Effects of Echinacea Compound on the lmmune Function of Weaned Piglets

Yanfang FU，Shaopeng MA，Weiping YAO，Guisheng GAO，Qiumei SHl*，Yanying ZHANG，Guangping GAO

1.Animal Husbandry and Veterinary Station of Hebei Province，Shijiazhuang 050031，China；

2.Xinji Bureau of Animal Husbandry，Xinji 052360，China；

3.Funing Bureau of Animal Husbandry and Aquatic Products，Funing 066300，China；

4.Key Laboratory of Preventive Veterinary Medicine of Hebei Province/College of Animal Science and Technology，Hebei Normal University of Science and Technology，Changli 066600，China

Effects of Echinacea Compound on the lmmune Function of Weaned Piglets

Yanfang FU1，Shaopeng MA2，Weiping YAO3，Guisheng GAO4，Qiumei SHl4*，Yanying ZHANG4，Guangping GAO4

1.Animal Husbandry and Veterinary Station of Hebei Province，Shijiazhuang 050031，China；

2.Xinji Bureau of Animal Husbandry，Xinji 052360，China；

3.Funing Bureau of Animal Husbandry and Aquatic Products，Funing 066300，China；

4.Key Laboratory of Preventive Veterinary Medicine of Hebei Province/College of Animal Science and Technology，Hebei Normal University of Science and Technology，Changli 066600，China

［Objective］This study aimed to investigate effects of Echinacea compound on the immune function of weaned piglets. ［Method］Eighty Duroc×Landrace× Yorkshire crossbred piglets were randomly divided into four groups:control group（drug-free group），1.5%dose group，1.0%dose group and 0.5%dose group.Blood samples of piglets were collected at 20，35，50，60，70 and 80 days old，respectively，to determine neutrophilleukocyte percentage in peripheralblood，lymphocyte transformation rate and the levels ofantibodies againstclassicalswine fever，porcine reproductive and respiratory syndrome disorder. ［Result］Applying a certain dose of Echinacea compound could significantly increase neutrophil leukocyte percentage in peripheral blood and lymphocyte transformation rate （P＜0.05），and extremely significantly improve the levels of antibodies against classical swine fever，porcine reproductive and respiratory syndrome disorder（P＜0.01）.［Conclusion］Echinacea compound has played a certain role in promoting nonspecific and specific immune function of piglets.

Echinacea compound；Immune function；Neutrophil leukocyte；Antibodies；Lymphocyte transformation rate

P olysaccharidesare a novel type of natural low-toxicity immune enhancers without drug residues.Modern medical research has found that Echinacea polysaccharides（EPS）and Astrgalus polysaccharides （APS）have immunomodulatory functions to improve the body’s specific and non-specific immune function，increase lymphocyte transformation rate and enhance the body’s immune response to the antigen［1-5］.Echinacea compound，composed of EPS and APS，has been widely applied in clinical practice as an immune enhancer or immunomodulatory agent.So far，a large number of studies have been reported on the effects of Echinacea compound on the immune function of mouse，chicken and other animals［6-9］，but little information is available about its effects on the immune function of pig.In this study，after the application of different doses of Echinacea compound，neutrophil leukocyte percentage in peripheral blood，lymphocyte transformation rate，and antibody titers against classical swine fever，porcine reproductive and respiratory syndrome disorder of weaned piglets were detected to analyze effects of Echinacea compound on the immune function of piglets.

Materials and Methods

Chinese herbal medicine and reagents

Echinacea and Astragalus mon-gholicus were purchased from Qinhuangdao Baicaotang Pharmaceutical Co.， Ltd. CSFV positive-indirect hemagglutination assay kit was purchased from Lanzhou Veterinary Research Institute，Chinese Academy of Agricultural Sciences，which was used to determine antibody titers against classical swine fever；96-well hemagglutination plates （V-type）were purchased from Jiangsu Kangjianhua Medical Apparatus Co.，Ltd.Colloidal gold test strips were purchased from Shanghai Quicking Biotech Co.，Ltd.，which were used to determine antibody titers against porcine reproductive and respiratory syndrome disorder.

Experimental animals

Eighty Duroc×Landrace×Yorkshire crossbred piglets were provided by a pig farm in Qinhuangdao City.

Preparation of the Chinese herbal compound

Qualified Chinese medicine raw material was soaked into water for 3 h，extracted three times （added with 10 times the amount of water and boiled for 1 h），and prepared into granular preparation through filtration-concentration-spray drying-dispensing（0.24 g of Chinese herbal compound was extracted from 1 g of raw material）.

Grouping and treatment of experimental animals

After clinical examination，80 healthy 15-day-old Duroc×Landrace× Yorkshire crossbred piglets were selected and randomly divided into four groups according to the gender and weight:controlgroup，1.5% dose group，1.0%dose group and 0.5% dose groups，20 piglets per group.

Detection of neutrophil leukocyte percentage in peripheral blood of piglets

Neutrophil leukocyte percentage in peripheral blood of piglets was detected with nitro blue tetrazolium（NBT）reduction test.First，0.5-1.0 ml of anticoagulated venous blood sample was coated on the slide，dried，fixed with methanol for 1-2 min and dried.Second，the blood smears were stained with Wright-Giemsa stain for 5 min，rinsed and dried.Neutrophil leucocyte were counted under oil immersion lens.Result evaluation:if nitro blue tetrazolium （NBT）was reduced by more than 10%of neutrophil leucocyte on the blood smears，the result of NBT test was positive；if the percentage was lower than 10%，the result of NBT test was negative.

Lymphocyte transformation assay

Lymphocyte transformation assay was conducted with morphological examination method，using nonspecific mitogen phytohemagglutinin（PHA）as a stimulus.Specific operations were performed in accordance with the method proposed by He et al［10］.

Determination of immune function

Healthy piglets were immunized with vaccines against classical swine fever，porcine reproductive and respiratory syndrome disorder at 20 and 50 days old，respectively.Ten piglets were randomly selected from each group at 20，30，40，50，60，70 and 80 days old，respectively；3 ml of whole blood was collected to separate serum that was preserved at-20℃.Antibody titers against classical swine fever were determined with indirect hemagglutination method；antibody titers against porcine reproductive and respiratory syndrome disorder were determined with colloidal gold test strips. Specific operations were performed in accordance with kit instructions.

Data processing

Experimental data were analyzed using SPSS 11.3 GLM statistical software.

Results and Analysis

Effects of Echinacea compound on neutrophil leukocyte percentage in peripheral blood of piglets

As shown in Table 1，at 40 days old，neutrophil leukocyte percentage in peripheral blood of piglets in experimental groups was higher than that in controlgroup，exhibiting extremely significant differences（P＜0.01）.Among various experimentalgroups，neutrophil leukocyte percentage in peripheral blood of piglets in 1.0%dose group was higher than that in 0.5% dose group and lower than that in 1.5%dose group，exhibiting no significant differences（P＞0.05）.At 50 days old，neutrophil leukocyte percentage in peripheral blood of piglets in experimental groups was higher than that in control group.Specifically，neutrophil leukocyte percentage in peripheral blood of piglets in 1.5%dose group was significantly higher than that in control group （P＜0.05）；no significant difference was observed in neutrophil leukocyte percentage in peripheral blood of piglets among other groups（P＞0.05）.

Table 1 Effects of Echinacea compound on neutrophil leukocyte percentage in peripheral blood of piglets %

Effects of Echinacea compound on T lymphocyte transformation rate of piglets

As shown in Table 2，at 20，30 days old，T lymphocyte transformation rate of piglets in four groups exhibited no significant difference （P＞0.05）.At 40，50 days old，compared with control group，T lymphocyte transformation rate of piglets in 0.5%dose group was improved by 20.48%and 19.19%，respectively （P＜0.01）；T lymphocyte transformation rate of piglets in 1.0% dose group was improved by 13.34% and 20.19%，respectively（P＜0.01）；T lymphocyte transformation rate of piglets in 1.5%dose group was improved by 16.79%and 18.47%，respectively （P＜0.01）.At 80 days old，compared with control group，T lymphocyte transformation rate of piglets in 1.5%dose group was improved by 13.36%（P＜0.01）.

Table 2 Effects of Echinacea compound on T lymphocyte transformation rate of piglets %

Determination of serum antibody titers against classical swine fever after immunization

Serum antibody titers against classical swine fever were determined according to the instructions of CSFV positive-indirect hemagglutination assay kit.Hemagglutination inhibition antibody titers refer to the maximum dilution multiple of the serum when 50% of erythrocytes agglutinated.Hemagglutination inhibition antibody titers higher than 1∶16 indicated qualified immunization；hemagglutination inhibition antibody titers lower than 1∶16 indicated unqualified immunization.

As shown in Table 3，the proportion of qualified immunized piglets increased since 60 days old（secondary immunization）and reached the qualification level（1∶16）.Therefore，newborn piglets immunized at 20 days old should be immunized once again at 45-50 days old to achieve the qualification level of antibody titers after secondary immunization.Adding Echinacea compound into feed before and after immunization led to better results. Specifically，the immunity of piglets was improved remarkably in 1.5% dose group；at 70-90 days old，the indirect hemagglutination antibody titer was improved by 1.3-1.9 log2 compared with control group.

Determination of serum antibody titers against porcine reproductive and respiratory syndrome disorder

Serum antibody titers against porcine reproductive and respiratory syndrome disorder were determined with colloidal gold strips.As shown in Table 4，the proportion of qualified immunized piglets increased since 60 days old （secondary immunization）and reached the qualification level（1∶16）.Therefore，newborn piglets immunized at 20 days old should be immunized once again at 45-50 days old to achieve the qualification level of antibody titers after secondary immunization.Adding Echinacea compound into feed before and after immunization resulted in better results.Specifically，the immunity of piglets was improved significantly in 1.5% dose group；at 70-90 days old，the neutralizing antibody titer was improved by 2.0-2.1 log2 compared with control group.

According to the results，after inoculation of vaccines against classical swine fever，porcine reproductive and respiratory syndrome disorder，drug administration by mixing 1.5%Echinacea compound into feed could significantly improve the levels of IHA and NA antibodies，exhibiting significant differences compared with control group（P＜0.05）.

Table 3 Effects of Echinacea compound on antibody titers against classical swine fever

Conclusion and Discussion

At the beginning of the experiment，neutrophil leukocyte percentage in peripheral blood of piglets in various group exhibited no significant difference.At 40 days old，neutrophil leuko-cyte percentage in peripheral blood of piglets in experimental groups was extremely significantly higher than that in control group.Since 50 days old，neutrophil leukocyte percentage in peripheral blood of piglets in experimental groups was higher than that in control group.Therefore，Echinacea compound had played a certain role in promoting nonspecific and specific immune function of piglets.

Table 4 Effects of Echinacea compound on antibody titers against porcine reproductive and respiratory syndrome disorder

At 40 days old，lymphocyte transformation rate of piglets in 1.0%and 1.5%dose groups was significantly and extremely significantly higher than that in control group，respectively，but lymphocyte transformation rate of piglets in 0.5%dose group exhibited no significantdifference compared with control group.At 50 days old，lymphocyte transformation rate of piglets in 1.0%and 1.5%dose groups was significantly higher than that in control group，but there was no significant difference in lymphocyte transformation rate of piglets between 0.5%dose group and control group，which indicated that applying a certain dose of Echinacea compound can increase lymphocyte transformation rate and improve the immune function of piglets.Similarly，Ma et al.［11］added two different refined Chinese herbal feed additives into basal diet and found that Chinese herbal additives could improve the growth performance of weaned piglets，promote the generation of immune globulin，complement and IL-2，and enhance the immune function.

According to the antibody levels of piglets after inoculation of vaccines against classical swine fever，porcine reproductive and respiratory syndrome disorder，there was no significant difference in the levels of antibodies against classical swine fever among various groups.At 50 and 70 days old，the levels of antibodies against classical swine fever，porcine reproductive and respiratory syndrome disorder in experimental groups were extremely significantly higher compared with control group.Therefore，applying a certain dose of Echinacea compound could significantly improve the levels of antibodies againstclassicalswine fever，porcine reproductive and respiratory syndrome disorder，which suggested that Echinacea compound had played a certain role in promoting immune function of piglets.Liu et al.［12］and Wang［13］found that Chinese herbal compound could significantly enhance T lymphocyte transformation rate and leukocyte phagocytosis rate in blood of piglets and increase the levels of antibodies against classical swine fever to improve cellular immunity and humoral immunity，thereby improving the immune function and disease resistance of piglets.

In this study，based on determination of neutrophil leukocyte percentage in peripheral blood，lymphocyte transformation rate and the levels of antibodies againstclassicalswine fever，porcine reproductive and respiratory syndrome disorder，the effects of Echinacea compound on nonspecific immunity，cellular immunity and humoral immunity of piglets were preliminarily analyzed，which laid a solid foundation for further investigating the mechanism of the effects of Echinacea compound on immune function of pigs.

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Responsible proofreader：Xiaoyan WU

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紫锥菊复方对仔猪免疫功能的影响

付艳芳1，马少朋2，姚维平3，高桂生4，史秋梅4*，张艳英4，高光平4（1.河北省畜牧站，河北石家庄050031；2.辛集市畜牧局，河北辛集052360；3.抚宁县畜牧水产局，河北抚宁066300；4.河北省预防兽医学重点实验室，河北科技师范学院动科院，河北昌黎066600）

［目的］研究紫锥菊复方对仔猪免疫机能的影响。［方法］试验将三元杂交仔猪随机分为4组，对照组（无药组）、试验Ⅰ、Ⅱ、Ⅲ组（紫锥菊复方1.5%组、1.0%组、0.5%组）。并分别于20、35、50、60、70、80日龄采血检测仔猪外周血嗜中性白细胞百分数、淋巴细胞转化率、猪瘟、猪呼吸繁殖障碍综合征抗体水平等免疫指标。［结果］应用一定剂量的紫锥菊复方能显著提高仔猪外周血嗜中性白细胞百分数和淋巴细胞转化率（P＜0.05），极显著提高猪瘟、猪呼吸繁殖障碍综合征疫苗的抗体水平 （P＜0.01）。［结论］紫锥菊复方对仔猪的免疫功能具有一定的促进作用。

紫锥菊复方；免疫功能；嗜中性白细胞；抗体；淋巴细胞转化率

国家自然基金（31472230）；河北省自然基金 （C2014407068）；河北省科技厅（14966610D）； 石 家 庄 市 科 技 局（131200063A）；河北省教育厅：百名优秀创新人才支持计划（Ⅱ）（ZH2011244）。

付艳芳（1978-），女，河北新河人，高级兽医师，人要从事畜牧兽医技术推广工作，E-mail：fyf781018@163.com。*通讯作者，博士，教授，主要从事动物传染病学研究，E-mail：shiqiumei@126.com。

2015-10-27

Supported by National Nature Science Foundation of China（31472230）；Nature Science Foundation of Hebei Province （C2014407068）；Project of Science and Technology Department of Hebei Province（14966610D）；Project of Shijiazhuang Municipal Science and Technology Bureau（131200063A）；Support Program of 100 Outstanding Innovative Talents of Hebei Education Department（ZH2011244）.

*Corresponding author.E-mail:shiqiumei@126.com

Received:October 27，2015 Accepted:November 26，2015

修回日期 2015-11-26