Chinese Herbal Ingredients Are Effective Immune Stimulators for Chickens Infected with the Newcastle Disease Virus

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1 Chinese Herbal Ingredients Are Effective Immune Stimulators for Chickens Infected with the Newcastle Disease Virus X.-F. Kong,* Y.-L. Hu,* 1 Y.-L. Yin, G.-Y. Wu, 1 R. Rui,* D.-Y. Wang,* and C.-B. Yang *Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P.R. China, ; Key Laboratory of Subtropical Agro-ecology, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, P.R. China, ; and Department of Animal Science, Texas A&M University, College Station, TX ABSTRACT This study was conducted to determine the castle disease virus-specific serum hemagglutination inhibition efficacy of 4 Chinese herbal ingredients (CHI) as immune stimulators for an active vaccine in chickens using both in vitro and in vivo assays. The CHI used were Astragalus polysaccharide (APS), Isatis root polysaccharide (IRPS), antibody (Ab) production in immunized chickens was quantified using established methods. The results indicate that a majority of the CHI used at appropriate concentrations were effective in enhancing in vitro proliferation of chick embryo fibroblasts in response to the Propolis polysaccharide, and Epimedium flavone at various concentrations. Two hundred 14-d-old male White NDV infection. In vivo administration of CHI to vaccinated chickens (7.25 to 58 mg/kg of BW, depending on Roman chickens were randomly divided into 10 groups. type) increased serum anti-ndv hemagglutination inhibition Ab titer concentrations, compared with the admin- Chickens in groups 1 to 9 were inoculated with the Newcastle disease virus (NDV) strain IV vaccine by intranasal istration the NDV alone. For all CHI, a beneficial effect and intraocular administration. Chickens in groups 1 to on the Ab production was observed on d 21 after the 8 were also administered subcutaneously on the dorsal initiation of the vaccination. On the basis of the in vivo region of the neck with 0.5 ml of the corresponding CHI doses used, Propolis polysaccharide and Epimedium flavone were more potent than APS and IRPS in promoting at 2 doses: 29 and 58 mg/kg of BW for APS and IRPS and 7.25 and 14.5 mg/kg of BW for the others, once daily the humoral immune response in the young birds (P < for 3 successive days. In group 9 (CHI-free control) and group 10 (both vaccine- and CHI-free control), chickens were injected with 0.5 ml of physiological saline. New- 0.05). Collectively, these findings suggest that appropriate doses of CHI can be used as novel, effective immune stimulators for chickens. Key words: Chinese herbal ingredient, viral infection, serum antibody, immune stimulator 2006 Poultry Science 85: INTRODUCTION Infectious diseases are a worldwide concern because they usually cause great losses in domestic animals and fowls (Yin and Liu, 1997). Although strict preventative programs have been implemented at the farm level, some infectious diseases are still hard to control due to the variation of microorganisms and occurrence of immunosuppressive diseases and other factors (Xie, 1995). It is thought that the application of immune stimulators with vaccine could improve the efficacy of vaccination (Liu et al., 2002). Many Chinese herbal medicines (CHM) or Chinese herbal ingredients (CHI) have been reported to have positive effects on immune enhancement (Hu, 1997) 2006 Poultry Science Association Inc. Received May 20, Accepted August 10, Corresponding authors: ylhu@njau.edu.cn and g-wu@tamu.edu and great potential for practical application as immune stimulators due to their convenient preparation, wide availability, as well as minimal toxicity and side effects (Liang et al., 1998; Liu et al., 2002). Animals treated with CHM or CHI before or after vaccination have shown a reduced incidence of infectious diseases and an increased immune response (Hu, 1997). However, because of the crude nature of the traditional CHM, their isolation procedures and preparations are inconvenient, and they are often unstable (Meng and Chen, 1990). At present, there are few reports comparing the immune-enhancing efficacy between CHM and CHI. In addition, existing immune stimulators from CHM are mainly used as inactive vaccines (Gao et al., 1998). Approximately 50% of the vaccines presently used in domestic animals and fowls are active (Yin and Liu, 1997), which restricts their practical application. Therefore, it is important to develop novel immune stimulators for active vaccines with high efficiency and low toxicity. Further investigations of CHI are expected to provide a new, effective solution. 2169

2 2170 KONG ET AL. Table 1. Preparation of Chinese herbal ingredients (CHI) 1 Content Concentration CHI Extraction method Measurement method of net content (%) (mg/ml) APS Water extraction and ethanol precipitation Vitriol anthracene ketone with IRPS glucose as the standard control PPS EF Polyacrylamide chromatography Rutin test with bioflavonoid, 3,3,4,5,7-pentahydroxy flavone-3-rutinoside as the standard control 1 The CHI were extracted from herbs using water extraction and ethanol precipitation for Astragalus polysaccharide (APS), Isatis root polysaccharide (IRPS), and Propolis polysaccharide (PPS), or by using polyacrylamide chromatography for Epimedium flavone (EF) (Kong et al., 2004). The diluted preparations were analyzed for the content of total polysaccharides and sterilized by pasteurization. Previous studies have shown that many Chinese herbal medicinal ingredients affect humoral and cellular immunity in chickens, mice, and rabbits (Liang et al., 1998; Tang et al., 1998; Chu et al., 2004). Several Chinese herbal medicinal ingredients, Astragalus polysaccharide (APS), Isatis root polysaccharide (IRPS), Propolis polysaccharide (PPS), and Epimedium flavone (EF), were found to have strong immune-enhancing effects (Liang et al., 1998; Kong et al., 2004). The objective of this study was to investigate the possibility of using CHI as immune stimulators for an active vaccine. Two strategies were used in our experiments. First, we determined the effects of CHI on the cell proliferation of the chick embryo fibroblast (CEF) in response to Newcastle disease virus (NDV) using the neutral red assay (Wen et al., 2000). We expected NDV to reduce CEF proliferation but the CHI to have a protective effect. Second, the dynamic variation of NDVspecific serum hemagglutination inhibition (HI) antibody (Ab) titer was measured after chickens were inoculated with the NDV and administered s.c. low and high dosages of the CHI (Kong et al., 2004). MATERIALS AND METHODS Preparation of CHI Four CHI listed in Table 1 were prepared as previously described (Liu et al., 2002; Kong et al., 2004). The diluted preparations were sterilized by pasteurization and stored at 4 C for the immune-enhancing experiments in vivo. At the same time, the sterilized solution of CHI was diluted into 5 concentration levels (listed in Table 2) with minimum essential medium (MEM) supplemented with 2% fetal bovine serum, 100 IU/mL of benzylpenicillin, 100 IU/mL of streptomycin, and 0.03% glutamine (maintenance media) for analyzing the direct effect of CHI on CEF proliferation in vitro. Each concentration of the CHI was lower than their maximal safe concentrations (Liu et al., 2002). Preparation of the NDV and NDV-CHI Mixtures The NDV IV strain vaccine (lot no.760-3) was provided by Nanjing Pharmaceutical and Instrument Manufacturer (Nanjing, China). Median tissue culture infective dose (TCID 50 ) of the virus was tested on CEF using the method of Reed-Muench (Yin and Liu, 1997) after successive transfer cultures in the chorioallantoic fluid of a 9-d-old specific pathogen free chick embryo twice. Then, the virus was diluted into 100 TCID 50 /ml with the 4 CHI at 5 concentrations and the maintenance media of MEM, respectively, and stored at 4 C. After the diluted NDV preparation was incubated with the CHI for 144 or 272 h, the solution was used for the experiment. Less than 0.5% of the CHI extraction diluents were present in the cultures and were included as a contamination control in all assays. In Vitro Experiments to Determine a Direct Effect of CHI on CEF Proliferation Chick embryo fibroblasts were prepared according to the published method (Xu, 1990) and then diluted to 5 Table 2. Effects of 4 Chinese herbal ingredients (CHI) on chick embryo fibroblast (CEF) proliferation (OD 570 ) 1 CHI concentration ( g/ml) Group 1,600 1, APS 0.25 ± ± ± ± ± ± IRPS 0.25 ± ± ± ± ± ± PPS 0.47 ± ± ± ± ± ± 0.04 EF 0.46 ± ± ± ± ± ± Data are means ± SEM, n = 6. Astragalus polysaccharide (APS), Isatis root polysaccharide (IRPS), Propolis polysaccharide (PPS), or Epimedium flavone (EF) was incubated with CEF for 72 h before cell proliferation assay using the neutral red method. The culture medium did not contain Newcastle disease virus. Light absorbance was measured at 570 nm (OD 570 ). 2 Means are different (P < 0.05) from those for the control (0 g/ml) group.

3 HERBAL INGREDIENTS AND IMMUNITY cells/ml with MEM supplemented with 5% fetal bovine serum, 100 IU/mL of benzylpenicillin, 100 IU/mL of streptomycin, and 0.03% glutamine (growth media). One hundred microliters of the diluted CEF cell suspension was added into each well of a 96-well tissue culture plate ( cells/well) and incubated under the conditions of 37 C and 5% CO 2 for 36 h. After washing 3 times with maintenance media, wells were assigned to the following treatments: CHI-NDV groups (a mixture of NDV and CHI containing 100 L of 100 TCID 50 /ml of NDV and 5 CHI concentrations); CON CHI groups (CHI at 5 concentrations without NDV); NDV group (100 L of 100 TCID 50 /ml of NDV without CHI); and MM group (only the maintenance medium without NDV or CHI). Each sample was seeded in 4 wells (100 L per well). In all the assays, culture plates containing only maintenance medium served as zero-resetting blanks when light absorbance was measured at 570 nm. After a 72-h culture, cell proliferation was determined using the neutral red assay, which is based on the principle that the absorption of the dye by live cells is proportional to their number (Gao et al., 1998; Wen et al., 2000). Results were expressed as light absorbance at 570 nm (OD 570 ). Briefly, 2 h before the cell proliferation assay, 50 L of the neutral red solution (0.05% solution containing 153 mmol/l NaCl, sterilized by pasteurization; lot no ; Shanghai Third Reagent Factory, P.R. China) was added into each well. After the wells were washed 3 times with Ca and Mgfree PBS (ph 7.4), 200 L of destaining liquid (50% ethanol solution containing 0.05 mol/l of NaH 2 PO 4 ) was added into each well, and the plates were shaken for 10 min to dissolve the precipitation completely (Gao et al., 1998; Wen et al., 2000). The OD 570 was measured with an ELISA reader (model DG-3022, East China Vacuum Tube Manufacturer, Nanjing, China). The linear range (in the optical density value unit) was between 0.05 and The effect of CHI on CEF proliferation in response to NDV infection was evaluated according to the difference in OD 570 between the CHI-NDV group and the NDV group, as well as between the CON CHI groups and MM group. When there were no significant differences in OD 570 between CON CHI and MM groups, and when the OD 570 value of the CHI-NDV group was higher (P < 0.05) than that of the NDV group, we considered that the CHI enhanced CEF proliferation in response to NDV infection. In Vivo Experiments with CHI as Immune Stimulators Birds and Housing. One-day-old male White Roman chickens (egg-type; Tangquan Poultry Farms, Nanjing, China) were brooded in wire cages ( cm) in airconditioned rooms at 37 C and with light for 24 h at the beginning of the pretrial period. The temperature was gradually reduced to room temperature, and the light time was reduced to 12 h per day and then kept constant for the remainder of the experiment. The chickens were fed for 13 d with a commercial starter diet (Feed Factory, Jiangsu Academy of Agricultural Sciences, P.R. China) to allow them to adapt to the experimental conditions. The average titer of maternal HI Ab against the NDV vaccine was 5 log 2 in 7-d-old chickens. The NDV vaccine was administered when the chicks were 14 d of age, at which time concentrations of maternal anti-ndv HI antibodies were expected to be low. In Vivo Treatments with CHI. Two hundred 14-d-old chickens were randomly divided into 10 groups with an average BW of 69 ± 8 g. Chickens in groups 1 to 9 were inoculated with the NDV IV strain vaccine by intranasal and intraocular administration, following the recommendation of the manufacturer. Chickens in groups 1 to 8 were also administered subcutaneously on the dorsal region of the neck with 0.5 ml of the corresponding CHI at 2 levels: 58 mg/kg of BW (high dosage) and 29 mg/ kg of BW (low dosage) for APS and IRPS and 14.5 mg/ kg of BW (high dosage) and 7.25 mg/kg of BW (low dosage) for the others, once daily for 3 successive days. In group 9 (CHI-free control) and group 10 (both vaccineand CHI-free control), chickens were injected with 0.5 ml of physiological saline. Chinese herbal ingredients were administered at a different site because they could inactivate the virus if they were mixed with the NDV immediately before vaccination (Kong et al., 2004). On d 7, 14, 21, and 35 after initiation of the vaccination, 6 chickens were sampled randomly from each group for determination of anti-ndv serum HI Ab titer using the micromethod (Xu, 1998). The isotype of the HI Ab is IgG. The assay is based on the principle that an NDV antigen can form aggregates with chicken erythrocytes, which can be inhibited by NDV-specific antibodies in serum (Maas et al., 2003). Assay of Serum HI Ab. Blood samples (1.5 ml per chicken) were collected from the main brachial vein into Eppendorf tubes without sodium heparin. After collection, the blood samples were placed at 37 C for 2 h and then centrifuged at 1,500 g for 15 min. Serum samples were collected and frozen at 20 C for assays. Briefly, 2- fold serial dilutions of serum were made in a 96-well, V- shaped bottom microtiter plate containing 50 L ofca and Mg-free PBS in all wells and then 50 L of the NDV Ag (4 hemagglutination units, Jiangsu Academy of Agricultural Sciences, P.R. China) was added into all of the wells except for the last row, which served as the controls. Serum dilutions ranged from 1:2 to 1:2,048. The Ag-serum mixture was incubated for 10 min at 37 C. Then, 50 L of a 1% rooster erythrocyte suspension was added to each well and reincubated for 30 min. A positive serum, negative serum, erythrocytes, and Ag were also included as controls. The highest dilution of serum causing complete inhibition was considered as the end point. The geometric mean titer was expressed as reciprocal log 2 values for the highest dilution that displayed HI (Gao et al., 1998; Xu, 1998; Wen et al., 2000). Statistical Analysis Data, expressed as mean ± SEM, were analyzed by 1- way and 2-way ANOVA for 1-factorial and 2-factorial

4 2172 KONG ET AL. Figure 1. Effects of Astragalus polysaccharide (APS) mixture with Newcastle disease virus on chick embryo fibroblast (CEF) proliferation (OD 570 ). Newcastle disease virus strain IV was incubated with 5 concentrations of APS) at 4 C for 144 or 272 h. The viral solution was then added to the CEF for a 72-h culture, followed by a cell proliferation assay using the neutral red method. Light absorbance was measured at 570 nm (OD 570 ). Data are means ± SEM,n=6.Asterisks (*) indicate means are different (P < 0.05) from those for the virus- and Chinese herbal ingredients-free control (0 g/ml) group. OD = optical density. experimental designs, respectively, using SAS (SAS Institute Inc., Cary, NC). In the case of 2-way analysis, interactions between treatment factors were also assessed using the SAS program. Differences among treatment means were determined by the Student-Newman-Keuls multiple comparison test. The 95% confidence intervals are mean ± 2 SEM. Probability values <0.05 were taken to indicate statistical significance. RESULTS In Vitro Effects of CHI on CEF Proliferation in the Absence of ND Virus Effects of the CHI on CEF proliferation in the absence of the NDV are summarized in Table 2. There were no differences (P > 0.05) in OD 570 values between APS at 1,200, 600, or 300 g/ml and the control (0 g/ml) group. The OD 570 values of IRPS at 300, 150, 120, and 80 g/ml did not differ (P > 0.05) from those for the control group. There were no differences (P > 0.05) in OD 570 values between PPS or EF at each of the 5 concentrations and the control group. However, APS at 150 and 1,600 g/ml and IRPS at 1,600 g/ml reduced (P < 0.05) CEF proliferation. In Vitro Effects of CHI on CEF Proliferation in the Presence of NDV In the absence of CHI, increasing the time of NDV incubation from 144 to 272 h increased (P < 0.01) CEF proliferation 180% on the basis of an increase in OD 570 from 0.10 ± 0.02 to 0.28 ± In the presence of all the CHI tested, increasing the time of NDV incubation increased (P < 0.01) CEF proliferation 2.5- to 4-fold, depending on CHI type and dose (Figures 1, 2, 3, and 4). Figure 2. Effects of Isatis root polysaccharide (IRPS) mixture with Newcastle disease virus chick embryo fibroblast (CEF) proliferation (OD 570 ). Newcastle disease virus strain IV was incubated with 5 concentrations of IRPS at 4 C for 144 or 272 h. The viral solution was then added to the CEF for a 72-h culture, followed by a cell proliferation assay using the neutral red method. Light absorbance was measured at 570 nm (OD 570 ). Data are means ± SEM,n=6.Asterisks (*) indicate means are different (P < 0.05) from those for the virus- and Chinese herbal ingredients-free control (0 g/ml) group. OD = optical density. There was a difference (P < 0.05) in the response to the NDV among the CHI treatment groups, in that the addition of APS (600 g/ml), IRPS (150 g/ml), PPS (20 g/ml), and EF (20 g/ml) maximally increased (P < 0.05) OD 570 values by 33.1 ± 2.7, 41.7 ± 3.9, 74.1 ± 5.1, and 57.2 ± 4.6%, respectively (mean ± SEM, n = 6), compared with the corresponding controls (0 g/ml). After a 144-h or 272-h incubation period, the OD 570 values at 1,200, 600, and 300 g of APS/mL were greater (P < 0.05) compared with the control (0 g of APS/mL) group (Figure 1). Similarly, after a 144-h or 272-h incubation period, the values for the groups of IRPS at 300, 150, 120, and 80 g/ml were greater (P < 0.05) compared with the control (0 g of IRPS/mL) groups (Figure 2). The values of PPS at 10 g/ml after a 144-h incubation period and at all concentration levels after a 272-h incubation period were greater (P < 0.05) than those of the control (0 g of PPS/mL) groups (Figure 3). The values of EF at 10 g/ml after a 144-h incubation period and at 20 to 2.5 g/ml after a 272-h incubation period were greater (P < 0.05) than those of the control groups (0 g of EF/ ml; Figure 4). There were no significant interactions (P > 0.05) between the mixing time and the CHI dose. Dynamic Changes of Serum HI Ab Titer The dynamic changes of serum HI Ab titer are summarized in Table 3. On each day of the blood sampling, serum HI Ab titer levels did not differ (P > 0.05) between the NDV and control groups. However, all CHI were

5 HERBAL INGREDIENTS AND IMMUNITY 2173 Figure 3. Effects of Propolis polysaccharide (PPS) mixture with Newcastle disease virus on chick embryo fibroblast (CEF) proliferation (OD 570 ). Newcastle disease virus strain IV was incubated with 5 concentrations of PPS at 4 C for 144 or 272 h. The viral solution was then added to the CEF for a 72-h culture, followed by a cell proliferation assay using the neutral red method. Light absorbance was measured at 570 nm (OD 570 ). Data are means ± SEM,n=6.Asterisks (*) indicate means are different (P < 0.05) from those for the virus- and Chinese herbal ingredients-free control (0 g/ml) group. OD = optical density. effective in increasing (P < 0.05) serum HI Ab titer, depending on dose and day after initiation of the vaccination. On d 7 following the vaccination, the HI Ab titers Figure 4. Effects of Epimedium flavone (EF) mixture with Newcastle disease virus on chick embryo fibroblast (CEF) proliferation (OD 570 ). Newcastle disease virus strain IV was incubated with 5 concentrations of EF at 4 C for 144 or 272 h. The viral solution was then added to the CEF for a 72-h culture, followed by a cell proliferation assay using the neutral red method. Light absorbance was measured at 570 nm (OD 570 ). Data are means ± SEM, n = 6. Asterisks (*) indicate means are different (P < 0.05) from those for the virus- and Chinese herbal ingredients-free control (0 g/ml) group. OD = optical density. of the high dosage group (58 mg/kg of BW) in APS and low dosage group (29 mg/kg of BW) in IRPS were higher (P < 0.05) than those of the NDV and control groups. On d 21, the titers of the low dosage APS group, high dosage PPS group, and low and high dosage IRPS or EF groups were greater (P < 0.05) compared with the NDV and control groups. On d 35, the titers of both dosage groups for APS and IRPS and the low dosage PPS group were higher (P < 0.05) in comparison with the NDV and control groups. The titers of most treatment groups reached a peak value on d 14, whereas those of the high dosage IRPS group and low dosage EF group reached a peak value on d 21. In addition, the titers of some treatment groups were positively correlated with CHI dosages. DISCUSSION Viral infection of cells is complex and results in many changes to the host (Gao et al., 1998). Results from the in vitro study showed that the majority of CHI treatments could enhance CEF proliferation in response to the NDV. These included APS at 600 and 300 g/ml after a 144- h incubation period and at 1,200, 600, and 300 g/ml after a 272-h incubation period; IRPS at 300, 150, 120, and 80 g/ml after a 144-h or 272-h incubation period; PPS at 10 g/ml after a 144-h incubation period and at all the 5 concentrations after a 272-h incubation period; and EF at 10 g/ml after a 144-h incubation period and at 20 to 2.5 g/ml after a 272-h incubation period. For APS, PPS, and EF, the number of effective concentration groups was higher with a 272-h incubation period than with a 144-h incubation period. These results indicate that the stimulatory effect of most CHI on CEF proliferation was stronger after a 272-h incubation period with the ND virus than after a 144-h incubation period. Morphologic observations and prothrombin tests are commonly used in the study of immune-enhancing effects of medicines (Gao et al., 1998; Kong et al., 2004). However, these tests are only qualitative or semiquantitative and suffer from the shortcomings of greater subjectivity and lower sensitivity (Yin and Liu, 1997). The neutral red absorbance assay adopted in our experiment was used to quantitatively determine the direct effect of CHI on CEF proliferation in vitro. In the present study, the analysis of the CHI activity on CEF proliferation was measured according to differences in OD 570 values among the various CHI concentrations. Interestingly, the results of our present study indicate that the effects of CHI on CEF proliferation critically depend on their doses used (Figures 1 to 4). Under our experimental conditions, there could have been spontaneous loss of NDV infectivity when the virus was incubated with CHI at 4 C for 6 to 12 d. Thus, our results may not necessarily reflect the optimal effect of CHI on increasing CEF proliferation in response to NDV infection. Serum HI Ab titer is a valid indicator of the humoral immunity in chickens (Kong et al., 2004). Previous studies have shown that the HI Ab is directly effective against NDV in chickens (Mtambo et al., 1999; Roy et al., 1999;

6 2174 KONG ET AL. Table 3. The dynamic variation of anti-newcastle disease virus (NDV) hemagglutination inhibition (HI) antibody (Ab) titer (log 2 ) 1 Anti-NDV HI Ab titer (log 2 ) Dosage Group (mg/kg of BW) d 7 d 14 d 21 d 35 APS ± ± ± ± ± ± ± ± IRPS ± ± ± ± ± ± ± ± PPS ± ± ± ± ± ± ± ± EF ± ± ± ± ± ± ± ± 0.8 NDV ± ± ± ± 0.8 Control ± ± ± ± Data are means ± SEM, n = 6. NDV = the Chinese herbal ingredients- (CHI) free group administered with NDV alone; Control = the vaccine- and CHI-free control group. In all groups except for the control, chicks were inoculated with the NDV-IV vaccine at 14 d of age. Astragalus polysaccharide (APS), Isatis root polysaccharide (IRPS), Propolis polysaccharide (PPS), or Epimedium flavone (EF) was administered subcutaneously to 14-dold vaccinated chickens, once daily for 3 consecutive days. On d 7, 14, 21, and 35 after initiation of the vaccination, blood samples were obtained from chickens for the analysis of HI Ab titer. 2 Means are different (P < 0.05) from those for the NDV- and CHI-free control groups. Maas et al., 2003). At a low dose of the NDV, we were not able to demonstrate an increase in serum HI Ab titer levels. Importantly, our results showed that the HI Ab titers in some treatment groups at d 7, 24, and 35 were higher than the NDV group (Table 3), suggesting that CHI could enhance the humoral immunity. Indeed, on d 7 after vaccination, the anti-ndv HI Ab titers of the high dosage APS group (58 mg/kg of BW) and the low dosage IRPS group (29 mg/kg of BW) were higher compared with the control group, indicating a more rapid response to the treatments. On d 21, the HI Ab titers of the lowdosage APS group (29 mg/kg of BW), high-dosage PPS group (14.5 mg/kg of BW), and 2 dosage IRPS or EF groups (7.24 and 14.5 mg/kg of BW) were substantially higher than those of the NDV group, indicating their potent effects in increasing the production of anti-ndv HI antibodies in the vaccine-treated host. On d 35, the HI Ab titers of the 2 dosage APS or IRPS groups (29 and 58 mg/kg of BW) and the low dosage PPS group (7.25 mg/kg of BW) were also higher in comparison with the NDV group, suggesting that their treatment effects were sustained for a prolonged period. These findings provide evidence for the use of CHI as effective herbal medicinebased immune stimulators. An important observation from the present study is that the effects of CHI on CEF proliferation and immune response were related to their dosages used. However, it should be kept in mind that such effects are not necessarily increased with increasing extracellular concentrations of CHI. Results from our in vitro and in vivo studies suggest that the interactions between CHI and NDV are likely complex in nature. Thus, an appropriate, effective dosage should be taken into consideration in development of the CHI as immune stimulators for active vaccine. Compared with CHI, the immune-enhancing mechanism of CHM is more complex because of their multiple components. In this regard, the use of CHI as immune stimulators for chickens offers a distinct advantage over CHM. Ultimately, the direct in vitro activity of CHI on CEF proliferation in combination with data on the in vivo immune response to their administration should be used in defining the CHI effects on viral infection. In summary, the CHI used in the present study exhibit potent immune-enhancing effects. Almost all of the CHI used in this study substantially enhanced in vitro CEF proliferation and promoted the humoral immunity in response to NDV infection in vivo. Notably, the immunological effect of CHI is related to their dosages. Our findings indicate that CHI can be used as novel immune stimulators for chickens. ACKNOWLEDGMENTS The research was supported by grants from the National Natural Science Foundation of China ( , , and ), the Chinese Academy of Sciences Knowledge Innovation Project (KSCX2-SW-323), the Outstanding Overseas Chinese Scholars Fund of The Chinese Academy of Sciences ( and ), the National Basic Research Program of China (2004CB117502), and Texas Agricultural Experiment Station (H-8200). We are thankful to all staff members in the Institute of Traditional Chinese Veterinary Medicine of Nanjing Agricultural University for assistance in this study and Todd Rideout (University of Guelph, Ontario, Canada) for critical reading of the manuscript. REFERENCES Chu, Y. F., X. R. Li, and Y. L. Hu Effects of Chinese herbal medicinal ingredient on cells mediated immunity in mice. J. Nanjing Agric. Univ. 1: Gao, Y. J., Y. Z. He, H. Shen, X. L. Chui, and Y. L. Jiang Application of neutral red absorb assay to study of antiviral Chinese herbal medicine. Chin. Herbal Med. Pharmacodyn. Clin. 14:45 47.

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