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Enhancement of immune responses to Newcastle disease vaccine by a supplement of extract of Momordica cochinchinensis (Lour.) Spreng. seeds
IMMUNOLOGY, HEALTH, AND DISEASE Enhancement of immune responses to Newcastle disease vaccine by a supplement of extract of Momordica cochinchinensis (Lour.) Spreng. seeds C. Xiao,*† G. Bao,† and S. Hu*1 *Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310029, P. R. China; and †Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, P. R. China 2,5-diphenyl tetrazolium bromide method. Results indicated that humoral immune response was enhanced by ECMS 14 d postimmunization. Eighty micrograms of ECMS was the best dose with the ND vaccine and was significantly different from the other groups 21 d after immunization. No significant differences were found in the cellular immune response, whereas the 80 μg of ECMS group had higher values than the other groups 35 d after immunization. No side effect was found on the growth performance during the experiment.
Key words: extract of Momordica cochinchinensis (Lour.) Spreng. seeds, adjuvant, saponin, vaccine, Newcastle disease 2009 Poultry Science 88:2293–2297 doi:10.3382/ps.2009-00059
INTRODUCTION Newcastle disease (ND) is a serious avian disease with worldwide distribution that can cause substantial economic losses and remains a major threat to the poultry industry around the world. The causative agent of the disease, ND virus, is a member of the genus Avulavirus in the family Paramyxoviridae (Mayo, 2002). The first case of ND in China was found in village chicken flocks in 1946, and since then, it has been regarded as an endemic disease (Liu et al., 2003). In the past decades, an intensive vaccination program against ND has been practiced in both large-scale poultry operations and village poultry farming. There are 2 kinds of ND vaccines available in China, attenuated vaccines and inactivated whole-virus vaccines (Hu et al., 2008). Most commercially available ND vaccines are inactivated whole-virus preparations containing oil emulsions as an adjuvant to improve their efficacy (Gao et al., 2008). However, the vaccines have been reported to induce
©2009 Poultry Science Association Inc. Received February 3, 2009. Accepted March 12, 2009. 1 Corresponding author: [email protected]
poor immune responses (Liu et al., 2008). Therefore, there is a need to improve currently available ND vaccines to effectively protect animals from ND infection. Our previous research has demonstrated that a supplement of saponin extracted from the bark of Quillaja saponaria Molina (Quil A) in vaccines significantly enhances immune responses to foot-and-mouth disease vaccination (Xiao et al., 2007b). However Quil A may be limited in veterinary use because of its hemolytic activity (Oda et al., 2000). Another study has shown that an extract from a traditional Chinese medicine, the seeds of Momordica cochinchinensis (Lour.) Spreng. (ECMS), has an adjuvant effect on the immune responses elicited by ovalbumin, with a much lower hemolytic activity (Xiao et al., 2007a). Extract of Momordica cochinchinensis (Lour.) Spreng. seeds has also been proven to be effective at enhancing immune responses to vaccination against avian influenza (H5N1) in chickens (Rajput et al., 2007). The medical use of the seeds has been described in ancient Chinese medical literature, Kai Bao Materia Medica, from the Song Dynasty (793 AD; Gao, 2005) and is currently included in both the Chinese pharmacopeia (Chinese Pharmaceutical Codex Evaluation Committee, 2005) and the Chinese veterinary pharmacopeia (Chinese Veterinary Pharmaceutical Codex Evaluation Committee, 2000).
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Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
ABSTRACT The study evaluated the immunological effect of extract of Momordica cochinchinensis (Lour.) Spreng. seeds (ECMS) on the immune response against Newcastle disease (ND) in chickens. Forty-eight chickens were divided into 4 groups (n = 12). Each chicken was immunized with ND vaccine mixed with 0, 20, 40, or 80 μg of ECMS on d 35. Blood samples were collected on d 0, 7, 14, 21, 28, and 35 postimmunization. Humoral and cellular immune responses were evaluated by indirect ELISA assay and the 3-(4,5-dimethyl thiazol-2-yl)-
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Traditionally, the seeds are used for the treatment of inflammatory swelling, scrofula, tinea, and diarrhea as well as suppurative skin infections, such as sores, carbuncles, furuncles, and boils, in humans and animals (Gao, 2005). This study was designed to evaluate the supplement of ECMS in oil-emulsified ND vaccine for its immunopotentiation in chickens.
MATERIALS AND METHODS Experimental Birds
Adjuvant and Vaccine The ECMS was extracted by the following procedure (Xiao et al., 2007a): 10 kg of dried Cochinchina momordica seeds were crushed into a powder and then immersed in 50% ethanol for 24 h. The powder and ethanol mixture was placed into a round-bottomed flask and the mixture was refluxed 3 times at 90°C, with each reflux taking 2 h. The remaining ethanol was removed using a R502B rotary evaporator (Shenko Tech Co. Ltd., Shanghai, China).The extract was then dissolved in water and washed with diethyl ether to remove the ether-soluble substance. After that, the saponin fraction was extracted with water-saturated n-butanol. The butanol-soluble fraction was purified by passage through a chromatography column with macroporous resin D101A (HaiGuang Chemical Co. Ltd., Tianjin, China). Purified ECMS was obtained by evaporating the liquid eluted from the column. Newcastle disease vaccine (La Sota strain) mixed with an oil emulsion as an adjuvant is available commercially in China (Harbin Veke Bio. Co. Ltd., Heilongjiang, China).
Experiment Design All of the birds were randomly divided into 4 groups with each consisting of 12 chickens. The birds were immunized by a single neck hypodermic injection of an ND vaccine (0.3 mL) mixed with ECMS (0, 20, 40, or 80 μg per dose). Blood samples were collected before and 1, 2, 3, 4, and 5 wk after immunization for ELISA assay. Five weeks after immunization, blood samples
ND-Specific IgG in Chicken Serum samples were analyzed for IgG antibodies by an ELISA using Flock Check Idexx ELISA Test Kit (Idexx Laboratories, Beijing, China; Rajput et al., 2007). The assay was performed according to the instructions provided by the company. Samples of test or standard sera were diluted (1:500) into test tubes. One hundred microliters of diluted serum samples were dispensed in duplicate to appropriate wells of ND antigen coated microtiter plates and incubated for 30 min at room temperature. Liquid content of all wells was aspirated and then wells were washed 6 times with 350 μL/well of distilled water. After that, 100 μL of goat anti-chicken/turkey horseradish peroxidase conjugated second antibody was added for 30 min at room temperature. After washing the wells 6 times, 100 μL of 3,3,5,5-tetramethyl-benzidine solution was added and incubated for 15 min at room temperature. The reaction was stopped by adding 100 μL of 2 M H2SO4 to each well. The optical density (OD) of the plate was read by an automatic ELISA plate reader at 630 nm.
Lymphocyte Proliferation Assay Five weeks after immunization, blood samples were collected from 5 chickens in each group and sodium citrate was used as anticoagulant. In vitro mitogen-induced responses of peripheral blood lymphocytes were measured as described previously (Finkelstein et al., 2003) with some modification. Briefly, 2 mL of blood sample was diluted with the same volume of sterilized PBS. Two milliliters of lymphocyte separation medium was added in a glass test tube. Diluted blood was carefully layered onto the surface of lymphocyte separation medium in the test tube. The glass tube was centrifuged for 30 min at 450 × g at room temperature. A buffy coat of lymphocytes was pipetted carefully to another screw-capped, V-shaped glass tube and diluted with PBS. After washing 2 times with PBS, the cells were added to 96-well plates (Costar, Shanghai, China) with 100 μL in each well (5 × 106/mL). One hundred microliters of stimulators, concanavalin A (5 μg/mL), lipopolysaccharide (10 μg/mL), or phytohemagglutinin (10 μg/ml), was added to the wells. After 48 h of incubation in 5% CO2, 50 μL of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added for another 4 h of incubation. Then the plates were centrifuged at 450 × g for 20 min. After removal of untransformed 3-(4,5-dimethyl thiazol-2-yl)2,5-diphenyl tetrazolium bromide, 150 μL of dimethyl sulfoxide with 4% 1 N HCl was dispensed to each well to dissolve the formed formazan crystals for 10 min in the dark. Optical density value was measured at 490 nm. The stimulation index was calculated as the mean
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
Forty-eight 1-d-old Hy-Line White layer chickens (male) were purchased from Hangzhou Layer Experimental Farm (Hangzhou, China). Different experimental groups were housed in separate wire cages in an air-conditioned room at 37°C and lighted for 24 h at the beginning of the pretrial period. The temperature was gradually decreased to room temperature and the light time to 12 h per day, both of which were kept constant in the following days. Chickens were fed with commercial starter diet, purchased from the same company mentioned above. The birds were given food and water ad libitum.
were collected from the heart for lymphocyte stimulation test.
ENHANCEMENT OF IMMUNE RESPONSES BY SAPONIN
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OD value of mitogen culture divided by the OD of nonstimulated cultures.
Effect of ECMS on Mitogen-Stimulated Lymphocyte Proliferation
Effect on Growth Performance
The effects of ECMS on mitogen-stimulated lymphocyte proliferation in chickens immunized with ND are depicted in Figure 2. The stimulation index of birds receiving 40 and 80 μg of ECMS and stimulated with concanavalin A, lipopolysaccharide, or phytohemgglutinin was numerically higher than in other groups but without significance.
Body weight of the chickens (n = 12 chickens/group) was measured before and after immunization at weekly intervals (wk 1, 2, 3, 4, and 5) with the help of a digital balance. The BW gain of each chicken was obtained (BW after immunization − BW before immunization) and compared among the groups.
Statistical Analysis
RESULTS Serum IgG Antibody Response Serum samples collected from chickens were analyzed for IgG antibody level to ND using the ELISA test. The results are depicted in Figure 1. On d 7, no change in IgG antibody level between groups was observed after immunization. At d 14, 21, and 28, antibody level of birds receiving 20, 40, and 80 μg/dose of ECMS was significantly higher than the control (P < 0.05). At d 35, antibody level of birds receiving 40 and 80 μg/dose of ECMS was significantly higher than the control (P < 0.05). There was a dose-dependent relation between ECMS and IgG level in chickens. Compared with the 20- and 40-μg groups, the 80-μg group had significantly higher IgG levels on d 21, 28, and 35 (P < 0.05).
Changes in live BW gain during the experimental period are shown in Figure 3. Average BW gain of the birds receiving 20 and 40 μg/dose of ECMS was numerically higher (insignificant) on d 21, 28, and 35.
DISCUSSION Adjuvants have significant effects on the nature of the immune responses and can tilt the immune system in favor of T-helper 1 and T-helper 2-type immune responses (Cox and Coulter, 1997). The choice of adjuvants is currently limited because the toxicity is a primary issue in adjuvant development. Extract of Momordica cochinchinensis (Lour.) Spreng. seeds has already been proven for its efficient adjuvant activity with low hemolytic activity (Xiao et al., 2007a,c). In this study, we have evaluated the utility of ECMS as an adjuvant in ND vaccination. The present results show that incorporation of ECMS into ND vaccine enhanced the serum IgG responses (Figure 1). From the results presented here, it is clear that immunization with formulations of ND vaccine containing 20, 40, and 80 μg/dose of ECMS yield significantly higher IgG antibody levels when compared with the controls on 14, 21, 28, and 35 d postimmunization. The birds receiving 80 μg/dose of ECMS had
Figure 1. Newcastle disease virus-specific antibody response in chickens (n = 12/group) immunized with Newcastle disease vaccine (0.3 mL) mixed with extract of Momordica cochinchinensis (Lour.) Spreng. seeds (ECMS; 0, 20, 40, or 80 μg). Blood samples were collected before and 1, 2, 3, 4, and 5 wk postimmunization for ELISA assay. Data are expressed as means ± SD. Bars with different letters at the same time point are statistically significantly different (P < 0.05). OD = optical density.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
Statistical analysis was performed using SPSS 10.0 for Windows (SPSS Inc., Chicago, IL). Data are expressed as means ± SD. Bonferroni’s multiple-range test was used to compare the parameters between groups. A probability of less than 0.05 was considered significantly different.
Effect on Growth Performance
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higher values of antibody level than the 20 and 40 μg/ dose. However, the best dose of ECMS as an adjuvant in influenza vaccination was 20 μg/dose (Rajput et al., 2007), which suggests that the effectiveness of ECMS as an adjuvant is antigen-specific. Figure 2 shows that lymphocyte proliferation response was numerically but not statistically highest in chickens receiving 80 μg of ECMS as compared with the control at 5 wk after immunization. Liu et al. (2008) reported that the cellular immune response reached a peak 3 wk after ND immunization. This suggests that 5
wk postimmunization may not have been an optimum time point for evaluation of this response. To study any adverse effect on growth performance, live BW was also recorded during the study period and no adverse effect was observed. Comparison of weight gain on d 0, 7, 14, 28, and 35 in all groups is depicted in Figure 3. Results revealed that the ECMS has no effect on growth performance in chicken. In ruminants and other domestic animals including chickens, the dietary saponins have significant effects on all phases of metabolism (Cheeke, 1996). Positive
Figure 3. Growing performance of chickens. Body weight of the chickens (n = 12 chickens/group) was measured before and after immunization at weekly intervals (wk 1, 2, 3, 4, and 5) with the help of a digital balance. The BW gain of each chicken was obtained (BW after immunization − BW before immunization) and compared among the groups. Data are expressed as means ± SD. No significant difference was found between groups. ECMS = extract of Momordica cochinchinensis (Lour.) Spreng. seeds.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
Figure 2. Mitogen-induced proliferative responses of lymphocytes isolated from peripheral blood in chickens (n = 5/group) immunized with Newcastle disease vaccine (0.3 mL) mixed with extract of Momordica cochinchinensis (Lour.) Spreng. seeds (ECMS; 0, 20, 40, or 80 μg). Blood samples were collected before and 1, 2, 3, 4, and 5 wk postimmunization for lymphocyte proliferation test. Data are expressed as means ± SD. No significant difference was found between groups. ConA = concanavalin A; LPS = lipopolysaccharide; PHA = phytohemagglutinin .
ENHANCEMENT OF IMMUNE RESPONSES BY SAPONIN
or negative effects of dietary saponins have been well documented (Ishaaya et al., 1969; Sim et al., 1984). The current study suggests that ECMS has vaccine adjuvant efficacy and deserves further study.
REFERENCES
VII Newcastle disease virus generated by reverse genetics. Vaccine 27:904–910. Ishaaya, I., Y. Birk, A. Bondi, and Y. Tencer. 1969. Soyabean saponins. IX. Studies of their effect on birds, mammals and coldblooded organisms. J. Sci. Food Agric. 20:433–436. Liu, R. S., Z. L. Xue, S. B. Zhang, and B. H. Wang. 2008. Adjuvant effect of Chinese retard compound medicine on the immune response to ND vaccination. Chin. J. Vet. Med. 44:27–28. Liu, X. F., H. Q. Wan, X. X. Ni, Y. T. Wu, and W. B. Liu. 2003. Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985–2001. Arch. Virol. 148:1387–1403. Mayo, M. A. 2002. A summary of taxonomic changes recently approved by ICTV. Arch. Virol. 147:1655–1663. Oda, K., H. T. Matsuda, S. Murakami, T. O. Katayama, and M. Yoshikawa. 2000. Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biol. Chem. 381:67–74. Rajput, Z. I., C. W. Xiao, and S. H. Hu. 2007. To improve the efficacy of influenza vaccination (H5N1) in chicken by using extraction of Cochinchina momordica seed (ECMS). J. Zhejiang Univ. Sci. B 8:331–337. Sim, J. S., W. D. Kitts, and D. B. Bragg. 1984. Effect of dietary saponin on egg cholesterol level and laying hen performance. Can. J. Anim. Sci. 64:977–984. Xiao, C. W., Z. I. Rajput, and S. H. Hu. 2007a. Adjuvant effect of an extract from Cochinchina momordica seeds on the immune responses to ovalbumin in mice. Front. Agric. China 1:90–95. Xiao, C. W., Z. I. Rajput, and S. H. Hu. 2007b. Improvement of a commercial foot-and-mouth disease vaccine by supplement of QuilA. Vaccine 25:4795–4800. Xiao, C. W., Z. I. Rajput, D. W. Liu, and S. H. Hu. 2007c. Enhancement of serological immune responses to foot-and-mouth disease vaccine by a supplement made of extract of Cochinchina momordica seeds. Clin. Vaccine Immunol. 14:1634–1639.
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Cheeke, P. R. 1996. Biological effects of feed and forage saponins and their impact on animal production. Pages 377–386 in Saponins Used in Food and Agriculture. G. R. Waller and Y. Yamasaki, ed. Plenum Press, New York, NY. Chinese Pharmaceutical Codex Evaluation Committee. 2005. Mu Bie Zi (Semen momordicae). Page 44 in Chinese Pharmaceutical Codex Edition 2005. Part I. Chemical Industry Press, Beijing, China. Chinese Veterinary Pharmaceutical Codex Evaluation Committee. 2000. Mu Bie Zi (Semen momordicae). Pages 49–50 in Chinese Veterinary Pharmaceutical Codex Edition 2000. Part II. Chemical Industry Press, Beijing, China. Cox, J. C., and A. R. Coulter. 1997. Adjuvants–A classification and review of their modes of action. Vaccine 15:248–256. Finkelstein, M., K. A. Grasman, D. A. Croll, B. Tershy, and D. R. Smith. 2003. Immune function of cryopreserved avian peripheral white blood cells: Potential biomarkers of contaminant effects in wild birds. Arch. Environ. Contam. Toxicol. 44:502–509. Gao, X. M. 2005. Mu Bie Zi (Semen momordicae). Pages 601–602 in Chinese Materia Medica. China Traditional Chinese Materia Medica Press, Beijing, China. Gao, G. H., Y. Li, C. H. Jin, and H. W. Ge. 2008. Serum analysis after ND vaccination. Chin. J. Anim. Husbandry Vet. Med. 4:31–32. Hu, T. J., Y. Y. Gao, J. R. Chen, and X. H. Shuai. 2008. Enhancement of immune responses to Newcastle disease vaccine by a supplement APS. Guang Xi Vet. Anim. Sci. 24:150–151. Hu, S. L., H. L. Ma, Y. T. Wu, W. B. Liu, X. Q. Wang, Y. L. Liu, and X. F. Liu. 2008. A vaccine candidate of attenuated genotype
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Key words: extract of Momordica cochinchinensis (Lour.) Spreng. seeds, adjuvant, saponin, vaccine, Newcastle disease 2009 Poultry Science 88:2293–2297 doi:10.3382/ps.2009-00059
INTRODUCTION Newcastle disease (ND) is a serious avian disease with worldwide distribution that can cause substantial economic losses and remains a major threat to the poultry industry around the world. The causative agent of the disease, ND virus, is a member of the genus Avulavirus in the family Paramyxoviridae (Mayo, 2002). The first case of ND in China was found in village chicken flocks in 1946, and since then, it has been regarded as an endemic disease (Liu et al., 2003). In the past decades, an intensive vaccination program against ND has been practiced in both large-scale poultry operations and village poultry farming. There are 2 kinds of ND vaccines available in China, attenuated vaccines and inactivated whole-virus vaccines (Hu et al., 2008). Most commercially available ND vaccines are inactivated whole-virus preparations containing oil emulsions as an adjuvant to improve their efficacy (Gao et al., 2008). However, the vaccines have been reported to induce
©2009 Poultry Science Association Inc. Received February 3, 2009. Accepted March 12, 2009. 1 Corresponding author: [email protected]
poor immune responses (Liu et al., 2008). Therefore, there is a need to improve currently available ND vaccines to effectively protect animals from ND infection. Our previous research has demonstrated that a supplement of saponin extracted from the bark of Quillaja saponaria Molina (Quil A) in vaccines significantly enhances immune responses to foot-and-mouth disease vaccination (Xiao et al., 2007b). However Quil A may be limited in veterinary use because of its hemolytic activity (Oda et al., 2000). Another study has shown that an extract from a traditional Chinese medicine, the seeds of Momordica cochinchinensis (Lour.) Spreng. (ECMS), has an adjuvant effect on the immune responses elicited by ovalbumin, with a much lower hemolytic activity (Xiao et al., 2007a). Extract of Momordica cochinchinensis (Lour.) Spreng. seeds has also been proven to be effective at enhancing immune responses to vaccination against avian influenza (H5N1) in chickens (Rajput et al., 2007). The medical use of the seeds has been described in ancient Chinese medical literature, Kai Bao Materia Medica, from the Song Dynasty (793 AD; Gao, 2005) and is currently included in both the Chinese pharmacopeia (Chinese Pharmaceutical Codex Evaluation Committee, 2005) and the Chinese veterinary pharmacopeia (Chinese Veterinary Pharmaceutical Codex Evaluation Committee, 2000).
2293
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
ABSTRACT The study evaluated the immunological effect of extract of Momordica cochinchinensis (Lour.) Spreng. seeds (ECMS) on the immune response against Newcastle disease (ND) in chickens. Forty-eight chickens were divided into 4 groups (n = 12). Each chicken was immunized with ND vaccine mixed with 0, 20, 40, or 80 μg of ECMS on d 35. Blood samples were collected on d 0, 7, 14, 21, 28, and 35 postimmunization. Humoral and cellular immune responses were evaluated by indirect ELISA assay and the 3-(4,5-dimethyl thiazol-2-yl)-
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Xiao et al.
Traditionally, the seeds are used for the treatment of inflammatory swelling, scrofula, tinea, and diarrhea as well as suppurative skin infections, such as sores, carbuncles, furuncles, and boils, in humans and animals (Gao, 2005). This study was designed to evaluate the supplement of ECMS in oil-emulsified ND vaccine for its immunopotentiation in chickens.
MATERIALS AND METHODS Experimental Birds
Adjuvant and Vaccine The ECMS was extracted by the following procedure (Xiao et al., 2007a): 10 kg of dried Cochinchina momordica seeds were crushed into a powder and then immersed in 50% ethanol for 24 h. The powder and ethanol mixture was placed into a round-bottomed flask and the mixture was refluxed 3 times at 90°C, with each reflux taking 2 h. The remaining ethanol was removed using a R502B rotary evaporator (Shenko Tech Co. Ltd., Shanghai, China).The extract was then dissolved in water and washed with diethyl ether to remove the ether-soluble substance. After that, the saponin fraction was extracted with water-saturated n-butanol. The butanol-soluble fraction was purified by passage through a chromatography column with macroporous resin D101A (HaiGuang Chemical Co. Ltd., Tianjin, China). Purified ECMS was obtained by evaporating the liquid eluted from the column. Newcastle disease vaccine (La Sota strain) mixed with an oil emulsion as an adjuvant is available commercially in China (Harbin Veke Bio. Co. Ltd., Heilongjiang, China).
Experiment Design All of the birds were randomly divided into 4 groups with each consisting of 12 chickens. The birds were immunized by a single neck hypodermic injection of an ND vaccine (0.3 mL) mixed with ECMS (0, 20, 40, or 80 μg per dose). Blood samples were collected before and 1, 2, 3, 4, and 5 wk after immunization for ELISA assay. Five weeks after immunization, blood samples
ND-Specific IgG in Chicken Serum samples were analyzed for IgG antibodies by an ELISA using Flock Check Idexx ELISA Test Kit (Idexx Laboratories, Beijing, China; Rajput et al., 2007). The assay was performed according to the instructions provided by the company. Samples of test or standard sera were diluted (1:500) into test tubes. One hundred microliters of diluted serum samples were dispensed in duplicate to appropriate wells of ND antigen coated microtiter plates and incubated for 30 min at room temperature. Liquid content of all wells was aspirated and then wells were washed 6 times with 350 μL/well of distilled water. After that, 100 μL of goat anti-chicken/turkey horseradish peroxidase conjugated second antibody was added for 30 min at room temperature. After washing the wells 6 times, 100 μL of 3,3,5,5-tetramethyl-benzidine solution was added and incubated for 15 min at room temperature. The reaction was stopped by adding 100 μL of 2 M H2SO4 to each well. The optical density (OD) of the plate was read by an automatic ELISA plate reader at 630 nm.
Lymphocyte Proliferation Assay Five weeks after immunization, blood samples were collected from 5 chickens in each group and sodium citrate was used as anticoagulant. In vitro mitogen-induced responses of peripheral blood lymphocytes were measured as described previously (Finkelstein et al., 2003) with some modification. Briefly, 2 mL of blood sample was diluted with the same volume of sterilized PBS. Two milliliters of lymphocyte separation medium was added in a glass test tube. Diluted blood was carefully layered onto the surface of lymphocyte separation medium in the test tube. The glass tube was centrifuged for 30 min at 450 × g at room temperature. A buffy coat of lymphocytes was pipetted carefully to another screw-capped, V-shaped glass tube and diluted with PBS. After washing 2 times with PBS, the cells were added to 96-well plates (Costar, Shanghai, China) with 100 μL in each well (5 × 106/mL). One hundred microliters of stimulators, concanavalin A (5 μg/mL), lipopolysaccharide (10 μg/mL), or phytohemagglutinin (10 μg/ml), was added to the wells. After 48 h of incubation in 5% CO2, 50 μL of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added for another 4 h of incubation. Then the plates were centrifuged at 450 × g for 20 min. After removal of untransformed 3-(4,5-dimethyl thiazol-2-yl)2,5-diphenyl tetrazolium bromide, 150 μL of dimethyl sulfoxide with 4% 1 N HCl was dispensed to each well to dissolve the formed formazan crystals for 10 min in the dark. Optical density value was measured at 490 nm. The stimulation index was calculated as the mean
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
Forty-eight 1-d-old Hy-Line White layer chickens (male) were purchased from Hangzhou Layer Experimental Farm (Hangzhou, China). Different experimental groups were housed in separate wire cages in an air-conditioned room at 37°C and lighted for 24 h at the beginning of the pretrial period. The temperature was gradually decreased to room temperature and the light time to 12 h per day, both of which were kept constant in the following days. Chickens were fed with commercial starter diet, purchased from the same company mentioned above. The birds were given food and water ad libitum.
were collected from the heart for lymphocyte stimulation test.
ENHANCEMENT OF IMMUNE RESPONSES BY SAPONIN
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OD value of mitogen culture divided by the OD of nonstimulated cultures.
Effect of ECMS on Mitogen-Stimulated Lymphocyte Proliferation
Effect on Growth Performance
The effects of ECMS on mitogen-stimulated lymphocyte proliferation in chickens immunized with ND are depicted in Figure 2. The stimulation index of birds receiving 40 and 80 μg of ECMS and stimulated with concanavalin A, lipopolysaccharide, or phytohemgglutinin was numerically higher than in other groups but without significance.
Body weight of the chickens (n = 12 chickens/group) was measured before and after immunization at weekly intervals (wk 1, 2, 3, 4, and 5) with the help of a digital balance. The BW gain of each chicken was obtained (BW after immunization − BW before immunization) and compared among the groups.
Statistical Analysis
RESULTS Serum IgG Antibody Response Serum samples collected from chickens were analyzed for IgG antibody level to ND using the ELISA test. The results are depicted in Figure 1. On d 7, no change in IgG antibody level between groups was observed after immunization. At d 14, 21, and 28, antibody level of birds receiving 20, 40, and 80 μg/dose of ECMS was significantly higher than the control (P < 0.05). At d 35, antibody level of birds receiving 40 and 80 μg/dose of ECMS was significantly higher than the control (P < 0.05). There was a dose-dependent relation between ECMS and IgG level in chickens. Compared with the 20- and 40-μg groups, the 80-μg group had significantly higher IgG levels on d 21, 28, and 35 (P < 0.05).
Changes in live BW gain during the experimental period are shown in Figure 3. Average BW gain of the birds receiving 20 and 40 μg/dose of ECMS was numerically higher (insignificant) on d 21, 28, and 35.
DISCUSSION Adjuvants have significant effects on the nature of the immune responses and can tilt the immune system in favor of T-helper 1 and T-helper 2-type immune responses (Cox and Coulter, 1997). The choice of adjuvants is currently limited because the toxicity is a primary issue in adjuvant development. Extract of Momordica cochinchinensis (Lour.) Spreng. seeds has already been proven for its efficient adjuvant activity with low hemolytic activity (Xiao et al., 2007a,c). In this study, we have evaluated the utility of ECMS as an adjuvant in ND vaccination. The present results show that incorporation of ECMS into ND vaccine enhanced the serum IgG responses (Figure 1). From the results presented here, it is clear that immunization with formulations of ND vaccine containing 20, 40, and 80 μg/dose of ECMS yield significantly higher IgG antibody levels when compared with the controls on 14, 21, 28, and 35 d postimmunization. The birds receiving 80 μg/dose of ECMS had
Figure 1. Newcastle disease virus-specific antibody response in chickens (n = 12/group) immunized with Newcastle disease vaccine (0.3 mL) mixed with extract of Momordica cochinchinensis (Lour.) Spreng. seeds (ECMS; 0, 20, 40, or 80 μg). Blood samples were collected before and 1, 2, 3, 4, and 5 wk postimmunization for ELISA assay. Data are expressed as means ± SD. Bars with different letters at the same time point are statistically significantly different (P < 0.05). OD = optical density.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
Statistical analysis was performed using SPSS 10.0 for Windows (SPSS Inc., Chicago, IL). Data are expressed as means ± SD. Bonferroni’s multiple-range test was used to compare the parameters between groups. A probability of less than 0.05 was considered significantly different.
Effect on Growth Performance
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higher values of antibody level than the 20 and 40 μg/ dose. However, the best dose of ECMS as an adjuvant in influenza vaccination was 20 μg/dose (Rajput et al., 2007), which suggests that the effectiveness of ECMS as an adjuvant is antigen-specific. Figure 2 shows that lymphocyte proliferation response was numerically but not statistically highest in chickens receiving 80 μg of ECMS as compared with the control at 5 wk after immunization. Liu et al. (2008) reported that the cellular immune response reached a peak 3 wk after ND immunization. This suggests that 5
wk postimmunization may not have been an optimum time point for evaluation of this response. To study any adverse effect on growth performance, live BW was also recorded during the study period and no adverse effect was observed. Comparison of weight gain on d 0, 7, 14, 28, and 35 in all groups is depicted in Figure 3. Results revealed that the ECMS has no effect on growth performance in chicken. In ruminants and other domestic animals including chickens, the dietary saponins have significant effects on all phases of metabolism (Cheeke, 1996). Positive
Figure 3. Growing performance of chickens. Body weight of the chickens (n = 12 chickens/group) was measured before and after immunization at weekly intervals (wk 1, 2, 3, 4, and 5) with the help of a digital balance. The BW gain of each chicken was obtained (BW after immunization − BW before immunization) and compared among the groups. Data are expressed as means ± SD. No significant difference was found between groups. ECMS = extract of Momordica cochinchinensis (Lour.) Spreng. seeds.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
Figure 2. Mitogen-induced proliferative responses of lymphocytes isolated from peripheral blood in chickens (n = 5/group) immunized with Newcastle disease vaccine (0.3 mL) mixed with extract of Momordica cochinchinensis (Lour.) Spreng. seeds (ECMS; 0, 20, 40, or 80 μg). Blood samples were collected before and 1, 2, 3, 4, and 5 wk postimmunization for lymphocyte proliferation test. Data are expressed as means ± SD. No significant difference was found between groups. ConA = concanavalin A; LPS = lipopolysaccharide; PHA = phytohemagglutinin .
ENHANCEMENT OF IMMUNE RESPONSES BY SAPONIN
or negative effects of dietary saponins have been well documented (Ishaaya et al., 1969; Sim et al., 1984). The current study suggests that ECMS has vaccine adjuvant efficacy and deserves further study.
REFERENCES
VII Newcastle disease virus generated by reverse genetics. Vaccine 27:904–910. Ishaaya, I., Y. Birk, A. Bondi, and Y. Tencer. 1969. Soyabean saponins. IX. Studies of their effect on birds, mammals and coldblooded organisms. J. Sci. Food Agric. 20:433–436. Liu, R. S., Z. L. Xue, S. B. Zhang, and B. H. Wang. 2008. Adjuvant effect of Chinese retard compound medicine on the immune response to ND vaccination. Chin. J. Vet. Med. 44:27–28. Liu, X. F., H. Q. Wan, X. X. Ni, Y. T. Wu, and W. B. Liu. 2003. Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985–2001. Arch. Virol. 148:1387–1403. Mayo, M. A. 2002. A summary of taxonomic changes recently approved by ICTV. Arch. Virol. 147:1655–1663. Oda, K., H. T. Matsuda, S. Murakami, T. O. Katayama, and M. Yoshikawa. 2000. Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biol. Chem. 381:67–74. Rajput, Z. I., C. W. Xiao, and S. H. Hu. 2007. To improve the efficacy of influenza vaccination (H5N1) in chicken by using extraction of Cochinchina momordica seed (ECMS). J. Zhejiang Univ. Sci. B 8:331–337. Sim, J. S., W. D. Kitts, and D. B. Bragg. 1984. Effect of dietary saponin on egg cholesterol level and laying hen performance. Can. J. Anim. Sci. 64:977–984. Xiao, C. W., Z. I. Rajput, and S. H. Hu. 2007a. Adjuvant effect of an extract from Cochinchina momordica seeds on the immune responses to ovalbumin in mice. Front. Agric. China 1:90–95. Xiao, C. W., Z. I. Rajput, and S. H. Hu. 2007b. Improvement of a commercial foot-and-mouth disease vaccine by supplement of QuilA. Vaccine 25:4795–4800. Xiao, C. W., Z. I. Rajput, D. W. Liu, and S. H. Hu. 2007c. Enhancement of serological immune responses to foot-and-mouth disease vaccine by a supplement made of extract of Cochinchina momordica seeds. Clin. Vaccine Immunol. 14:1634–1639.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 29, 2015
Cheeke, P. R. 1996. Biological effects of feed and forage saponins and their impact on animal production. Pages 377–386 in Saponins Used in Food and Agriculture. G. R. Waller and Y. Yamasaki, ed. Plenum Press, New York, NY. Chinese Pharmaceutical Codex Evaluation Committee. 2005. Mu Bie Zi (Semen momordicae). Page 44 in Chinese Pharmaceutical Codex Edition 2005. Part I. Chemical Industry Press, Beijing, China. Chinese Veterinary Pharmaceutical Codex Evaluation Committee. 2000. Mu Bie Zi (Semen momordicae). Pages 49–50 in Chinese Veterinary Pharmaceutical Codex Edition 2000. Part II. Chemical Industry Press, Beijing, China. Cox, J. C., and A. R. Coulter. 1997. Adjuvants–A classification and review of their modes of action. Vaccine 15:248–256. Finkelstein, M., K. A. Grasman, D. A. Croll, B. Tershy, and D. R. Smith. 2003. Immune function of cryopreserved avian peripheral white blood cells: Potential biomarkers of contaminant effects in wild birds. Arch. Environ. Contam. Toxicol. 44:502–509. Gao, X. M. 2005. Mu Bie Zi (Semen momordicae). Pages 601–602 in Chinese Materia Medica. China Traditional Chinese Materia Medica Press, Beijing, China. Gao, G. H., Y. Li, C. H. Jin, and H. W. Ge. 2008. Serum analysis after ND vaccination. Chin. J. Anim. Husbandry Vet. Med. 4:31–32. Hu, T. J., Y. Y. Gao, J. R. Chen, and X. H. Shuai. 2008. Enhancement of immune responses to Newcastle disease vaccine by a supplement APS. Guang Xi Vet. Anim. Sci. 24:150–151. Hu, S. L., H. L. Ma, Y. T. Wu, W. B. Liu, X. Q. Wang, Y. L. Liu, and X. F. Liu. 2008. A vaccine candidate of attenuated genotype
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