Adjuvant Effects Of Momordica Cochinchinensis Seed Extract On The Immune Responses To Vaccination Against Foot-and-Mouth Disease, Avian Influenza And Infectious Bursal Disease

Vaccination is one of the most important and cost-effective methods to prevent the infectious diseases in animals. Potent and efficient vaccines against infectious diseases are always in great demand. Adjuvants have a crucial contribution in the improvement and development of vaccine immunogenicity. Many substances have been found to have adjuvant activity but only few are permitted. Among those, a large numbers of adjuvants have the strong immunological effects however toxicity is the major obstacle in the use of those adjuvants. To explore new potent adjuvants with minimal toxicity, extract of cochinchina momordica seed (ECMS) was evaluated for its adjuvant effects on humoral and cellular immune responses. The seed of cochinchina momordica (Latin Momordica cochinchinensis) is a traditional Chinese medicine and has been used for more than one thousand year. According to our knowledge, medicinal and therapeutic use of the seed is well documented however; few reports have been found on its adjuvant properties. In present study, adjuvant potentials of ECMS have been explored. Crude extraction of the seeds was obtained by ethanol extraction method and endotoxin level was measured by a gel clot Limulus amebocyte lysate assay. The endotoxin level was less than 0.5 EU (endotoxin units)/ml which excluded the influence caused by endotoxin in the experiments. ECMS in saline was prepared for the study on laboratory animals, ruminants and poultry. Mice, cattle and chickens were used to evaluate the adjuvant effect of ECMS on the immune responses to the vaccination against foot-and-mouth disease (FMD) avian influenza (AI) and infectious bursal disease (IBD).First experiment in mice was conducted for the evaluation of adjuvant effect of ECMS. Twenty eight ICR mice were distributed in 4 groups and immunized with FMDV (Asia-1) antigen alone (n=9) serving as control or emulsified with 50 (n=5),200 (n=9) or 800 (n=5)μg/dose of ECMS on days 1 and 21. Blood samples were collected on day 15, 30 and 45 after boosting. Levels of serum immunoglobulin (Ig) G and subclasses were measured by Enzyme-linked immunoabsorbent assay (ELISA). The total IgG level was significantly increased (P< 0.05 or P< 0.01) in 200μg/dose of ECMS group during whole period of the study when compared with control. On days 30 and 45 after boosting, significant increase(P< 0.05) of total IgG was recorded in group received 800μg/dose of ECMS as compared to control. Numerical but insignificant increase of total IgG in 50μg/dose of ECMS group was observed throughout the study period when compared with control. Significant enhancement (P< 0.05) in serum IgGl on day 15 and 30 was observed in ECMS immunized group (800μg/dose) as compared to control. No significant difference in serum IgGl was found in 50 and 200μg/dose of ECMS groups when compared with control. Significant enhancement (P< 0.05 or P< 0.01) in serum IgG2a antibody was recorded in 800 (day 15 and 30) and 50μg/dose of ECMS group (day 30 and 45) as compared to control. No significant change in IgG2a antibody level was recorded in group received 200μg/dose of ECMS. The mean IgG2b antibody level on day 15 was significantly higher in 800μg/dose ECMS group (P< 0.05 or P< 0.01) when compared with all immunized groups while antibody level in group received 200μg/dose of ECMS was also significantly increased (P< 0.05) as compared to control on same day. On day 30, significant enhanced IgG2b level was recorded in group received 800μg/dose of ECMS (P< 0.05). No significant differences of IgG2b level were observed among all immunized groups on day 45. Significant enhancement during whole period of the study in serum IgG3 level was recorded in group immunized with 800 jig/dose of ECMS as compared to control (P< 0.05 or P< 0.01). Level of IgG3 in 800μg/dose of ECMS group was also significantly higher (P< 0.05) than other ECMS groups on day 15. Significant increase (P < 0.05) in IgG3 antibody levels was also observed in groups 50μg/dose (on day 15 and 45) and 200μg/dose of ECMS (on day 15) as compared to control. Three weeks after boosting, four mice in each of control and 200μg/dose of ECMS groups were sacrificed and splenocytes proliferation assay was conducted for the detection of cellular immune responses. Antigen stimulated splenocyte proliferation in the mice immunized with FMDV (Asia-1) antigen emulsified in ECMS was significantly higher (P< 0.01) than that of the antigen control group.Second experiment was conducted for the evaluation of synergistic effect of ECMS and oil in mice. Twenty-three ICR mice were distributed in 3 groups and immunized with FMDV (Asia-1) antigen+oil (n=9) serving as control or emulsified in 200 (n=9) or 800 (n=5)μg/dose of ECMS on days 1 and 21. Blood samples were collected on day 15,30 and 45 after boosting. Three weeks after booster immunization, four mice in each of control and 200μg/dose of ECMS groups were sacrificed and splenocytes proliferation assay was performed for the detection of cellular immune responses. Significant increase (P< 0.05) in total IgG level in 800μg/dose ECMS-oil group was observed on day 15 and 30. All ECMS groups showed slightly higher level of IgGl antibody but no significant differences were recorded during whole period of the study. While significant enhancement (P< 0.05) in serum IgG3 antibody level was recorded in 200μg/dose of ECMS group when compared with control on day 15. Numerical but not significant increase in cellular immune responses was observed in the group received 200μg/dose of ECMS emulsified in oil and Asia-1 antigen as compared to control.ECMS was studied for its effect on the immune responses against FMDV (Asia-1 and O) in heifers, calves and lactating cows. Eighteen heifers were distributed into three groups (n=6) and immunized with commercially available bivalent FMDV (Asia-1 and O) vaccine alone or combined with 0.5 or 2 mg/dose of ECMS. Humoral immune responses were evaluated by indirect haemagglutination antibody (IHA) test, sandwich ELISA and VP-1 assay. Significantly higher antibody titers (P＜0.05) were found in 2 mg ECMS group on day 60 and 90 than that of the control. The anti-VP1 antibody in 0.5 mg/dose of ECMS group was significantly higher (P＜0.05) at day 30 while numerical but insignificant increase was recorded in 2 mg/dose of ECMS group on same day than the control. On day 60 and 90, the anti-VP1 antibodies in 2 mg/dose of ECMS group were significantly higher (P＜0.05), while numerical but insignificant increase was recorded in 0.5 mg/dose of ECMS group on same days when compared with control. The IgG levels against FMDV (Asia-1) detected by ELISA were significantly higher (P＜0.05) in the animals received vaccine containing 2 mg/dose of ECMS in its formulation on day 30 and 60. The levels of total IgG against FMDV (type-O) were significantly higher (P<0.05) in 2 mg/dose of ECMS group on day 60 and 90.The adjuvant effect of ECMS was also studied in calves when twelve calves (n=6) were immunized with vaccine alone or containing 2mg/dose of ECMS. Humoral immune responses were estimated on day 0,30,60, and 90 by IHA and ELISA, while the cellular immune responses were detected using FMDV (Asia-1, type-O) antigen and mitogen (Con-A, PHA) by lymphocytes proliferation assay. The IHA titers of all ECMS groups were significantly higher (P＜0.05; P＜0.01) when compared with the control during whole period of the study. The IgG antibody levels against both serotype of FMDV were significantly higher (P＜0.05) in the animals received vaccine containing ECMS in its formulation on day 60 and 90. Significant differences (P＜0.01) among ECMS and control groups were also detected in the cell-mediated immune responses when lymphocytes were stimulated with antigen or mitogen.The adjuvant effect of ECMS was also studied in 14 lactating cows (n=7) immunized with FMDV (Asia-1 and O) vaccine alone or containing 2mg/dose of ECMS. To compare the humoral immune responses of both groups, total IgG level in serum against both serotypes was detected by sandwich ELISA on day 0,30,60 and 90. The IgG antibody level against FMDV serotype Asia-1 was significantly higher (P＜0.05; P＜0.01) in the animals received vaccine containing ECMS in its formulation as compared to control on day 60 and 90. While significant enhancement of total IgG level (P＜0.05) against FMDV serotype-O was recorded in animals belonging to ECMS group when compared with vaccine alone, during whole period of the study.The study also evaluated the immunological effect of ECMS on the immune response to FMDV in milking cows immunized with different doses of bivalent vaccine at acupuncture site. In first experiment,21 lactating cows were distributed equally into three groups (n=7) and immunized with 2 ml FMDV (Asia-1 and O) vaccine alone (in neck) or combined with 2 mg/dose of ECMS (in neck or acupoint-Hou hai). Antibody titers in serum against FMDV (type-O) were measured by IHA on day 0,30,60,90 or 120. Results revealed the significant enhancement (P<0.05; P<0.01) of antibody titers in serum of both ECMS groups (neck or acupoint) at different time points as. compared to vaccine (alone) group. Antibody titer in the serum of ECMS group immunized at acupoint was insignificantly higher during whole period of the study except on day 120 when significant difference (P<0.05) was recorded as compared with ECMS group immunized at conventional site (neck). Cell mediated immune response was also evaluated by stimulating the peripheral blood lymphocytes with FMDV Asial and O type antigen. Proliferative response of lymphocytes to antigen in both ECMS groups was significantly higher (P<0.05; P＜0.01) than the vaccine group. Numerical but insignificant proliferative response was recorded in ECMS group immunized at acupoint as compared to the ECMS group injected at neck. In second experiment, two groups of twelve milking cows (n=6) were immunized for 90 days at acupuncture site (Hou hai) with half dose (1 ml) of FMDV bivalent (Asia-1 & O) vaccine alone or combined with 2 mg/dose of ECMS. Serum IgG level against FMDV (Asia-1) in ECMS group was insignificantly higher whole period of the study but significant difference (P<0.05) was recorded on day 60 as compared to the group received vaccine alone. The level of total IgG against FMDV (type-O) in ECMS group was also insignificantly higher whole period of the study but significant differences (P<0.05 or P<0.01) were observed on day 60 and 90 when compared with control. Antibody titer measured by IHA in ECMS group was also significantly higher (P＜0.05) than control on day 30.Adjuvant effect of ECMS on immune responses was evaluated in chickens immunized against avian influenza (AI). In first experiment, total sixty,2 weeks old chickens (n=10) were immunized with influenza vaccine (H5N1) alone or combined with ECMS (5,10,20,40 and 80μg/dose) and serum samples were collected on day 0,7,14 and 28. ELISA results revealed that the antibody levels against AI in all ECMS groups were insignificantly higher throughout study while significant enhancement (P<0.05) was recorded in 10 and 20μg/dose of ECMS groups on day 28, as compared with control. Average daily weight gain in 20μg/dose ECMS group was significantly higher (P＜0.05) than control reflecting no adverse effect of ECMS on growth performance. Adjuvant effect was also confirmed in second experiment when HI results showed the significant enhancement (P＜0.05) of antibody titer at certain time points studied in fifty chickens (n=25) immunized with 20μg/dose of ECMS mixed with AI vaccine of Vaccine alone.Adjuvant effect of ECMS on humoral and cellular immune responses was evaluated in chickens immunized against infectious bursal disease virus (IBDV). Fifty two chickens (n=13) were immunized with commercially available oil adjuvanted IBDV vaccine alone or emulsified with ECMS (20,40 and 80μg/dose). ELISA results showed that the antibody levels against IBDV in all ECMS groups were insignificantly higher throughout the study period except 20μg/dose of ECMS group in which significant enhancement (P< 0.05) of total IgG level was recorded on day 14,21,28 and 35 as compared to control. Significant increase (P< 0.05; P< 0.01) of mitogenic stimulated lymphocytes proliferation was also observed in ECMS groups when compared with control. To study any adverse effect of ECMS on growth performance, average weight gain was also compared. No adverse effect of ECMS on growth performance was observed even ECMS groups showed significantly higher (P< 0.05; P< 0.01) growth performance at certain time points and doses as compared to control.Taken together our data suggest that ECMS has the potential adjuvant effects to enhance the immune responses to foot-and-mouth disease in mice, and cattle and to avian influenza as well as to infectious bursal disease in chickens. Hence ECMS deserve for further studies as an adjuvant.