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Effects of antibac...

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

doi:10.2527/jas2014-7933

INTRODUCTION

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

Materials

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).

Lymphocyte Proliferation

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

follows:

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

Data Analysis

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.

RESULTS

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 +

DISCUSSION

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.

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猪防御素与六种抗生素联合使用的抑菌情...

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,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

及延迟抗生素耐药性的发生,能更好地治疗混合性感染,这也为寻找新的用药方案提供了良好的铺垫。

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,预防及延迟抗生素耐药性的发生,能更好地治疗混合性感染,这也为寻找新的用药方案提供了良好的铺垫。

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。

 

1.2菌种及培养基

1.21菌种

 

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。

 

1.22 LB固体培养基

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.

 

 菌悬液的制备。

 

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

1.4主要仪器

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。

实验结果分析

 

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

 

研究表明,抗菌肽的确对动物的常见致病菌具有强烈的抑制作用,并且对动物的生长性能也有促进作用。在养殖行业的疾病防御期间,如果在饲料中添加抗菌肽,能够避免致病菌产生耐药性,其使用效果稳定,属无毒副作用、无残留、无致细菌耐药性的一类环保型

饲料添加剂

在畜产品生产行业当中,尤其是在疾病治疗控制期间,如果让抗菌肽和抗生素混合合理使用,这样不仅能更好地提高用药效果,还可以降低因药物过度使用造成的经济损失,并且合理减少用药剂量以避免达到毒性剂量的危险,最重要的是利用抗菌肽和抗生素的合理搭配使用还可以预防及延迟抗生素耐药性的发生,能更好地治疗混合性感染。因此抗菌肽的确是寻找新的药物使用替代方案的理想候选。

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韩国Park博士《不使用促生长抗生素...
韩国养猪协会理事,首尔大学动物营养学博士Jong-ho Park博士讲述了韩国禁用抗生素后的饲料发展策略,他主要从动物肠道健康原理,动物营养策略以及抗生素替代物应用策略等方面对禁用抗生素后的饲料策略进行全面细致的分析,Park博士认为在乳仔猪腹泻的问题上应该从增强肠道健康,使用替代性抗菌物质以及现场治疗三个方面入手解决,而其中传统饲料配方中的部分因素会直接成为影响肠道健康的关键因素,比如高蛋白问题,可溶性多糖问题,乳清粉问题等,此外在改善肠道健康同时还必须做好防控工作,使用一些功能性添加剂以清除肠道有害微生物,比如酸化剂,微生态制剂,抗菌肽等物质,最后Park博士提出肠道问题是系统问题需要多方面多层次解决,并且在使用添加剂策略方面应该慎重,尽力避免为了解决一个现有问题而添加新产品,并引入新问题。
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抗菌肽在家禽养殖中的应用前景
抗菌肽普遍存在于自然界的生物体中,目前发现的抗菌肽大多数来源于动物,它是免疫多肽,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,是脊椎动物长期进化保留下的有效防御机制。本文主要针对抗菌肽的作用以及抗菌肽在家禽方面的一些实验研究实例来说明抗菌肽在家禽养殖方面是非常有潜力的。
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抗菌肽在产业化养殖中的应用
近年来,随着规模化养殖的快速发展,畜禽和水产养殖中普遍存在抗生素和农药等化学药物的滥用,从而导致农产品的耐药性增强以及药物残留,不仅威胁人类健康,还造成生态环境的恶化。因此,人们迫切希望寻找一种新的药物,其不仅需要具有抗菌和杀虫的作用,还要对宿主没有危害并且对环境、动物和人类的消费产品没有副作用。抗菌肽的诸多特性使其成为了规模化养殖的的最适候选者。
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