded with the amino acid composition of rabbit defensin NP-2 propeptide by 1 N-terminal residue. The NP-2 is composed of signal, propeptide, and mature peptide. The mature peptide has antibiotic, antifungal, and antiviral activity. It also inhibits corticotropin-stimulated corticosterone production. But the function of signal and propeptide is still unclear. Interested fractions were collected and were vacuum frozen dry before being stored at –20°C.

Antimicrobial Assay
The RSRP elution was analyzed by agarose diffusion assay (Lehrer et al., 1991) and pasteurella cuniculicida was used as a test organism. Briefly, a single colony of pasteurella cuniculicida was grown overnight at 37°C in 25 mL of Trypticase soy broth (TSB; 30 g/L). A 25-µL aliquot of the overnight growth was then grown in 25 mL of TSB for 2 h at 37°C. The bacteria were pelleted by centrifugation, and the bacterial pellet was resuspended in 10 mL of cold sterile 10 mM sodium phosphate, pH 7.4. The bacterial concentration was determined by measuring the absorbance at 620 nm, assuming that an absorbance of 1 equals 2.5 x 108 cfu/mL. Then, 2.5 x 106 cfu was mixed with 10 mL of warmed (to 55°C) underlay agarose [1% agarose (low EEO; Sigma, St. Louis, MO), 0.03% TSB, 0.02% Tween 20, and 10 mM sodium phosphate, pH 7.4]; this mixture was poured into 15-mm square Petri dishes and allowed to harden. Sample wells were made by punching holes with a 3-mm agar punch (BioRad). A 5-µL sample was added to each well. The plate was incubated at 37°C upright for 3 h to dry the sample and maximize the specific killing of bacteria by the applied samples before addition of 10 mL of overlay agarose [an autoclaved solution of 60 g of TSB per liter, 10 mM sodium phosphate (pH 7.4), and 1% agarose], which had been warmed to 42°C prior to pouring. The agarose was allowed to harden before incubation of the plates overnight at 37°C. Antimicrobial activity was quantitated by measuring the area of the circular clear zones on the opaque background of bacterial growth.

Animals and Experimental Treatment
One-day-old healthy Leghorn chicks were randomly allocated into 2 groups of 45 birds, which were housed in separate isolators. Both groups received the same diet. All chickens were not vaccinated, and feed and water were given ad libitum. On d 7, 14, 21, and 28, the animal received 0.1 mg of RSRP dissolved in 0.5 mL of physiological saline by gavage, and the control groups received same dose of physiological salt solution on the same day.

Sample Collection
On d 28, 42, and 56, fifteen chickens from each group were killed, and then duodenum, jejunum, and ileum were immediately collected and fixed in 10% (vol/vol) neutral buffered formalin. The tissue blocks were dehydrated with graded ethanol (50, 70, 80, 90, 95, and 100% ethanol by turns), then embedded in paraffin, and serial sections (5 µm thick) were made.

Histological Examination for Height of Villus
After staining with hematoxylin-eosin, the values of the height of the villus were obtained using Motic Med 6.0 CMIAS [MicroOptic Industrial Group Co. Ltd. (China), Guangzhou, China]. For villus height measurement, 10 villi having the lamina propria were selected per section. The villus length was measured from the villus tip to the bottom, not including the intestinal crypt. An average of these 10 villi per section was expressed as a mean villus height for each section. A total of 8 sections were counted per chicken. Then, an average of these 8 sections was expressed as a mean villus height for each chicken. Finally, these 15 mean villus heights from 15 chickens were expressed as a mean villus height for 1 group.

Histological Examination for Intestine Intraepithelial Lymphocytes
Hematoxylin-eosin staining were applied and then sealed with a cover slip. The number of intestine intraepithelial lymphocyte (iIEL) cells at 5 different microscope fields of intestinal villi in each bird was counted under the microscope for the statistical analysis of the data.

Immunohistochemical Examination for IgA-Secreting Cells
Sections were stained for S-IgA-containing cells in the lamina propria of the intestine. Mouse anti-chick IgA (Southern Technology Inc., Longwood, FL) American and Histostain-Plus Kits [SP-9002, Beijing Zhongshan Goldenbridge Co., Ltd., Beijing, China including IgG/Bio, S-A/horseradish peroxidase, blocking buffer (normal goat serum)] and 3, 3-diaminobenzidin (Beijing Zhongshan Goldenbridge Co., Ltd.) were used in an indirect immuno-peroxidase technique for IgA staining (Duncker et al., 2006). The paraffin sections were removed and then immersed in the distilled water following routine methods. Afterwards, rinsing the paraffin sections (3 x 5 min) in PBS-T (0.01 M PBS pH 7.4: KH2PO4 0.02%, N2HPO4 0.29%, KCl 0.02%, 0.8% NaCl, 0.05% BSA, Tween-20 0.05%, 0.0015% Triton X-100), and then blocked with 3% peroxide-methanol at room temperature for endogenous peroxidase ablation. All following steps were carried out in a moist chamber:

  1. Incubation with blocking buffer (normal goat serum) at room temperature for 20 min.
  2. Discarding the goat serum and dropping the mouse anti-chicken IgA, which was diluted with PBS (0.01 M PBS, pH 7.4).
  3. Incubating the sections for 2 h at 37°C.
  4. Rinsing in PBS-T (3 x 5 min).
  5. Dropping the goat anti-mouse IgG/Bio and incubating the sections for 30 min at 37°C.
  6. Rinsing in PBS-T (3 x 5 min).
  7. Incubation with the S-A/horseradish peroxidase at 37°C for 30 min.
  8. Rinsing (3 x 5 min) in PBS-T.
  9. Coloration with 3, 3-diaminobenzidin, kept at room temperature without light for 10 min.
  10. Finishing coloration with the distilled water.
  11. Hematoxylin stained.
  12. Dehydration, clearing, and mounting with neutral gums.
  13. The negative control group was carried out with the same steps as described above, but the mouse anti-chicken IgA was replaced by PBS-T.

The areas of IgA-secreting cells in 5 different microscope fields of intestinal villus in each tissue were counted under the microscope for the statistical analysis of the data. The area of each field of the intestinal villus was measured using the Motic Med 6.0 CMIAS (MicroOptic Industrial Group Co. Ltd.). Then, the number of IgA-positive cells was divided by each area (cells/µm2).

Statistical Analysis
Experimental data were analyzed by 1-way ANOVA of the SAS (SAS Institute Inc., Cary, NC) statistical program. The results were expressed as means and standard errors. A P-value < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histological Analysis of Villus Height
A comparison of villus heights is given in Figure 1Go. The RSRP increased the villus height of the duodenum (P < 0.01) and jejunum (P < 0.05) at the age of 28, 42, and 56 d. However, there was no significant change in height of the villus in the ileum between RSRP and control chickens.


Figure 1
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  		Figure 1. The height of villi of different parts of intestine in chicken. *Significantly differs from control at P < 0.05. **Significantly differs from control at P < 0.01. C: control group; T: rabbit sacculus rotundus antimicrobial peptides group.

 
The Number Changes of iIEL
The numbers of iIEL in the different parts of the small intestine gradually increased as animals grew. As shown in Figure 2Go, the numbers in duodenum and jejunum of RSRP group were increased significantly from than that of control (P < 0.01) on d 28, 42, and 56. The numbers in the ileum of the RSRP group were also higher than that of the control group (P < 0.05).


Figure 2
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  		Figure 2. The number of intestine intraepithelial lymphocytes (iIEL) in different parts intestine in chickens. *Significantly differs from control at P < 0.05. **Significantly differs from control at P < 0.01. C: control group; T: rabbit sacculus rotundus antimicrobial peptides group.

 
The Area Changes of IgA Secreting Cells in Intestine
In each fragment of intestine samples, IgA-secreting cells were recognized as lymphocytes by their characteristic morphology: rounded, with a nucleus surrounded by a ring of yellow-brown cytoplasma. In the duodenum, the areas of IgA-secreting cells of RSRP group were increased significantly from that of the control group (P < 0.01) during the whole experimental period. The areas of IgA-secreting cells in the jejunum of RSRP group were increased significantly from that of the control group (P < 0.01) on d 28 and 42. The area changes of defensin group in the ileum were the same as that in the duodenum (Figure 3Go).


Figure 3
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  		Figure 3. The area of IgA-secreting cells in different parts intestine (µm2). The area of IgA-secreting cells in different parts intestine. **Significantly differs from control at P < 0.01. C: control group; T: rabbit sacculus rotundus antimicrobial peptides group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Defensins are an efficient part of the first line of host defense because of their ability to recognize and neutralize invading microorganisms quickly and specifically (Lehrer and Ganz, 1999). Numerous studies demonstrate that defensins are able to recruit and promote other elements of host immunity, particularly innate immunity (Bals et al., 1999; Nizet et al., 2001). However, a few studies have been conducted on the effects of defensins on intestinal mucosal local innate immunity. The aim of this study was to assess the effect of RSRP on the intestinal structure and mucosal IgA-producing cells and iIEL of chickens.

The intestinal mucosa is the first barrier encountered by the microorganism; consequently, it could be exposed to high amounts of dietary antigens. The digestive function of the small intestine is closely related to mucosal architecture, in particular to the villi density, shape, and size (Thomsen et al., 2006). The iIEL are the immuno-competent cells, which first encounter the antigen in the mucosal immune system. It is well known that iIEL were programmed for cytokine production to protect against bacteria and viruses (Yang et al., 2003). It has been reported that the characteristics, distribution, and number of iIEL relied on the mucosal immunization in different species, suggesting that iIEL may play a supporting role in mucosal immune response (Sim, 1995; Inagaki-Ohara et al., 2005; Duncker et al., 2006). The IgA can neutralize viruses, block adherence of microbes to mucus membranes, and enhance the bactericidal effects of other immune factors (Mestecky and McGhee, 1987). The IgA-secreting cells in the lamina propria tissue of the intestine are the important effector molecules to protect mucosal surfaces. Now, the change of IgA-secreting cells in the intestine is one of the standards to estimate intestine mucosal immunity (Zhang et al., 2007).

The results of the experiment showed that RSRP could effectively enhance the height of the intestinal villus of the duodenum and jejunum, but there was no significant effect on the ileum. It seems that RSRP can account for the integrity of intestinal surfaces. Significantly increased number of iIEL and area of IgA-secreting cells in the duodenum, jejunum, and ileum of RSRP group were found. These results suggested that RSRP could drive mucosal immune response; the differences only existed in different fragments of intestine and response extension. And, especially, the effects lasted for an extended period. These findings demonstrated the regulative activity of RSRP in improving the structure of intestine and in promoting intestinal mucosal immunity during the chicken growth period. However, further studies will be required to define the precise advanced mechanisms that are responsible for these observations. And more functions of RSRP will be found and lead to more extensive usage in the future.


   ACKNOWLEDGMENTS
 
The authors thank National Natural Science Foundation of China (grant no. 30471301) for financial support.

Received for publication August 23, 2007. Accepted for publication October 17, 2007.


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 ABSTRACT
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