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Poult Sci 2006. 85:2169-2175
© 2006 Poultry Science Association
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IMMUNOLOGY, HEALTH, AND DISEASE

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

X.-F. Kong*,{dagger}, Y.-L. Hu*,1, Y.-L. Yin{dagger}, G.-Y. Wu{dagger},{ddagger},1, R. Rui*, D.-Y. Wang* and C.-B. Yang{dagger}

* Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P.R. China, 210095; {dagger} Key Laboratory of Subtropical Agro-ecology, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, P.R. China, 410125; and {ddagger} Department of Animal Science, Texas A&M University, College Station, TX 77843

1 Corresponding authors: ylhu{at}njau.edu.cn and g-Wu{at}tamu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was conducted to determine the 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), Propolis polysaccharide, and Epimedium flavone at various concentrations. Two hundred 14-d-old male White Roman chickens were randomly divided into 10 groups. Chickens in groups 1 to 9 were inoculated with the New-castle disease virus (NDV) strain IV vaccine by intranasal and intraocular administration. 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 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 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-castle disease virus-specific serum hemagglutination inhibition 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 NDV infection. In vivo administration of CHI to vaccinated chickens (7.25 to 58 mg/kg of BW, depending on type) increased serum anti-NDV hemagglutination inhibition Ab titer concentrations, compared with the administration the NDV alone. For all CHI, a beneficial effect on the Ab production was observed on d 21 after the initiation of the vaccination. On the basis of the in vivo doses used, Propolis polysaccharide and Epimedium flavone were more potent than APS and IRPS in promoting the humoral immune response in the young birds (P < 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 immuno-suppressive 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) 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.

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 NDV-specific 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of CHI
Four CHI listed in Table 1Go 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 2Go) 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).


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  		Table 1. Preparation of Chinese herbal ingredients (CHI)1
 

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  		Table 2. Effects of 4 Chinese herbal ingredients (CHI) on chick embryo fibroblast (CEF) proliferation (OD570)1
 
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 (TCID50) 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 TCID50/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 x 105 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 (5 x 104 cells/well) and incubated under the conditions of 37°C and 5% CO2 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 µL of 100 TCID50/mL of NDV and 5 CHI concentrations); CON CHI groups (CHI at 5 concentrations without NDV); NDV group (100 µL of 100 TCID50/mL of NDV without CHI); and MM group (only the maintenance medium without NDV or CHI). Each sample was seeded in 4 wells (100 µL 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 (OD570). Briefly, 2 h before the cell proliferation assay, 50 µL of the neutral red solution (0.05% solution containing 153 mmol/L NaCl, sterilized by pasteurization; lot no. 080525; Shanghai Third Reagent Factory, P.R. China) was added into each well. After the wells were washed 3 times with Ca and Mg-free PBS (pH 7.4), 200 µL of destaining liquid (50% ethanol solution containing 0.05 mol/L of NaH2PO4) 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 OD570 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 1.00.

The effect of CHI on CEF proliferation in response to NDV infection was evaluated according to the difference in OD570 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 OD570 between CON CHI and MM groups, and when the OD570 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 (60 x 100 cm) in air-conditioned 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 log2 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 vaccine-and 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 micro-method (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 x 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 µL of Ca and Mg-free PBS in all wells and then 50 µL 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 µL 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 log2 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 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 x SEM. Probability values <0.05 were taken to indicate statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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 2Go. There were no differences (P > 0.05) in OD570 values between APS at 1,200, 600, or 300 µg/mL and the control (0 µg/mL) group. The OD570 values of IRPS at 300, 150, 120, and 80 µg/mL did not differ (P > 0.05) from those for the control group. There were no differences (P > 0.05) in OD570 values between PPS or EF at each of the 5 concentrations and the control group. However, APS at 150 and 1,600 µg/mL and IRPS at 1,600 µg/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 OD570 from 0.10 ± 0.02 to 0.28 ± 0.03. 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 1Go, 2Go, 3Go, and 4Go). 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 µg/mL), IRPS (150 µg/mL), PPS (20 µg/mL), and EF (20 µg/mL) maximally increased (P < 0.05) OD570 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 µg/mL).


Figure 1
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  		Figure 1. Effects of Astragalus polysaccharide (APS) mixture with Newcastle disease virus on chick embryo fibroblast (CEF) proliferation (OD570). 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 (OD570). 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 µg/mL) group. OD = optical density.

 

Figure 2
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  		Figure 2. Effects of Isatis root polysaccharide (IRPS) mixture with Newcastle disease virus chick embryo fibroblast (CEF) proliferation (OD570). 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 (OD570). 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 µg/mL) group. OD = optical density.

 

Figure 3
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  		Figure 3. Effects of Propolis polysaccharide (PPS) mixture with New-castle disease virus on chick embryo fibroblast (CEF) proliferation (OD570). 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 (OD570). 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 µg/mL) group. OD = optical density.

 

Figure 4
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  		Figure 4. Effects of Epimedium flavone (EF) mixture with Newcastle disease virus on chick embryo fibroblast (CEF) proliferation (OD570). 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 (OD570). 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 µg/mL) group. OD = optical density.

 
After a 144-h or 272-h incubation period, the OD570 values at 1,200, 600, and 300 µg of APS/mL were greater (P < 0.05) compared with the control (0 µg of APS/mL) group (Figure 1Go). Similarly, after a 144-h or 272-h incubation period, the values for the groups of IRPS at 300, 150, 120, and 80 µg/mL were greater (P < 0.05) compared with the control (0 µg of IRPS/mL) groups (Figure 2Go). The values of PPS at 10 µg/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 µg of PPS/mL) groups (Figure 3Go). The values of EF at 10 µg/mL after a 144-h incubation period and at 20 to 2.5 µg/mL after a 272-h incubation period were greater (P < 0.05) than those of the control groups (0 µg of EF/mL; Figure 4Go). 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 3Go. 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 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 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.


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  		Table 3. The dynamic variation of anti-Newcastle disease virus (NDV) hemagglutination inhibition (HI) antibody (Ab) titer (log2)1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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 µg/mL after a 144-h incubation period and at 1,200, 600, and 300 µg/mL after a 272-h incubation period; IRPS at 300, 150, 120, and 80 µg/mL after a 144-h or 272-h incubation period; PPS at 10 µg/mL after a 144-h incubation period and at all the 5 concentrations after a 272-h incubation period; and EF at 10 µg/mL after a 144-h incubation period and at 20 to 2.5 µg/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 OD570 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 1Go to 4GoGoGo). 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; 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 3Go), 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 low-dosage 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 medicine-based 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 (30070566, 30528006, and 30671517), the Chinese Academy of Sciences Knowledge Innovation Project (KSCX2-SW-323), the Outstanding Overseas Chinese Scholars Fund of The Chinese Academy of Sciences (2005-1-4 and 2005-1-7), 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.

Received for publication May 20, 2006. Accepted for publication August 10, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chu, Y. F., X. R. Li, and Y. L. Hu. 2004. Effects of Chinese herbal medicinal ingredient on cells mediated immunity in mice. J. Nanjing Agric. Univ. 1:97–100.

Gao, Y. J., Y. Z. He, H. Shen, X. L. Chui, and Y. L. Jiang. 1998. Application of neutral red absorb assay to study of antiviral Chinese herbal medicine. Chin. Herbal Med. Pharmacodyn. Clin. 14:45–47.

Hu, Y. L. 1997. Progress in the study of immunopharmacology of Chinese herbal medicine. Chin. J. Immunol. 13:96–98.

Kong, X. F., Y. L. Hu, R. Rong, D. Y. Wang, and X. R. Li. 2004. Effects of Chinese herbal medicinal ingredients on peripheral lymphocyte proliferation and serum Ab titer after vaccination in chicken. Int. Immunopharmacol. 4:975–982.[ISI][Medline]

Liang, R., K. K. Guo, L. Yi, Y. X. Zhang, J. Q. Li, and Z. Q. Yue. 1998. Study of Chinese herbal medicinal immune stimulator on immune enhancement in chickens. Chinese J. Vet. Sci. Technol. 28:11–13.

Liu, J. G., Y. L. Hu, Y. K. Chen, B. K. Zhang, and X. F. Kong. 2002. The safe concentrations of several components in natural drugs on CEF. Prog. Vet. Med. 23:88–91.

Maas, R. A., M. Komen, M. van Diepen, H. L. Oei, and I. J. T. M. Claassen. 2003. Correlation of haemagglutinin-neuraminidase and fusion protein content with protective antibody response after immunization with inactivated Newcastle disease vaccines. Vaccine 21:3137–3142.[ISI][Medline]

Meng, X. S., and F. K. Chen. 1990. Analysis of Chinese Patent Medicine. People Sanitary Press. Beijing, China.

Mtambo, M. M. A., E. J. Mushi, L. D. B. Kinabo, A. Maeda-Machangu, G. L. M. Mwamengele, M. G. S. Yongolo, and R. P. C. Temu. 1999. Evaluation of the efficacy of the crude extracts of Capsicum frutescens, Citrus limon and Opuntia vulgaris against Newcastle disease in domestic fowl in Tanzania. J. Ethnopharmacol. 68:55–61.[ISI][Medline]

Roy, P., A. T. Venugopalan, and A. Koteeswaran. 1999. Efficacy of live adjuvanted mesogenic Newcastle disease vaccine in chickens. Vaccine 17:2674–2676.[ISI][Medline]

Tang, X. M., Y. L. Hu, B. K. Zhang, and J. G. Liu. 1998. Effect of astragalus polysaccharide on T lymphocyte transformation function of peripheral blood in chickens. Chin. J. Vet. Sci. 3:269–270.

Wen, P., Y. He, Q. L. Ye, and G. S. Dai. 2000. Test of cell proliferation by neutral red absorb assay. J. Zhenjiang Med. Coll. 10:161–163.

Xie, Q. G. 1995. Pages 143–168 in Progress in the Study of Grave Epidemic Diseases Immune Prevention and Cure of Domestic Animals and Poultry. Chin. Agric. Sci. Technol. Press, Beijing, China.

Xu, W. Y. 1998. Pages 222–225 in Veterinary Virology. Chin. Agric. Press, Beijing, China.

Yin, Z., and J. H. Liu. 1997. Pages 204–246 in Animal Virology. Sci. Press, Beijing, China.




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