Effects of Polysaccharide Extracted from Traditional Chinese Medical Herbs on Lymphocyte Transformation Rate and AI-HI Antibody Titer in Chicks

Object]: Detect whether different concentrations of Chinese herbs compound polysaccharides (CPS), astragalus polysaccharides (APS) and angeulica polysaccharides (ASP), epimedium herb polysaccharides (EPS) have effects on the immunity function of healthy Roman chicken. [Method]: 260 one-day-old chickens were divided into thirteen groups randomly, 20 birds each group. The physiological saline, Chinese herbs compound polysaccharides, APS, ASP or EPS had been hypodermically injected for seven days continuously, and the blood was drawn on the 7, 14, 21, 28, 35, 42, 49 and 56 day to evaluate the activity of the translation rate of blood lymphocyte and AI-HI antibody titers in chickens. [Result]: The results of the experiment showed that the translation rate of blood lymphocyte and AI-HI antibody titers increased markedly after the use of Chinese herbs compound polysaccharides, APS, ASP and EPS to the chickens. Chinese herbs compound polysaccharides had more effective function improving the translation rate of blood lymphocyte and AI-HI antibody titers than others. [Conclusion]: The Chinese herbs compound polysaccharides, APS, ASP and EPS could promote the immunity function of the chickens. The Chinese herbs compound polysaccharides were the strangest one among them.


Introduction
Chicken is sensitive to many infectious diseases, such as Newcastle Disease, Avian Influenza (AI) and Infectious Bursa Diseases, which caused high mortality and economic loss world widely.But there are no effective drugs to cure these diseases and the most effective way is to use Vaccines to prevent the occurrence of these.But them another problem comes which is that, vaccines is not always as effective as people expected.
Sometimes, because of weak immunity, poor quality, short protective terms or virus mutation immunity failure which often appears all can cause immunity failure (Chen X, 2002, p18-20).One way to solve it is to find a safe and effective immunopotentiator.Many papers in recent years showed that polysaccharides can enhance immunity and have no obvious side effect.So they are potential immunopotentiator.But the majority scholars only aim at single polysaccharide like angelica sinensis polysaccharide (Yang T H, 2005, p782-783), astragalus polysaccharide (Li S P, 2005, p51-54), lycium barbarum polysaccharide (Tao D Y, 2007, p6816-6818) and so on, to conduct the research, or several polysaccharide composed by a certain percentage of polysaccharide (Wang D Y, 2005, p3704-3708) conducted a study.The traditional Chinese medicine compound prescription polysaccharide (Yan G Q, 1998, p31-32) had not reported as the immunity intensifier's research.In this study, we focused on effect of compound polysaccharides (CPS), astragalus polysaccharides (APS), angeulica polysaccharides (ASP), epimedium herb polysaccharides (EPS) on immunity in health Roman chicken.Because the four selected polysaccharides are neglected by others, although other polysaccrides have been investigated by many studies.In the study, we check the effect of the four polysaccrides.In the next step our goal is to observe whether their application to the new discovered medicines can enhance their effects.

Reagent
The live vaccine of the Avian Influenza -Newcastle Disease reorganizes (rL-H5) was provided by the Shihezi veterinary station.Avian Influenza H5 the hypotype HI antigen was provided by the Xinjiang army corps raising livestock veterinary services main terminal.Cell nutrient fluid: takes the TC199 culture medium to dissolve according to the instruction booklet in the 1000ml doubled distilled water, filters after the fungus loads separately, restored at 4 .Joins 10% calf blood serum near the time (56 the 30min deactivation, filtration eliminates fungus) and penicillin 100U/ml, chain mildew element 100g/ml, and adjusts pH with 60.0g/L NaHCO3 to 7.2~7.4;The plant haemagglutinin (PHA) 10mg/ml filters has eliminated the fungus.

Equipment
DNP-9082 electric heating constant temperature incubator (Shanghai fine great test installation Limited company, Shanghai); PHS-3C precise PH counts (Shanghai instrument 3rd factories, Shanghai); DGW-99 table model high speed miniature centrifuge (Ningbo new iris biotechnology Limited liability company, Ningbo); HHS21-6 electric heating constant temperature water-bath (Beijing Chang An Scientific instrument factory, Beijing); OLYMPUS-CX21FS1 biology microscope (Guangzhou bright beautiful science and technology Limited company, Guangzhou); Ultra-clean work table.

Experimental design
Experimental chickens are from the chick's farm in Shihezi city, and their health is tested clinically.To divide 260 one-day-old chickens into 13 groups at random and each group has 20 chickens.Use the medication on them when they're one day old.The program is as follows: group 1 is the normal control, and receive 0.2ml/d normal saline, group 2~13 receive 0.2ml/d i.p. 12.5mg/ml, 25 mg/ml, 50 mg/ml compound polysaccharides(CPS), astragalus polysaccharides(APS), angeulica polysaccharides(ASP), epimedium herb polysaccharides(EPS) respectively.The inject continuously for 7 days.

Breeding and management
The breeding method, condition, environment, feeder quality and husbandry of four groups' chicks are unified.The chickling on 7th the age with the live vaccine (rL-H5) the drop nose, and the spot eye immunity, on 21st the age carries on the second immunity.(Lin Q H, 1999, p188-189) In the 7, 14, 21, 28, 35, 42, 49 and 56 days after treatment, we take 10 chickens from each group to prepare the blood stochastically separately, and antifreeze with the sodium citrate.Takes 0.1ml, add 1.8ml TC199 nutrient fluid, and PHA 0.1 ml, to 0.1ml anti-hemoglutination samples, and then mix uniform postpositioned 37 nurtures 72h warm.Shake them one time a day.Take them out after the raise had ended blot the majority of supernates, join 8.3g/L NH 4 Cl 4ml to mix uniform, and set them at 37 water bath 10min.After taking out, 2000r/min centrifugal 5min, abandons clear, the settling adds the 5ml fixture, sets at room temperature 10min.The offcenter abandons clear, takes 2~3 drop of settling in ice-cold and has on the moisture content slide glass, causes it to unfold evenly, after rapid drying up, the dye liquor dyes 15min with Giemsa Ranye.Then laundering, dry, oil mirror inspection.Observes 200 lymphocytes, the counting transformation cell number.According to the equation below equatation: % lymphocyte transformation rate= transformed lymphocyte/ (transformed lymphocyte + untransformed lymphocyte) ×100

AI-HI antibody examination
In experiments 7, 14, 21, 28, 35, 42, 49, 56 days, takes 10 chickens from each group to pick the blood stochastically, the separation blood serum, examines the AI-HI antibody titer with the -micromethod.The result expressed with the log2 geometry average value.

Statistical analysis
Statistical analyses were carried out by One-way-ANOVA, Tukey and Dunnett's posthoc tests by using SPSS 12.0.1 Statistical Package (SPSS Inc., Chicago, IL).P values less than 0.05 (p<0.05) were regarded as statistically significant.All the values are presented as mean± SEM (the standard error of the mean).

Results
The result of table 1: each polysaccride elevated lymphocyte transformation at the checkpoint 7 th , 14 th , 21 st , 28 th , and arrived at the perk at 28 th .Compared with control, by 56 th day, only 25mg/ml compound polysaccharides (CPS) showed significant difference (P<0.05).
The result of table 2: each polysaccride elevated AI-HI titers, AI-HI titers arrived at the perk at 28 th and 35 th .Compared with control group, even at 56 th , compound polysaccharides (CPS), 25mg/ml astragalus polysaccharides (APS) have the significant effect (P<0.01).

Discussion
In the study, compound polysaccharides (CPS), astragalus polysaccharides (APS), angeulica polysaccharides (ASP), epimedium herb polysaccharides (EPS) enhanced the proliferation of lymphocyte and AI-HI.This result is in accordance with others.The result showed that the four polysaccharides can enhance herb's immunity.The findings of Xu Y (Xu Y, 2000, p434-437), Wang D Y (Wang D Y, 2006, p194-196), Gu X L (Gu X L, 2005, p813-820) and Li N (Li N, 2004, p61-64) coincide with our experimental result, which shows that the four polysaccharides can enhance herb's immunity.
At the three checkpoints 7 th , 14 th , 21 st , the detection index of each experimental group was elevated which compared with the normal control group.At 28 th day, it reached the peak, in the experiment, effect of 25mg/ml groups was better than 12.5mg/ml and 50mg/ml groups in all of the four polysaccrides groups.It indicated that there was not a positive correlation between concentration and improvement of immunity and its mechanism is expected to be further studied.
Cell immunity is mediated by T cells; lymphocyte transformation rate was an important index for evaluating cell immunity.The results of this study indicated that compound polysaccharides(CPS), astragalus polysaccharides(APS), angeulica polysaccharides(ASP), epimedium herb polysaccharides(EPS) elevated lymphocyte transformation rate.By 49th day, lymphocyte transformation rate recovered to normal level in ASP, EPS and 12.5mg/ml APS, 50mg/ml APS groups.

Table 1 .
The effect of different density polysaccharides on the translation rate of blood lymphocyte Compared with the control group, in same Column,* P<0.05, ** P<0.01.

Table 2 .
The effect of different density polysaccharides on AI-HI antibody titers