ABSTRACT: The aim of this study was to evaluatethe effects of antibacterial peptide (ABP) suf?ciency oncellular immune functions by determining the spleencell cycle and apoptosis, peripheral blood T cell sub-sets, and T cell proliferation function in weaned piglets.A total of 90 piglets (Duroc × Landrace × Yorkshire)of both sexes were randomly allotted to 5 dietary treat-ments. Each treatment consisted of 3 replicates with 6piglets per replicate. The dietary treatments consistedof the negative control (NC; basal diet), positive control(PC; basal diet supplemented with 400 mg/kg Astraga-lus polysaccharide), and ABP (basal diet mixed with250, 500, and 1,000 mg/kg ABP). The experimentallasted for 28 d. Two piglets from each replicate wereselected randomly for blood samples extraction fromthe jugular vein to obtain peripheral blood T cell sub-sets, and T cell proliferation function analysis was per-formed on d 32, 39, 46, and 53. Two piglets from eachreplicate were selected and euthanized to observe the spleen cell cycle and apoptosis on d 39 and 53. InABP-suf?cient piglets, the G /G phase of the spleen cell cycle was much lower (P < 0.05) and the S and G +M phases and proliferation index (PI) were greater (P < 0.05) than in NC piglets. The percentage of apoptotic cells in the spleen signi?cantly decreased under ABP suf?ciency (P < 0.05). The proliferation function of peripheral blood T cells increased (P < 0.05) in ABP- suf?cient piglets. Percentages of CD + and CD CD ratios (d 39, 46, and 53) and CD CD ratios (d 32, 39, 46, and 53) increased remarkably (P < 0.05) under ABP suf?ciency compared with NC. These resultssuggest that ABP suf?ciency could increase the T cellpopulation and proliferation function of T cells andcould induce decreased percentages of apoptotic cells.Overall, the cellular immune function was evidentlyimproved in weaned piglets.We suggest optimal dosag-es of 500 mg/kg ABP for 4-wk addition and 1,000 mg/kg ABP for 2-wk addition.
Key words: antimicrobial peptide, apoptosis,
lymphocyte cycle, lymphocyte proliferation, T cell subset, weaned piglets
2015 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2015.93:127–134
In the modern pig industry, most piglets are
weaned at 3 to 4 wk of age; however, the immune func-tion of weaned piglets can be considered mature only at about 7 wk of age (Yang and Schultz, 1986; Kim et
al., 2004). During this critical period, some viral dis- eases, such as classical swine fever and porcine respi- ratory and reproductive syndrome, could cause great morbidity and mortality in weaned piglets, resulting in signi?cant economic loss. Therefore, improving the
immunity of weaned piglets is an important problem.Antimicrobial peptides represent a series of short- chain peptides composed of dozens of amino acid resi- dues. They are bioactive substances that are extracted, separated, and puri?ed from a variety of plants, ani- mals, and human tissues and cells in vivo (Wang and Wang, 2004). They have a broad range of functions, such as antibacterial (Koczulla and Bals, 2003), anti- viral (Huang et al., 2013), antifungal (Rossignol et al., 2011), antitumor (Yan et al., 2012), antiparasitic (Torrentet al., 2012), and immune function enhancing (Yu et al.,2010), among others. In some studies, dietary supple-mentation of different antibacterial peptides (ABP) to mice, chicken, rabbit, and piglet diets improved E ro- sette ratios (Geng et al., 2011) as well as levels of IgG, IgM, IgA, and alexin C (Lv et al., 2011; Guo et al.,
2012; Liu et al., 2012; S. D. Wu et al., 2012). To the best of our knowledge, few reports on theeffects of ABP on cellular immunity in weaned pigletsare available. In this study, we investigate the effectsof different concentrations of ABP on cellular immu-nity in weaned piglets.
MATERIALS AND METHODS
The antimicrobial peptide used in the present studywas provided by Rota Bioengineering Co., Ltd. (Sichuan,China).TheABPwas composed of swine defensin (DHY-ICAKKGGTCNFSPCPLFNRIEGTCYSGKAKCCIR)and a ?y antibacterial peptide (ATCDLLSGTGVKH- SACAAHCLLRGNRGGYCNGRAICVCRN)ata blending ratio of 50%. Astragalus polysaccharide (AP;net content 65%) was purchased from Centre BiologyCo., Ltd. (Beijing, China). All chemicals used were ofthe highest-purity grade available.
Animals and Experimental Design
Piglets (Landrace × Yorkshire × Duroc; 21 ± 2 d of age) were purchased from Xin Qiao Agricultural Sci- ence and Technology Development Co., Ltd. (Cheng- du, Sichuan, China) and were acclimated for 5 d be- fore the experiment. Weanling piglets (average BWof 8.24 ± 0.67 kg) were caged in elevated pens withwire ?ooring and fed a standard diet (Table 1; NRC,1998). The temperature (26°C to 27°C) and relativehumidity (65% to 70%) were kept constant. Food andwater were provided ad libitum during the acclimationperiod and throughout the study. All piglets used in this study were suitably healthy, and all experimen- tal manipulations were undertaken in accordance withthe Institutional Guidelines for the Care and Use ofLaboratory Animals. Ninety weanling piglets of both sexes were ran- domly allotted to 5 treatments in a randomized com- plete block design for 28 d. Each treatment consisted
of 3 replicates with 6 piglets per replicate. Dietarytreatments included the negative control (NC; basaldiet), positive control (PC; basal diet supplemented
with 400 mg/kg AP), and ABP (basal diet supplement- ed with 250, 500, or 1,000 mg/kg ABP). The NC diet
Table 1. Ingredient composition of diets, as-fed basis1
was considered to be a 0 mg/kg ABP treatment. Twopiglets from each replicate were selected randomly forblood extraction from the jugular vein to perform pe-ripheral blood T cell subset and T cell proliferationfunction analyses at 32, 39, 46, and 53 d of age. At39 and 53 d of age, 2 piglets from each replicate were selected from each treatment group and euthanized for spleen cell cycle and apoptosis determination via ?ow cytometry (Beckman Coulter Corp., Fullerton, CA).
At 32, 39, 46, and 53 d of age, 6 piglets from eachtreatment group were selected, and blood sampleswere obtained by puncturing the vena cava. Lympho- cyte proliferation was measured as described by Fan et al. (2012). Two milliliters of peripheral blood were collectedin 5-mL heparinized vacuum tubes (Vacutainer System, Becton Dickinson, Franklin Lakes, NJ), were mixed with an equal volume of Hanks’ solution (HyClone, Thermo Scienti?c, Logan, UT), and then were care-fully layered on the surface of the lymphocyte separa-tion medium (density of 1.077 ± 0.001 g/mL; JingyangCo., Tianjin, China). The vacuum tubes were then cen- trifuged at 3,000 × g for 20 min at room temperature.
Mononuclear cells were collected and washed 3 times with RPMI 1640 medium (Gibco BRL, Grand Island, NY) without fetal bovine serum. The resulting pelletwas resuspended to 2 × 10 cells/mL with complete RPMI 1640 medium for proliferation assay.
Suspensions of mononuclear cells (2 × 10 /well) were incubated in 96-well culture plates at 100 μL perwell; each sample was seeded in 6 wells. Exactly 100 μLof concanavalin A (ConA; 10 μg/mL; Sigma ChemicalCo., St. Louis, MO) were then added into each well. Theplates were incubated in a humid atmosphere of 5% CO2 for 44 h at 37°C. Then, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; 5 mg/ mL; Sigma Chemical Co.] were added to each well, and
the plates were reincubated for another 4 h. After incu- bation, 100 μL ofdimethyl sulfoxide (DMSO; Sigma Chemical Co.,) were added to each well.The plates were
shaken for 10 min to dissolve the precipitate completelyand then were placed in an automated ELISA reader(MQX200; BioTek Instruments Inc., Winooski, VT) for absorbance measurement at 570 nm. The stimulation in- dex (SI), which indicates the lymphocyte proliferationactivity, was calculated as follows: SI = OD (optical density) value of ConA-stimu-lating cells/OD value of ConA-free cells.
Spleen Cell Cycle
At 39 and 53 d of age, 6 piglets were euthanizedin each group to determine spleen cell cycle stages by ?ow cytometry, as described by B. Y. Wu (2012). Im- mediately after death, the spleen of each piglet (about0.2 cm ) was placed on a ring glass plate (diameter of6 cm) containing 0.5 mL of normal saline, was broken
into a single-cell suspension with ophthalmic scissors and then was transferred to a centrifuge tube contain- ing 1.5 mL of normal saline. The single-cell suspension
was ?ltered through a 300 mesh nylon screen. The cells were washed twice and diluted to 1.0 × 10 cells/mL with PBS. About 1 mL of the solution wastransferred
to another tube for centrifugation at 200 × g for 5 min. The supernatant was discarded, and 1 mL of propidium iodide (5 μL/mL propidium iodide, 0.5% Triton X-100, 0.5% RNase, PBS) was added to the pellet. Staining for20 min at room temperature was performed, followed by washing with PBS. The supernatant was discarded, and cells were resuspended in 0.5 mL of PBS. Cell phases were ?nally analyzed by ?ow cytometry. The proliferation index (PI) was calculated as
Annexin V Apoptosis Detection by Flow Cytometry
At 39 and 53 d of age, 6 piglets were euthanizedin each group to determine the percentage of apoptoticcells in the spleen, as described by Chen et al. (2013).The cells were consistent with the lymphocyte cycle ofthe spleen. About 100 μL of the cell suspension weretransferred to a centrifuge tube, and about 5 μL of V-FITC (BD Pharmingen, Sparks, Maryland, USA) and5 μL of propidium iodide (5 μL/mL propidium iodide,0.5% Triton X-100, 0.5% RNase, PBS) were mixed with this suspension for staining at room temperature for 15 min in the dark. About 400 μL of 1× binding buffer were added to each centrifuge tube, and ?ow cytometry assay was performed within 1 h.
T Cell Subsets
At 32, 39, 46, and 53 d of age, 6 piglets from eachtreatment group were selected, and blood samples were obtained by puncturing the vena cava. The percentages blood were determined via ?ow cytometry (BeckmanCoulter Corp.), as described by Chen et al. (2009).About 1 mL of peripheral blood was collected in5-mL heparinized vacuum tubes, was mixed with an
equal volume of PBS (0.01 M and pH 7.4), and wascarefully layered on the surface of the lymphocyteseparation medium. Centrifugation was done at 200 ×g for 20 min at room temperature. The lymphocyteswere collected, transferred to another centrifuge tube,
and then washed with PBS. The resulting pellet was resuspended at a concentration of 1 × 10 cells/mL with PBS. About 1 mL of cell suspension was trans-ferred to another tube for centrifugation at 200 × g for5 min. The supernatant was discarded. The cells were
stained with 10 μL of mouse anti-pig CD phytoery thrin (Southern Biotechnology Associates, Birming ham, AL), mouse anti-pig CD phycoerythrin (South-ern Biotechnology Associates), and mouse anti-pig CD a FITC (Southern Biotechnology Associates) for20 min at room temperature and then were washed with PBS. The supernatant was discarded, and cells were resuspended in 0.5 mL of PBS and analyzed by ?ow cytometry
Results are reported as mean ± SD. Statisticalanalysis was performed by 1-way ANOVA usingSPSS 19.0 software (International Business MachinesCorporation,Armonk, NY). Duncan’s test for multiplecomparisons was done, and P < 0.05 was consideredstatistically signi?cant.
Peripheral Lymphocyte Proliferation Assay
During the entire experimental period, the SI of pe-
the level of dietary ABP increased.ripheral blood T cells was greater (P < 0.05; Table 2)in piglets fed the PC and the 250, 500, and 1,000 mg/kg ABP diets than in the piglets fed the NC diet. Pigletsgiven the 250 mg/kg ABP diet on d 46 and 53 and the
500 and 1,000 mg/kg ABP diets on d 32, 39, 46, and 53had greater (P < 0.05) SI values for peripheral blood Tcells than piglets given the PC diet. Moreover, the SI ofperipheral blood T cells linearly improved (P < 0.05) as
Cell Cycle of Spleen
On d 39 and 53, the G /G phase distributions of spleen cells were lower (P < 0.05; Table 3 and Fig. 1) in piglets fed the PC and 250, 500, and 1,000 mg/kg ABP diets than in piglets fed the NC diet. The S and G + M phase cell distributions as well as PI gain were greater(P < 0.05) in the spleen cells of piglets fed the PC and
250, 500, and 1,000 mg/kg ABP diets than in piglets fed the NC diet. Also, piglets given the 1,000 mg/kg ABP
diet showed better (P < 0.05) overall G /G phases, Sphase cell distributions, and PI in spleen cells than pig-lets given the PC diet on d 39. On d 53, piglets given the250, 500, and 1,000 mg/kgABPdiets showed better (P < butions, and PI in spleen cells than piglets given the PCdiet. However, piglets given the 1,000 mg/kg ABP diet
had fewer (P < 0.05) G /G phases, G + M phases, and S phase cell distributions and a lower PI in spleen cellsthan piglets given the 250 and 500 mg/kg ABP diets.
Apoptosis of Spleen Cells
On d 39 and 53, piglets given the PC and 250,500, and 1,000 mg/kg ABP diets had lower (P < 0.05;Table 4 and Fig. 2) percentages of apoptotic spleencells than piglets fed the NC diet. The percentage ofapoptotic spleen cells was lower (P < 0.05) in pig-lets given the 1,000 mg/kg ABP diet on d 39 and the.
250, 500, and 1,000 mg/kg ABP diets on d 53 than in piglets given the PC diet. However, piglets given the 1,000 mg/kg ABP diet had greater (P < 0.05) percent- ages of apoptotic spleen cells than piglets given the250 and 500 mg/kg ABP diets on d 53.
(d 39, 46, and 53) and CD CD ratios (d 32, 39, 46, and 53) were greater (P < 0.05; Table 5) in piglets given the 250, 500, and 1,000 mg/kg ABP diets than in piglets fed the NC diet. The percentages of CD +
Lymphocyte proliferation is an indicator of thestate of cellular immunity. T and B lymphocytes playan important role in enhancing the immune functionsof various organisms (Minato et al., 2004). In our study, supplementation with AP and ABP enhanced the pro- liferation of T lymphocytes, although ABP showed bet- ter effects than AP. This result is consistent with Fan et al.’s (2012) study, in which 4.0 mg/mL AP was admin- istered to weaned piglets. In line with the results of the
present study, Wang and Li (2007) reported that oraladministration of 300 and 600 mg/kg ABP to broilers could promote (P < 0.05) proliferation of T lympho cytes in peripheral blood. Similarly, weaned piglets fed diets supplemented with 1,000 mg/kg ABP (lactofer- rin) were reported to have greater (P < 0.05) phytohe- magglutinin (PHA)–stimulated peripheral lymphocyte proliferation (Shan et al., 2007). Increased (P < 0.05) ANAE (acidalpha naphthyl aetate esterase) percent ages of T lymphocytes of the thymus, spleen, and bursaof Fabricius were also reported in chickens receivingdrinking water supplemented with 1 μg/mL ABP
that was isolated from African ostrich skin (Yang et al.,2009). In contrast to the results of the present study, Yang et al. (2006) reported no signi?cant differences in B lymphocyte proliferation in chickens receivingdrinking water supplemented with chickenintestinal ntimicrobial peptides (1 mg/mL) right after hatching.This discrepancy in the results may be attributed tovariations in the type of ABP used, the level of dietary
supplementation, and the mode of action of the ABP.Four major phases of the eukaryotic cell cycle have been described: the G phase, which occurs before DNA replication; the S phase, which describes periods of DNA synthesis; the G phase, which occurs before cell
division; and the M phase, which describes actual celldivision (Pines, 1995). To the best of our knowledge, noreports on the effects of ABP on the spleen cell cycle ofweanling piglets are yet available. In our study, supple-mentation of AP and ABP in piglets caused decreases. in G phase cells, which corresponds to increases in Sand G + M phase cells and PI in the spleen. Also, ABPshowed better effects than AP. Results showed that AP and ABP suf?ciency can cause developmental progres- sion of the spleen because of cell growth promotion in piglets. Shan et al. (2007) reported that supplement- ing 1,000 mg/kg antimicrobial peptide (lactoferrin) re- markably increases (P < 0.05) ConA and PHA-inducedspleen lymphocyte proliferation in weanling piglets.Themechanism of ABP in lymphocyte proliferation is not clear. Interleukin-2 is believed to play a central role in regulating host responses to pathogenic challenges and is known as the principal cytokine responsible for the
progression of T cells from the G phase to the S phase of the cell cycle (Bonham et al., 2002). Therefore, wesuppose that ABP could promote lymphocyte prolifera-
tion by improving production levels of the cytokine IL-2.
黏膜、免疫细胞以及免疫器官等部位，并且具有广谱的杀菌作用，越来越多的研究者认为抗菌肽是目前最有潜力的新抗菌药物。现在对于抗菌肽协同研究的报告不多，文中利用改良的肉汤稀释法对猪防御素和抗生素的最小抑菌浓度（MIC）做了相关研究，然后再通过相同浓度的猪防御素与不同浓度的六种抗生素进行联合抑菌试验，并观察抗生素 MIC值的变化情况。试验结果表明，猪防御素与其中五种抗生素的联合使用都能使抗生素的 MIC值降低，这证明猪防御素具备与抗生素协同抑菌的潜力，也为抗菌肽在配方饲料中的研究提供了良好的试验基础。
近些年来，畜产品中的抗生素药物残留问题越来越受到人们的重视，许多国家都出台了相应的政策来限制抗生素的添加，有的国家甚至禁止任何抗生素在饲料中的使用。此外，由于抗生素的使用不当 ,也造成了许多畜产品中耐药菌株的产生，并且其出现的频率已超过抗生素新药的开发速度 ,给疾病的治疗带来了很大的难度。因此，在畜产品生产中需要寻找一种新的途径来替代现有的传统生产方式。抗菌肽普遍存在于自然界的生物体中，是先天性免疫系统的重要组成成分，主要分布在黏膜，免疫细胞以及免疫器官等部位。抗菌肽不仅具有广泛的杀菌作用，还具有稳定性好，水溶性好、抗菌机理独特对高等动物正常细胞无害等特点。关于抗菌肽和抗生素联合抑菌的报道虽然不多，但已经有一些相关报道证实了抗菌肽和抗生素之间存在协同抑菌效果。本文利用改良的肉汤稀释法来判定猪防御素和六种抗生素之间联合。抑菌试验效果，为药物合理使用问题提供了一个科学的参考依据。如果抗菌肽和抗生素的联合抑菌存在协同作用或累加作用，不仅能更好地提高用药效果，而且合理减少用药剂量以避免达到毒性剂量的危险，预防
猪防御素：四川华德生物工程有限公司提供，其成分为抗菌肽；抗生素：硫酸黏杆菌素（ colistin sul? fate）、阿散酸（arsanilic acid）、替米考星（tilmicosin）、和6种抗生素的MIC值找出来，然后来确定每种药物的氟苯尼考（florfenicol）、盐酸多西环素（doxycycline hy?drochloride）、阿莫西林（amoxicillin）全部购自于中国兽医药品监察所。
大肠埃希氏菌（Escherichia coli）和金黄色葡萄球菌（Staphylococcus aureus）均购置于中国工业微生物菌种保藏管理中心。
蛋白胨 10 g/l、酵母浸粉 5 g/l、NaCl 10 g/l、琼脂 15 g/l，pH值自然，121 ℃条件下高压灭菌15 min。
1.2.3LB液体培养基蛋白胨 10 g/l、酵母浸粉 5 g/l、NaCl 10 g/l，自然，121 ℃条件下高压灭菌 15 min.
分别将大肠埃希氏菌（Escherichia coli）和金黄色葡萄球菌（Staphylococcus aureus）划线菌接种至LB琼脂平板上，37 ℃培养 24 h，挑取平板上的一个单菌落的所对接种至30 ml（250 ml三角瓶）LB液体培养基中的浓度值
高压蒸汽灭菌锅（上海申安LDZX-30KBS）、超净工作台（苏州净化SW-CJ-2FD）、可见分光光度计（上海佑科 721）、恒温振荡培养器（上海苏坤 SKY-200B）、隔水式培养箱（GNP-9080）、96孔酶标板。
抗菌肽的制备：精确称取抗菌肽，配制成浓度为0.1 g/ml的溶液，在2 000 r/min下离心 10 min，留上清液备用。
抗生素的制备：精确称取抗生素，用各自对应缓冲液将抗生素溶解配制成如表1对应浓度在2 000 r/min下离心 10 min，留上清液备用。
由于抗菌肽的自身结构的特殊性，所以试验在微量肉汤稀释法的基础上加以改进，这样能更准确地反应试验效果。试验前期先用微量稀释法把猪防御素依次对倍系列稀释，应用改良的肉汤稀释法观测 6种抗生素MIC的变化。该试验通过96孔酶标板作为细菌培养器皿，每排共有12孔，在每排的第1个孔和第 9个孔加入270μl含指示菌的LB营养液，剩余的孔全部加入含
150μl含指示菌的LB营养液。然后向每排的第一孔加入指定的样品溶液 30 μl（单品15 μl+对应单品溶解缓冲液 15 μl；单品15 μl+抗菌肽 15 μl）。然后每排用吸头从第一个孔开始吹打混匀后依次向下面一个孔加入150 μl混合液，直到加入每排第 8个孔有孔中的混合液按照 10 ~10 6个稀释梯度涂板计数，最后算出混合液菌含量。该实验最低抑菌浓度定义为抑制指示菌浓度对数。
猪防御素与六种抗生素联合使用后对不同指示菌MIC的变化情况以及对应的 FIC指数见表 2。由于硫酸黏杆菌素主要对革兰氏阴性菌存在抑菌作用，阿散酸、替米考星这两种抗生素主要对革兰氏阳性菌存在抑菌作用，所以试验中只有盐酸多西环素、氟苯尼考和阿莫西林同时做了革兰氏阳性菌和革兰氏阴性菌的抑菌试验。对于革兰氏阴性菌而言 ,从MIC的变化情况来看，该试验只有氟苯尼考和阿莫西林单独使用时或者与猪防御素联合其 MIC没有任何变化，替米考星的MIC降低得最多，是原来的 1/8倍，这些数据表明了猪防御素与六种抗生素的联合使用能提高大多数抗生素的抑菌效果。从不同指示菌角度来看，对于革兰氏阴性菌而言，抗生素联合使用时
硫酸黏杆菌素、盐酸多西环素、氟苯尼考、阿莫西林的 MIC分别为 0.187 5、2.5、6.25、12 μg/ml，单独使用时硫酸黏杆菌素、盐酸多西环素、氟苯尼考、阿莫西林对应的 MIC分别为 0.75、10、6.25、12 μg/ml，通过 FIC值的计算结果可以判断出猪防御素与硫酸黏杆菌素、盐酸多西环素存在协同抑菌效果，与氟苯尼考及阿莫西林联合抑制大肠埃希氏菌时存在无关作用。对于革兰氏阳性菌而言，猪防御素与盐酸多西环素、氟苯尼考、替米考星、阿散酸以及阿莫西林的联合使用所对应的 MIC分别为 0.125、3.125、6.25、25、15 μg/ml，这5种抗生素单独使用时 MIC分别为 0.25、6.25、50、100、30 μg/ml，通过计算FIC值可以判断出，猪防御素与阿散酸、替米考星具有协同抑制金黄色葡萄球菌的作用；此外，猪防御素与盐酸多西环素、氟苯尼考及阿莫西林具有累加抑菌效果。抗生素是通过破坏细菌细胞壁杀灭细菌的，抗菌肽是通过与细菌细胞壁结合，然后使细菌胞内物质溢出来杀灭细菌的，当抗菌肽和抗生素联合抑菌时，他们可能结合了各自的作用机理，使验结果分析可以看出，猪防御素的加入使盐酸多西环素、替米考星、氟苯尼考、阿散酸和阿莫西林的最低抑菌浓度都有不同程度的降低，间接表明了抗菌肽的加入使这几种某些抗生素的抑菌效果都提高了。本次研究表明了猪防御素和某些抗生素之间存在协同抑菌的作用，同时也体现出抗菌肽在配方饲料中的潜在应用价值。在本次试验研究的基础上，今后还需要付出更多的时间和精力去研究不同的抗生素与不同抗菌肽联合抑菌的相关机理，以及更进一步的动物体内、体外试验研究，为抗菌肽在新配方饲料方案中的大量使用奠定更加完善的试验基础。近年来有许多