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IMMUNOLOGY, HEALTH, AND DISEASE |
* College of Veterinary Medicine, China Agricultural University, Beijing, 100193, P. R. China; College of Veterinary Medicine, Anhui Agricultural University, Hefei, 230036, P. R. China; and Department 3, Command and Engineering College of Chemical Defense of Chinese PLA, Beijing 102205, P. R. China
1 Corresponding author: sheruiping{at}126.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: pig antibacterial peptide • growth performance • small intestine • mucosal immunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibacterial peptides extracted from the small intestine have the capacity to kill or inactivate a particular spectrum of bacteria, fungi, and some enveloped viruses in vitro (Boman 1995; Ganz and Lehrer, 1998). Recent research demonstrated that some antibacterial peptides have been shown to have a second major function—expediting the growth of animals (Liu et al., 2008). The use of antibiotics to promote growth has resulted in harmful residues in the food chain and induced the spreading of drug resistance genes (Witte, 1997). Antibacterial peptides may be used as feed additives, providing an alternative to antibiotics in animal feed. In this report, the effect of pig antibacterial peptides on the immunity of mucosa and growth performance of broiler chickens was investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pig antibacterial peptides (PABP) were isolated from pig small intestine as described previously (Ma et al., 2004). Briefly, mucosa tissues scraped from small intestines (obtained from healthy pigs) were washed with cold sterile saline (0.85% NaCl). The mucosa tissue homogenates were stirred and extracted in ice-cold aqueous 5% acetic acid (1:10 wt/vol) overnight. Extracts were placed in boiling water for 10 min and then cooled quickly. The precipitate was discarded after centrifugation at 6,440 x g for 30 min at 4°C. The supernatants were adjusted to pH 7.0 with 1 M sodium hydroxide and purified by size-exclusion column chromatography and preparation HPLC (Evans et al., 1994). The fractions of interest were enriched by bag filter (molecular weight cut-off 3,000, microdialysis chambers; ISC Bio-Express, Kaysville, UT) and Macrogo-20000 (Guangzhou Shengda Chemical Co., Guangzhou, China). The minimum bactericidal concentration was determined by bacteriolytic assay, and protein concentrations in 0.01% acetic acid were determined by spectrophotometric absorbance at 280 nm. The fractions having antibacterial activity were assessed by acid-urea PAGE. The purified peptides were subjected to NH2-terminal sequence determination by Edman degradation at Beijing SBS Genetech Co. Ltd. (Beijing, China) to confirm their composition. The result of amino acid analysis corresponded with the amino acid composition of pig defensin β-1 (Zhang et al., 1998, 1999). Peptides were stored as lyophilized powders at –20°C.
Assay for Antibacterial Activity
The antibacterial activity was tested against Staphylococcus aureus (ATCC 25923) and Escherichia coli (K88+) as described previously (Lehrer et al., 1991). Briefly, after cells were grown in nutrient broth for 16 h at 37°C with shaking, the cells were washed with 10 mM PBS buffer (pH 7.4) after centrifugation (6,000 x g for 10 min) and diluted in the same buffer to give approximately 2 x 106 cfu/mL. The cells were mixed with 10 mL of warmed (below 55°C) underlay agarose [1% agarose (low EEO, Sigma, St. Louis, MO), 0.06% nutrient broth, and 10 mM PBS buffer, pH 7.4]. This mixture was poured into 15-mm sterile flat plates and allowed to harden. Sample wells were made by punching holes with a 3-mm agar punch (BioRad Laboratories, Hercules, CA). Fifteen microliters of PABP was added to each well. The plates were incubated upright at 37°C for 2 h to allow dissemination of the samples and maximize the interaction with bacteria by the applied samples before addition of 10 mL of overlay agarose (1%). The plates were incubated overnight at 37°C after the overlay agarose solidified. Antibacterial activity was quantified by measuring the area of the circular clear zones on the opaque background of bacterial growth.
Birds
Three hundred 1-d-old Arbor Acre male broiler chicks (provided by Huadu broiler company, Beijing, China) of similar BW were selected. The chickens were 2 d old when the experiment was initiated, after a 1-d acclimation. The experiment lasted 42 d in total. The study was approved by the Institutional Animal Care and Use Committee at China Agriculture University.
Experimental Design and Feeding Conditions
The chickens were randomly assigned into 5 groups with 60 chickens per group, and 30 chickens per pen. The groups were control group, supplied in the drinking water with 20 mg of PABP/L of water, supplied in the drinking water with 30 mg of PABP/L of water, feed supplemented with 150 mg of PABP/kg of diet, and feed supplemented with 200 mg of PABP/kg of diet. Chickens of the control group were fed a corn-soybean basal diet. Two diets were used for all chickens, with the starter diet fed for experimental d 1 to 21, and the grower diet for experimental d 21 to 42. No chickens were vaccinated. Feed and water were provided ad libitum. Body weights were recorded weekly, and feed intakes were measured from d 1 to 42. The experiment ended on d 42. Average daily gain and feed conversion ratio were calculated. The room temperature was kept between 32 and 25°C with nearly continuous light (23L:1D); relative humidity was 50 to 70% (Arab et al., 2006).
Sampling
All experimental birds were sacrificed to determine the effects of PABP on villus morphology and mucosal immune status in the small intestine at 42 d of age. The birds were weighed before slaughter, and the intestinal tract was excised for sample collection. Two adjacent segments of intestinal samples (approximately 2 cm each) were removed, from the proximal duodenum (cut from approximately 4 cm after the pylorus) and proximal jejunum (cut from approximately 2 cm after the yolk stalk). Immediately after collection, each tissue sample was rinsed 3 times in 0.9% NaCl solution. Each sample divided into 3 parts (about 0.7 cm each) and then fixed in 80% alcohol fixative (80 mL of 100% alcohol, 20 mL of double-distilled water), formaldehyde and glutaraldehyde mixture fixative [50 mL of 0.2 M sodium phosphate buffer, pH = 7.4, 10 mL of 25% glutaraldehyde, 20 mL of 10% formaldehyde polymer, and 20 mL of double-distilled water], and Carnoy’s fixative (60 mL of 100% ethanol, 30 mL of chloroform, and 10 mL of glacial acetic acid), respectively, mixed and stored at 4°C for further analysis.
Measurement of Villus Morphology
Cross-sections of intestinal specimens preserved in formaldehyde and glutaraldehyde mixture fixative were routinely dehydrated and embedded in paraffin. Sections from each sample were cut at a thickness of 5 µm and stained with hematoxylin and eosin according to Mayer (Luna, 1968). The measurements of villus height and gut mucosa thickness were performed on the stained sections under the microscope with 40 x combined magnification and an ocular micrometer. At least 15 well-oriented, intact villi were measured in triplicate portions of the slide for each broiler chicken within each treatment. The length of the villus was measured from the crypt mouth to the villus tip, and all measurements were made (villus height and gut mucosa thickness) in 10-µm increments.
Measurement of Alkaline Phosphatase and Goblet Cells
Frozen sections (5 µm) were prepared from the samples (duodenum and jejunum) preserved in 80% alcohol fixative. The sections were stained with the Gomori’s calcium-cobalt amendment method (Yang, 1990) to reveal alkaline phosphatase (AKP) in the intestinal mucosa surface. The positive areas of AKP in 15 different microscope fields of well-oriented, intact villi in each tissue were measured using Motic Med 6.0 CMIAS (Micro-Optic Industrial Group Co. Ltd., Guangzhou, China).
Small intestine samples preserved in Carnoy’s fixative were washed in distilled water, dehydrated in alcohol (50, 70, 80, 90, 95, and 100%), cleared in dimethyl-benzene, and embedded in paraffin. Transverse sections were cut at 5 µm and stained using the periodic acid-Schiff method to visualize goblet cells (GC; Tokuhiro et al., 1978). The number of GC among 100 enterocytes in 15 different intestinal villi of each tissue was counted under the microscope for the statistical analysis of the data.
Measurement of Secreting IgA
The number of cells secreting IgA (sIgA) was revealed using the avidin-biotin complex (ABC) immunohistochemical method (Yang, 1990). The samples preserved in the formaldehyde and glutaraldehyde mixture fixative were paraffin-embedded. Serial 5-µm tissue sections were prepared and stained using the ABC immunohistochemical method. Rabbit monoclonal antibodies against chicken sIgA (Southern Biotechnology Inc., Birmingham, AL) and Histostain Bulk SP kit (Zymed Co., San Francisco, CA) were used. Sections were deparaffinized and rehydrated, rinsed in 0.01 M PBS (pH 7.4), and then incubated in 0.3% H2O2 for 30 min at room temperature to block endogenous peroxidase activity. After washing 3 times in PBS, sections were placed in 500 mL of 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven for antigen retrieval. The buffer was brought to 100°C over a period of approximately 5 min, and then left to cool for 15 min. After washing 3 times using PBS, sections were preincubated with goat serum for 20 min at room temperature to prevent nonspecific protein binding. Sections were then incubated for 3 h with primary antibodies in a moist chamber at 37°C. The sIgA antibody was diluted 1:200 (vol/vol) in PBS. After washing 3 times with PBS, sections were reacted with biotinylated goat anti-mouse Ig (Histostain Bulk SP kit) for 2 h at 37°C. The sections were washed in PBS 3 times and then incubated with peroxidase-conjugated streptavidin (Histostain Bulk SP kit) for 30 min at 37°C. After washing 3 times with PBS, the sections were incubated with 3,3-diaminobenzidine. Sections were then counterstained with Mayer’s hematoxylin for 3 min, dehydrated, cleared, and mounted. The areas of IgA-secreting cells in 15 different microscope fields of well-oriented, intact intestinal villi in each tissue was measured by Motic Med 6.0 CMIAS (Micro-Optic Industrial Group Co. Ltd.). The number of IgA positive cells was divided by each area (cells/µm2).
Statistics
Results were expressed as means or means ± standard deviation (SD). Villus morphology, number of GC, AKP, and sIgA data were analyzed by one-way ANOVA (SPSS 12.0, 2003, SPSS Taiwan Corp., Taibei, Taiwan). Once main effects were significant with P < 0.05, means were compared by Duncan’s test using the procedure of SPSS (2003, SPSS Taiwan Corp.); P-values 
0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effect of the PABP in water and in feed on BW, average daily gain, daily feed intake, and feed conversion ratio (FCR) at d 21 and 42 are shown in Table 1
. There was no difference in initial BW among groups before treatment. There was no mortality during the study. From d 1 to 42, water and feed intake were not different among the groups. At d 21 broilers administered 20 mg of PABP in water had the greatest BW (892 g; P < 0.001), followed by the birds administered 30 mg of PABP in water and 200 mg of PABP in the feed (869 and 868 g, respectively); the differences in BW attributable to PABP remained until d 42 (P < 0.01). Average daily gain was greater for birds in all the PABP groups compared with the control group (P < 0.01) in both the starter and grower periods. There was no effect of PABP on daily feed intake compared with control group at d 21 and 42. The FCR at d 21 was lower (P < 0.05) for birds administered PABP compared with the control group, but there was no significant difference in FCR at d 42 between the PABP groups and the control group.
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Compared with the controls, chickens administrated PABP had an increase in villus height and mucosa thickness in the duodenum (Table 2). In addition, administration of PABP in the drinking water increased villus height and mucosa thickness in the duodenum and jejunum (P < 0.05). Supplementation with PABP in feed at 150 mg/kg increased villus height and mucosa thickness in the jejunum (P < 0.05). However, there was no difference in villus height and mucosa thickness in the jejunum between the chickens on a feed supplemented with PABP at 200 mg/kg and the controls.
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The enzymatic activity of AKP was greater in the duodenum than in the jejunum (Figure 1A). A positive reaction of the AKP test showed as a black color and was displayed mainly in the mucosa epithelium surface (Figure 2A,B). In the duodenum and the jejunum, AKP activity was significantly greater for the PABP-treated groups compared with the control group (P < 0.05). The greatest AKP enzymatic activity in the duodenum and jejunum, respectively, was observed in birds receiving PABP at 20 and 30 mg/L in the drinking water. The AKP activity in the jejunum was greater in the PABP-treated groups than in the control group (P < 0.05). The greatest AKP activity was in the group administered PABP in the drinking water at 30 mg/L.
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Cells expressing sIgA stained brown and were displayed mainly in the mucosa epithelium and lymphocytes (Figure 2C, D
). In both the duodenum and jejunum, the area of positive sIgA was greater (P < 0.01 and < 0.05, respectively) in the PABP-treated groups compared with the control group. There was no significant difference among the PABP-treated groups (Figure 1B).
Effect of PABP on GC in the Small Intestine
The number of GC from all intestinal sections measured was higher (P < 0.01) in the PABP-treated groups than in the control group. The number of GC was higher in the 30 mg/L group than in the 20 mg/L group in both the duodenum and jejunum. The number of GC was higher in the 150 mg/kg group than in the 200 mg/kg group both in the duodenum and jejunum (Figure 1C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The increase of drug-resistant bacteria and the banning of antibiotic growth promoters worldwide make the search for novel means of preventing bacterial infections and promoting animal growth imperative (Van den Bogaard and Stobberingh, 2000). Antibacterial peptides are believed to be one of the ideal substitutes for bactericidal agents (Hancock and Lehrer, 1998). The present study demonstrated the effect of PABP as a potential feed additive on growth performance and mucous immunity in chickens. Diets supplemented with PABP can affect animal growth performance and nutrient digestion and absorption, and should be taken into account in diet formulations. Average daily gain was affected by PABP in both the starter and grower periods, and FCR was improved with administration of PABP during the starter period. The discrepancy of effects of PABP on growth performance could be ascribed to differences in dose and administration. It was important to analyze the morphology of broiler’s intestine and mucosal innate immunity to establish a possible mechanism of growth promotion.
The present results were in agreement with a previous study that reported that antibacterial peptides could stimulate growth performance in laying hens (Ma et al., 2004). To study the mechanism of action of PABP on the intestinal absorption, small intestine villus morphology was investigated in the current study. The intestine is the most important site of nutrient absorption, and its efficiency is correlated to its surface area, which is reflected by villus height and mucosa thickness (Hampson, 1986). In the present study, morphology of the duodenum and jejunum changed after treatment with PABP. Histological analysis showed that PABP increased villus height and mucosa thickness via administration in the drinking water or feed. The enhancement of growth performance observed in the study is consistent with other results (Liu et al., 2008). Peng et al. (2007) indicated that PABP can significantly promote growth performance and disease resistance in tilapia. Results of our study suggest that PABP administration can affect the rate and apparent energetic efficiency of the intestinal AKP activity (Figure 1A) and may increase the efficiency of nutritional utilization. Alkaline phosphatase is expressed mainly in the microvillus membrane of the intestine and exists both in soluble and membrane-bound forms (Torres et al., 2007). Alkaline phosphatase is an enzyme capable of hydrolyzing phosphate esters in an alkaline medium (Hirano et al., 1985) and is involved in digestive processes such as the absorption of cholesterol, lipids, vitamin D, calcium, amino acid, and glucose (Toofanian and Targowski, 1982). In the current study, the AKP activity was greater in PABP-treated groups than in the control group. This may imply that PABP potentiates the function of digestion and absorption by stimulating AKP activity. The result demonstrated that PABP could be more effective if administered in the drinking water. The notable effect of PABP administered in the drinking water on stimulation of the AKP supported our findings on growth.
sIgA and GC
For many years it has been appreciated that IgA antibodies in the intestinal (and other mucosal) secretions provide a first line of immunological defense against microbial pathogens by helping to prevent pathogens from adhering to and penetrating the mucosal epithelium (Russell and Kilian, 2005). There are 3 principal mechanisms by which sIgA is believed to aid mucosal immunity. The first is through synergic, nonspecific antimicrobial factors. The second mechanism is inactivation of bacterial enzymes and toxins. The third, and most important, is immune exclusion, where bacterial attachment to the mucus membrane is blocked (Bai et al., 2000). Little is known about the effects of dietary PABP in broiler mucosal immunity. It has been suggested that antibacterial peptides increase humoral immunity and antibody titers after vaccination in chickens (Yang et al., 2006). A recent study has shown that rabbit antibacterial peptides could increase the number of intestine intraepithelial lymphocyte and sIgA in the intestines of chickens (Liu et al., 2008). The intestine is relatively enriched in cells actively secreting IgA, and sIgA present in the lamina propria of villi in the duodenum and jejunum and in Peyer’s patches are important components of intestinal mucosal immunity (Mestecky et al., 1987). The results of this study showed that administration with PABP could enhance sIgA expression compared with the control group. This increase would strengthen the host’s defense mechanism in the intestinal mucosa (Brandtzaeg, 2007). There was a slight difference between the high dose and low dose when PABP was administered in the drinking water or in the feed. Further study is warranted to determine the biologically active concentration of PABP in broiler chickens.
Goblet cells play an important role in intestinal immune function. They can secrete mucus to the mucosa surface and participate in adjusting local intestinal immune function by a specific immune mechanism and nonspecific immune mechanism (Iijima et al., 2001). The protective effect of mucus is further evidenced by increased secretion on the mucosal surface (Tse and Chadee, 1991). The present study showed that PABP had a significant effect on the number of GC in all intestinal tracts evaluated. There were more GC in the duodenum and jejunum of the PABP-treated groups than in that of the control group.
Based on these results, the administration of PABP in the drinking water or feed is capable of promoting growth performance and intestine mucosal immunity. Supplementation with PABP induced a significant increase in GC, AKP, and sIgA in the duodenum and jejunum. However, further studies are required to determine the mechanisms that underlie these observations.
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ACKNOWLEDGMENTS |
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Received for publication August 6, 2008. Accepted for publication October 14, 2008.
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