| Newcastle disease(ND)is an acute,highly contagious fatal disease in poultry caused by the Newcastle disease virus,it is one of the main infectious diseases endangering poultry breeding.Currently,the most effective method for the prevention and control of this disease was the vaccination ND vaccine.However,the selection of vaccine types,inoculation methods,and inoculation time could affect the immune effect of the vaccine.Chickens were susceptible to bacterial infection after hatching because of their low immune function,which was a critical period for chicks to survive.Therefore,it is crucial to improve the early immune protection of hatched chicks.Studies have shown that an embryo immunization vaccine can establish immune protection against some viruses in advance,such as embryonic immunity to Marek's disease can establish good immune protection,but the effect of the ND vaccine on embryo immunity is not ideal.Therefore,it is of great significance to explore the use of immune adjuvants to enhance the immune effect of the ND vaccine embryos and improve the early immune protection of chicks.Considering that Astragalus polysaccharide(APS)can promote growth and development and enhance immunity,It is an ideal natural immune adjuvant,in this study,the combination of APS and VG/GA ND vaccine was used to immunize embryos.Compared with the ND vaccine alone,it not only promoted the early growth and development of chicks,but also enhanced the immune effect of the ND vaccine,established immune protection and provided a new immune method for the effective prevention and control of ND.At the age of 18.5 days,SPF eggs were injected into the amniotic cavity with normal saline(Control group),ND vaccine(NDV group),Astragalus polysaccharide injection(APS group),and APS and ND vaccine mixture(APS+NDV group).The effects of this method on the growth,development and immune function of chickens were investigated at 3,7,14,21 and 28 days of age,and the mechanism of action was preliminarily investigated.NDV challenge protection test was carried out at 14 days of age to explore the immune protective effect of NDV on chickens.The experiments were performed as follows:(1)Effects of embryo immunization with APS combined with ND vaccine on embryo egg hatching rate and chick growth and development.The chickens that immunization with APS combined with ND vaccine exhibited significantly enhanced hatching rates and caused no adverse effects on chick survival rate.The body weight and the weight gain of chicks in the APS+NDV group each week were significantly higher than chickens of the NDV group and control group.The feed conversion rate(FCR)of the APS+NDV group was significantly lower than the NDV group,and the Control group.The morphological and structural development of duodenum,jejunum,ileum in APS+NDV group were shown a significant enhancement.The villus height and crypt depth of duodenum,jejunum,ileum in the APS+NDV group were increased.However,there was no significant effect on the ratio of villus height to crypt depth.The development of spleen,thymus,bursa of Fabricius,and an organ indexes of chicks in the APS+NDV group were also shown a significant enhancement compared with the NDV group.The above results indicate that APS combined with ND vaccine for embryo immunization could promote the growth and development of chickens.(2)Immune function in blood and spleen of chickens treated with APS and ND vaccine through embryo delivery.The blood clotting inhibition(HI)analysis showed that the log2 of antibody titers in chicks of the APS+NDV group at 14 and 28 days of age were 6.50 and 6.10 respectively,whereas the titers of the NDV group were 6.10 and 6.00 respectively at 21 and 28 days of age,the immune protection was induced one week in advance,and a high antibody level was maintained for a longer period.ELISA analysis revealed that the levels of IFN-?,IL-2,IL-4,IL-6 in serum of the APS+NDV group chickens were significantly higher than the NDV group.The levels of IFN-?and IL-2 in the APS+NDV group show the first significant difference at the age of 3 days compared to the NDV group,and the levels of IL-4 and IL-6 showed the significant differences since day 7 day of age.Flow cytometry analyzed the T lymphocytes subgroup differentiation of the peripheral blood CD4~+and CD8~+in the immune chicks.At the age of 14,21 days,the results showed that the percentage of CD3~+CD4~+T cells in chicks of APS+NDV group were significantly higher than NDV group,about by 12%and 9.3%respectively,and it also showed that the percentage of CD3~+CD8~+T cell in chicks of APS+NDV group were greater than NDV,about by 10.6%and 6%.The proliferation of lymphocytes in the spleen was detected by a lymphocyte proliferation test.After stimulated with Con A and LPS,the stimulation index(SI)value of T lymphocytes and B lymphocytes in the APS+NDV group increased with the ages,which is higher than NDV group.The mRNA expression of IFN-?,IL-2,IL-4 and IL-6 in spleen was detected by Real-time Quantitation PCR,the mRNA expression of IFN-?,IL-2,IL-4 and IL-6 in APS+NDV group was all higher than the NDV group.The above results indicate that the immunization of APS combined with the ND vaccine could improve the levels of cellular immunity and humoral immunity.(3)Effects of embryo immunization with the APS combined with ND vaccine on intestinal immune functions of chicks and the TLR4 signal transduction pathwayThe mRNA expression of IFN-?,IL-2,IL-4 and IL-6 genes in duodenum and jejunum were detected by RT-PCR,compared with NDV group,the mRNA expression of IFN-?,IL-2,IL-4and IL-6 genes in duodenum and jejunum of chickens in the APS+NDV group were significantly increased.ELISA detection of the sIgA antibody levels in duodenal and jejunal and ileum lotions showed that s IgA value of chicks in the APS+NDV group was significantly higher than the NDV group.The s IgA value in duodenal lotions of chicks in the APS+NDV group was achieved the highest level at 14 days of age,the value of s IgA value in jejunum increased with ages and in ileum achieved the highest level at 21 days of age.Using immunohistochemical to examine the distribution of IgA~+cells,and revealed that IgA~+cell numbers in the duodenum,jejunum and ileum in chicks of the APS+NDV group at the ages of 7,14,21,28 days were significantly higher than NDV groups,expressed the most in the duodenum and least at ileum.At the age of day 3,the lg A~+cells were only detected in the duodenum.Meanwhile,a large number of IgA~+cells were found distributed surrounding the area of the lamina propria of the intestinal villi basal,and with the increase of age,it was detected at the tip and the upper part of the lamina propria of the villi.IgA~+cells in the chicks of the NDV and Control group were mainly distributed in the lamina propria of the intestinal villi basal.Real-time Quantitation PCR was applied for the detection of the mRNA expression of the intestinal TLR4,My D88,TRAF-6,NF-?B,TRIF,IRF3,IFN-?genes in chicks.Preliminary findings showed that the expression of TLR4,My D88 mRNA,TRAF-6,NF-?B,TRIF,IRF3,IFN-?mRNA expression in the APS+NDV group were all upregulated to different extents,suggesting that APS combined with NDV vaccine could facilitate trigger both the My D88-dependent and TRIF-dependent signaling pathways by the TLR4 receptor,meanwhile maintained the stable state of intestinal immune function.The above results indicate that the immunization of APS combined with ND vaccine could enhance the intestinal immune functions,up-regulate the TLR4 receptor,and activated the My D88-dependent and TRIF-dependent signaling pathways.(4)Immunological protective effect of APS combined with ND vaccine on chicks after embryo immunization.The immune protection rates of the APS+NDV group(98%)was higher than NDV group(85%).The value of EID50 of the APS+NDV group(1.56)was lower than the NDV group(3.36).The HI antibody titers of the APS+NDV group(7.80)was higher than the NDV group(5.80).The result of the loading of virus and the histopathology of the APS+NDV group was better than the NDV group.The results of this study showed that the immunization of APS combined with the ND vaccine could enhance the immune effect better.Based on the results of this study,APS combined with ND vaccine for embryo immunization promoted the growth and development and immune function,Immune protection was established in advance,provided a new idea for the use of traditional Chinese medicine adjuvant to enhance the immune effect of vaccine and prevent ND. |
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