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Poult Sci 2007. 86:2530-2535. doi:10.3382/ps.2007-00076
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IMMUNOLOGY, HEALTH, AND DISEASE

Immunopotentiating Effects of Four Chinese Herbal Polysaccharides Administered at Vaccination in Chickens

Y. Qiu*,{dagger}, Y. L. Hu{dagger}, B. A. Cui*,1, H. Y. Zhang*, X. F. Kong{dagger}, D. Y. Wang{dagger} and Y. G. Wang*

* College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, P. R. China; and {dagger} College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, P. R. China

1 Corresponding author: baoancui{at}henau.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was conducted to evaluate the effects of 4 Chinese herbal polysaccharides on the production of serum antibodies and the proliferation of peripheral T lymphocytes, including subpopulations in vaccinated chickens. A total of 450 chickens were randomly assigned to 9 groups at 14 d of age and vaccinated first with live Newcastle disease (ND)-infectious bronchitis virus vaccine, and second with ND-infectious bronchitis oil adjuvant vaccine at 28 d of age. At the same time as the first vaccination, the chickens in groups 1 to 8 were intramuscularly injected with 4 polysaccharides at high and low dosages, respectively, once a day for 3 successive days starting on the day of the first vaccination. Group 9 (control group) was injected in the same manner with saline instead of a polysaccharide. On d 7, 14, 21, 28, 35, 42, and 49 after the first vaccination, the temporal changes in serum ND hemagglutination inhibition antibody titer were determined by the micromethod. On d 10, 20, 30, 40, and 50 after the first vaccination, the proliferation of peripheral blood mononuclear cells in response to concanavalin A stimulation as well as the proportions of CD3+, CD4+, and CD8+ peripheral blood mononuclear cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and flow cytometry, respectively. The results showed that astragalus polysaccharide and isatis root polysaccharide at low dosages, and achyranthes root polysaccharide and Chinese yam polysaccharide at high dosages significantly enhanced the ND antibody titers, concanavalin A-induced proliferation of peripheral blood lymphocytes, and ratio of CD4+ to CD8+ (P <0.05). Collectively, these findings indicate that the 4 polysaccharides possess significant immune-enhancing properties in chickens. This finding may have direct application in vaccine design and other strategies designed to potentiate immune system development and function in chickens.

Key Words: Chinese herbal polysaccharide • vaccine • lymphocyte • antibody • chicken


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
With the breeding of poultry of superior genetic stock and with high production pressure, birds have become more susceptible to many diseases. As a result, many hitherto unknown and latent forms of infections have emerged in addition to the prevailing diseases. These changes in the host-parasite relationship have resulted in the emergence of variants of existing viruses or the development of new syndromes, impeding the profitability of the poultry industry, often as a result of the chickens’ increased susceptibility to secondary infections and suboptimal response to vaccinations. Among the various emerging diseases, viral diseases in general and immunosuppressive viruses in particular have been incriminated as the etiological agents for a variety of clinical conditions in poultry. The emergence of such diseases not only threatens the economy of poultry production but also poses a challenge to the scientific community (Bal and Kata, 2006). It is believed that the simultaneous application of a vaccine and an immunopotentiator could improve the efficacy of vaccination. However, some adjuvants commonly used in animal vaccines, such as oil emulsions and aluminum, ordinarily result in side effects or fail to increase the immunogenicity of weak antigens. Therefore, it is urgent that novel immunopotentiators with high efficiency, low toxicity, and extensive availability be developed (Sun, 1998). Medicinal plants using immunomodulation can provide an alternative to conventional chemotherapy for a variety of diseases, especially when the host’s defense mechanism has to be activated under conditions of impaired immune response or when selective immunosuppression is desired in situations such as autoimmune disorders.

Recent research has shown that many Chinese herbal medicines and their ingredients have immune enhancement properties (Hu, 1997). Polysaccharides, as one of the active ingredients in Chinese herbal medicine, can enhance cellular immunity and promote antibody production and secretion of cytokines (Chen, 2006). They are regarded as biological response modifiers and have attracted much attention because of their natural origin, low toxicity to humans and animals, and long-standing use as folk medicines (Lu et al., 2003; Yon et al., 2006).

On the basis of the foregoing information, we hypothesized that vaccination with Newcastle disease (ND)-infectious bronchitis (IB) vaccine, together with 4 kinds of herbal polysaccharides, astragalus polysaccharide (APS), isatis root polysaccharide (IRPS), achyranthes root polysaccharide (ARPS), and Chinese yam polysaccharide (CYPS), at high (H) and low (L) dosages would enhance various aspects of immunoactivities in chickens. To test this hypothesis and to examine the possibility of using Chinese herbal polysaccharides as novel immunopotentiators, a time-course study was conducted to examine serum ND antibody titers, mitogen-induced proliferation of peripheral blood lymphocytes, and their proportions among the peripheral blood lymphocyte population following vaccination and polysaccharide administration in chickens.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Chinese Herbal Polysaccharides
The 4 polysaccharides were extracted by the water decoction-ethanol precipitation method (Xue, 1985). The total polysaccharide was measured by the Vitriol-anthracene ketone method, with glucose without H2O as the standard control (Liu et al., 1994). The contents (%) of total APS, IRPS, ARPS, and CYPS (comparable with those of glucose) were 65.1, 56.4, 54.3, and 72.8%, respectively. Based on our previous studies and on the polysaccharide contents of the extracts prepared here, the 4 polysaccharides were diluted with deionized water into 2 concentrations (mg/mL): 4 and 2 for APS, 3 and 1.5 for IRPS, and 6 and 3 for ARPS and CYPS, respectively. The diluted preparations were sterilized by pasteurization and tested for endotoxins by pyrogen tests (Veterinary Pharmacopoeia Commission of the People’s Republic of China, 2000). Following confirmation that all polysaccharide extracts met the acceptable standard of the Chinese Veterinary Pharmacopoeia (less than 0.5 endotoxin units/mL), the preparations were stored at 4°C until use.

Reagents
Roswell Park Memorial Institute (RPMI)-1640 medium (no. 1335533, Gibco, purchased from Sino-American Biotechnology Co. Ltd., Beijing, China) was supplemented with 100 IU/mL of benzylpenicillin, 100 IU/mL of streptomycin, and 10% fetal bovine serum (no. 200511003, Zhengzhou Ben Bioengineering Co. Ltd., Zhengzhou, China) and was used for cell culture in vitro, washing and resuspending the cells, and mitogen dilution. Concanavalin A (Con A; no. 2005914, Sigma, purchased from Sino-American Biotechnology Co. Ltd., Beijing, China) was dissolved in RPMI-1640 medium to 0.025 mg/mL. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; no. 2005823, Amresco, purchased from Sino-American Biotechnology Co. Ltd.) was dissolved with calcium- and magnesium-free (CMF) PBS (pH 7.4) to 5 mg/mL. After filtering through a 0.22-µm syringe filter, the ConA solution was stored at –20°C, the MTT solution was stored at 4°C in dark bottles, and the RPMI-1640 medium was stored at 4°C. Mouse antichicken (mac) CD3-fluorescein isothiocyanate (no. 820002, Southern Biotechnology Associates, Birmingham, AL), mac CD4-phycoerythrin (PE; no. 821019, SBA), mac CD8a-PE (FITC; no. 822009, SBA), mouse IgG1-FITC (no. 010202, SBA), and mouse IgG1-PE (no. 010209, SBA) were purchased from Jingmei Bio Tech Co. Ltd., Shenzhen, China. Lymphocyte separation medium (no. 051106, Ficoll-Hypaque;{rho}: 1.077 ± 0.002) was a product of Hua-jing Biostix (Shanghai, China). Dimethyl sulfoxide was produced at the Zheng-xing Institute of Chemical Engineering in Suzhou, China.

Vaccine
Newcastle disease (Lasota strain)-IB (H120 strain) live virus vaccine (no. 315) and ND-IB oil adjuvant vaccine (no. 200551) were provided by the Institute of Veterinary Medicine, Animal Husbandry Bureau of Henan Province in Zhengzhou, China.

Birds and Housing
One-day-old White Roman male chickens (egg type), purchased from Ruixiang Co. Ltd. (Zhengzhou, China), were housed in wire cages (60 x 60 x 100 cm; 10 chickens per wire cage) in climate-controlled rooms at 36 ± 1°C and kept under 24-h light at the beginning of the pretrial period. The temperature was gradually reduced to room temperature in the spring and the photoperiod was reduced to 12 h/d, where it was kept constant over the following days. Chickens were fed with a commercial starter diet provided by the Feed Factory of the Animal Husbandry Bureau of Henan Province.

Experimental Design
At 14 d of age, a total of 450 chickens were vaccinated with live ND-IB virus vaccine by intranasal and intraocular administrations, and then randomly divided into 9 treatment groups of 50 chickens each, with 5 replicate cages per treatment. The average titer of maternal anti-body against the ND virus was 4.5 log2, and the average BW was 97.6 g. Each chicken in groups 1 to 8 was injected subcutaneously to the cervicum with 0.5 mL of 1 of the 4 polysaccharides at 1 of 2 concentrations, once a day for 3 successive days. In group 9, as the medicine-free control, each chicken was injected with 0.5 mL of saline at the same times. At 28 d of age, all chickens were vaccinated for a second time with ND-IB oil adjuvant vaccine by subcutaneous injection in the dorsal region of the cervix. On d 10, 20, 30, 40, and 50 after the first vaccination, 8 chickens were sampled randomly from each group to determine peripheral blood lymphocyte proliferation by MTT assay, as well as proportions of CD3+, CD3+CD4+, and CD3+CD8+ peripheral blood lymphocytes by flow cytometry and the double-color-staining method. On d 7, 14, 21, 28, 35, 42, and 49 after the first vaccination, 15 chickens were sampled randomly from each group for analysis of serum ND hemagglutination inhibition (HI) antibody titer by the micromethod (listed in Table 1Go).


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  		Table 1. Experimental design
 
Sample Collection and Assay
Peripheral Blood Lymphocyte Proliferation Assay.
Blood samples (5 mL/chicken) were collected from the heart and transferred immediately into aseptic capped tubes with sodium heparin, and then diluted with an equal volume of Hanks’ solution and carefully layered on the surface of the lymphocyte separation medium. After 20 min of centrifugation at 1,000 xg, a white layer of peripheral blood mononuclear cells (PMNC) at the plasma-Ficoll interface was collected and washed twice with RPMI-1640 medium without fetal bovine serum. The resulting pellet was resuspended to 5 x106/mL with RPMI-1640 medium and incubated in 96-well culture plates at 80 µL/well; ConA was then added at 20 µL/ well, seeded at 4 wells per sample. The final working concentration of ConA in each well was 5 µg/mL. The plates were incubated at 39.5°C for 48 h in a humid atmosphere with 5% CO2. After 44 h of incubation, 20 µL of MTT (5 mg/mL) was added to each well, and the plates were reincubated for 4 h. The supernatant was carefully removed and 100 µL of dimethyl sulfoxide was added to each well and shaken for 5 min to completely dissolve the precipitation. The absorbance of cells in each well was measured by a microliter ELISA reader (model 353, Thermo Labsystems, Helsinki, Finland) at a wavelength of 570 nm (Barta et al., 1992; Bao, 1998; Kong et al., 2004).

Cell Population Analysis.
Peripheral blood mononuclear cells were isolated as described above (peripheral blood lymphocyte proliferation assay) and adjusted to a concentration of 1 x107/mL with CMF-PBS. For each sample (8 PMNC suspensions), 3 tubes containing the following combinations of monoclonal antibodies were set up: 10 µL of mac CD3-FITC (amount used: 0.2 µg/106 cells) and 10 µL of mac CD4-PE (amount used: 1 µg/106 cells); 10 µL of mac CD3-FITC and 10 µL of mac CD8a-PE (amount used: 0.2 µg/106 cells); and 10 µL of mouse IgG1-FITC (amount used: 10 µL/106 cells) and 10 µL of mouse IgG1-PE (amount used: 10 µL/106 cells). Each tube received 50 µL of PMNC suspension, and the contents were gently mixed and then incubated at 4°C for 20 min in the dark. Then 500 µL of CMF-PBS solution was dispensed into each tube, gently mixed, and left for 10 min at room temperature out of direct light. After 10 min of centrifugation at 800 xg, the cells were resuspended with 500 µL of CMF-PBS solution, and the percentages of CD3+, CD3+CD4+, and CD3+CD8+ lymphocytes in the PMNC suspension were determined by flow cytometry (model EPICSXL, American Beckman Coulter, Fullerton, CA; Yang et al., 2005).

Serum HI Antibody Assay.
Blood samples (1.0 mL/ chicken) were drawn into Eppendorf tubes from the main brachial vein of the chicken and allowed to clot at 37°C for 2 h prior to collecting serum. Serum was separated by centrifugation and stored at –20°C for use. Briefly, a 2-fold serial dilution of serum, after inactivation at 56°C for 30 min, was made in a 96-well, V-shaped bottom microtiter plate containing 50 µL of CMF-PBS in all wells, and 50 µL of ND virus antigen (4 hemagglutination units) was added to all the wells except for the last row (the controls). Serum dilutions ranged from 1:2 to 1:2,048. The antigen serum mixture was incubated for 10 min at 37°C. Fifty microliters of a 1% rooster erythrocyte suspension was then added to each well and the wells were reincubated for 30 min. A positive serum, a negative serum, erythrocytes, and antigens were also included as controls. The highest dilution of serum causing complete inhibition of erythrocyte agglutination was considered the end point. The geometric mean titer was expressed as reciprocal log2 values of the highest dilution that displayed anti-ND-HI (Xu, 1990).

Statistical Analysis
Data are expressed as means ±SD. Standard deviations were calculated with Microsoft Office Excel 2003 (Microsoft, Redmond, WA). Duncan’s multiple range test was used to determine the differences among herbal polysaccharides and control groups. Differences between means were considered significant at P <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Peripheral Blood Lymphocyte Proliferation
Lymphocyte proliferation (optical density) in response to Con A stimulation is shown in Table 2Go. On d 10 after the first vaccination, the values of the APSL, IRPSL, ARPSH, and CYPSH groups were higher compared with the control group (P <0.05). On d 20, the values of the APSH, APSL, IRPSL, ARPSH, and CYPSH groups were higher than that of the control group (P <0.05); on d 30, the values of the other 6 treatment groups, except for the ARPSL and CYPSL groups, were higher in comparison with the control group (P <0.05). On d 40 and 50, the values of the APSL, IRPSL, ARPSH, and CYPSH groups were higher than that of the control group (P <0.05).


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  		Table 2. T lymphocyte proliferation (OD570 value)1
 
Proportions of CD3+CD4+ and CD3+CD8+ Lymphocyte Populations
On d 10 and 40 after the first vaccination, the ratios of CD4+ to CD8+ in the APSL, IRPSL, ARPSH, and CYPSH groups were higher than those of the control group (P <0.05). On d 20, the ratios of the APSH, APSL, IRPSL, and CYPSH groups were higher than those of the control group (P <0.05). On d 30, the ratios in the other 6 treatment groups, except for the ARPSL and CYPSL groups, were higher than those of the control group (P <0.05). On d 50, the ratios of the APSL, IRPSH, IRPSL, ARPSH, and CYPSH groups were higher than those of the control group (P <0.05; listed in Table 3Go).


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  		Table 3. Ratio of T lymphocytes CD4+:CD8+ 1
 
Dynamic Changes in Serum Antibody Titer
The dynamic changes in ND antibody titer are listed in Table 4Go. On d 7 after the first vaccination, the titers among the 9 treatment groups showed no significant difference (P >0.05). On d 14, the titers in the APSL and IRPSL groups were higher compared with the control group (P <0.05). On d 21, the titers in the APSL, IRPSL and CYPSH groups were higher than that of the control group (P <0.05). On d 28, the titers in the APSL, IRPSH, IRPSL, ARPSH, ARPSL, and CYPSH groups were higher compared with those of the control group (P <0.05). On d 35, the titers in the APSH, APSL, IRPSH, IRPSL, ARPSH, and CYPSH groups were higher than those of the control group (P <0.05). On d 42 and 49, the titers in the APSL, IRPSL, ARPSH, and CYPSH groups were higher than those of the control group (P <0.05).


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  		Table 4. Dynamic changes of Newcastle disease hemagglutination inhibition antibody titer (log2)
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Lymphocytes are important immune cells that play a critical role in maintaining immune functions, and optical density value is an indicator of lymphocyte proliferation. The high dosage of ARPS and CYPS, as well as APS and IRPS at both dosage levels, resulted in higher Con A-stimulated lymphocyte proliferation compared with control values. For APS and IRPS, this effect was more pronounced at the low dosage level. Considering that Con A is primarily a T-cell mitogen, these 4 polysaccharides appear to improve the ability of T cells to respond to a proliferation stimulus in vitro. Our findings are in agreement with reports that several polysaccharides have been shown to have a mitogenic function, particularly in stimulating proliferation in lymphocytes and enhancing Con A-induced lymphocyte proliferation either in vitro or in vivo (He and Wu, 2001). Hence, it appears that at a suitable dosage, the Chinese herbal polysaccharides tested here have enhancing effects on this aspect of cell-mediated immune activity.

CD3 is the cell surface marker for identifying T lymphocytes, which are the key cells of cell-mediated immune activity. CD4 and CD8 are 2 important surface markers of T lymphocytes that allow the 2 subsets of mature T cells to be distinguished. CD4+ T lymphocytes (T-helper cells) can induce and enhance the immune response by secreting cytokines. CD8+ T lymphocytes (cytotoxic cells) can mediate cytotoxic killing of target cells (Summerfield et al., 1996). Hence, CD4+ and CD8+ lymphocytes represent key functional subsets of adaptive cell-mediated immunity. The ratio of CD4+ to CD8+ has been shown to be indicative of the general state of immune functioning [e.g., a high CD4+:CD8+ ratio may be indicative of improved immune activity (Hu et al., 2003; Yang et al., 2005)]. In the present study, the proportions of CD3+ lymphocytes were not affected by treatment. However, within the T-cell compartment, the ratios of CD4+ to CD8+ T lymphocytes in 8 treatment groups were higher than that in the control group, especially in the APSL, IRPSL, ARPSH, and CYPSH groups, indicating that polysaccharides at a suitable dosage may enhance cellular immunity by promoting the differentiation or proliferation of CD4+ T lymphocytes. In addition, CD4+ T lymphocytes help in the initiation and progression of the B-cell response; thus, the increased proportions of CD4+ T lymphocytes may also have beneficial effects on humoral immunity (Jiang et al., 2006).

The serum antibody titer is an indicator of humoral immunity. In this experiment, the anti-ND virus HI antibody titers in most treatment groups were higher than that of the control group at nearly all time points. From d 21 to d 35, the titers in each group reached higher levels, but in the APSL, IRPSL, ARPSH, and CYPSH groups, the titers reached 11.5 to 12.3 log2, whereas in the control group, the titers reached 10.6 to 11.0 log2. On d 49, the titer in the control group dropped to 8.3 log2, whereas in the APSL, IRPSL, ARPSH, and CYPSH groups, the titers dropped to 9.1 and 9.6 log2. These findings indicate that at a suitable dosage, the 4 polysaccharides could promote specific antibody production earlier and maintain it longer, and thus improve the immune effect of the vaccine.

In summary, this research confirms that the 4 Chinese herbal polysaccharides could enhance aspects of cellular and humoral immune activities and would be expected to be effective as component drugs of a new immunopotentiator. More studies are needed to examine the observed dosage effect and to determine the immunofunctional effects and mechanisms of these Chinese herbal polysaccharides more comprehensively before their development as Chinese herbal medicinal immunopotentiators in poultry.


   ACKNOWLEDGMENTS
 
This research was supported by grants from National Food Safety Foundation of China, Zhengzhou of Henan province (no. 2001BA804A30-11). The authors are grateful to all other staff in the Veterinary Microbiology Laboratory of Henan Agricultural University for their assistance in the experiments.

Received for publication February 14, 2007. Accepted for publication July 3, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Barta, O., V. Barta, and F. W. Pierson. 1992. Optimum conditions for the chicken lymphocyte transformation test. Avian Dis. 36:945–955.[CrossRef][ISI][Medline]

Chen, H. L. 2006. Studies on the extraction, immunomodulating activities of Chinese herbal polysaccharides and approach to the mechanism. Chin. Acad. Agric. Sci. 6:23–25.

He, Y., and X. Z. Wu. 2001. Immune regulation of Chinese herbal polysaccharide. J. Sichuan Trad. Chin. Med. 192:15–17.

Hu, Y. J., Y. C. Lin, G. L. Zhou, and D. Q. Yu. 2003. Effect of Chinese extracts on performance and T lymphocyte cell subset of yellow broilers. China Poult. 12:14–17.

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Kong X. F., Y. L. Hu, and R. Rui. 2004. Effects of Chinese herbal medicinal ingredients on peripheral lymphocyte proliferation and serum antibody titer after vaccination in chicken. J. Int. Immunopharmacol. 4:975–982.[CrossRef]

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Summerfield, A., H. J. Rziha, and A. Saalmüller. 1996. Functional characterization of porcine CD4+CD8+ extrathymic T lymphocytes. Cell. Immunol. 168:291–296.[CrossRef][ISI][Medline]

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Xue, Y. L. 1985. The extracts method of plants. Pages 136–138 in Handbook of Laboratory Experiments in Plant Physiology. Shanghai Sci. Technol. Press, Shanghai, China.

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Yon Z. T., Z. h. Lina, and C. K. Peter. 2006. Physicochemical properties and antitumor activities of water-soluble native and sulfated hyperbranched mushroom polysaccharides. Carbohydr. Res. 341:2261–2269.[CrossRef][ISI][Medline]





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