| Avian leukosis is caused by the viral infections of avian leukosis virus(ALV) and avian sarcoma virus(ASV). The disease has occurred in laying hens, broiler chickens and our local strains, resulting in lower flock performance, harm the immune suppression, cancer death, etc.The infection of ALV causes poor performance of infected chickens and immune suppression.And thus, the chickens are more susceptible to other viruses and bacteria, causing significant economic losses poultry industry. There is no effective drugs and vaccines for the prevention of ALV, except for population purification, but in the past few decades, due to failure to take effective measures to purify, the prevalence and incidence of leukemia has been very extensive in China. This situation cause of the purification process is more time-consuming.Therefore, it is an urgent need to explore new ways to control the spread of ALV, reduce subclinical infection caused by ALV infection, and reduce the harm of the disease. As a new additive, variety of plant polysaccharides have been demonstrated antiviral, anti-tumor, antioxidation, anti-radiation, and other biological effects. Our previous studies have found Taishan pine pollen polysaccharide(TPPPS) could significantly improve the effects of different vaccines, such as Proteus mirabilis, the rabbit hemorrhagic disease, and the recombinant Bordetella avium omp A subunit vaccines. However, until now the composition of TPPPS has not been determined, and the biological activity of each composition is also unknown.Based on the above considerations, we analyzed the TPPPS composition, physicochemical properties, and biological activity of each identified component.And the characteristics of inhibition to ALV-B proliferation are further analyzed. Finally, through the establishment of chicken immunosuppression model caused by infection of ALV-B, we explores TPPPS’conditioning effectes for ALV-B-induced immune suppression and immune restoration effect.The research lay the foundation for exploring and developing new ways of prevention and control of avian leukemia. This study was divided into the following three parts:1. Physical and chemical properties and biological activity of TPPPS1.1 Column chromatographic purification of TPPPS and the molecular weight and monosaccharide composition of various componentsTo further study of the mechanism of the function and play of TPPPS, crude polysaccharide was further purified by DEAE-cellulose column and Sephadex G-200 gel column in this study. The results showed that the crude polysaccharides including three components, named: TPPPS1, TPPPS2 and TPPPS3, and isolated a homogeneous polysaccharide of these three components.Molecular weight and monosaccharide composition of the three polysaccharides were detected by high performance gel permeation chromatography(HPGPC) and high performance liquid chromatography(HPLC). The results show that the molecular weight of TPPPS1, TPPPS2 and TPPPS3 respectively is 56,25 and 128 k Da. TPPPS was composed by mannose, ribose, xylose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose,the kind and proportion of each component monosaccharide between TPPPS1-3 were significantly different. These results suggest that the three components may have different biological properties.1.2 Detection of inoxidizability, immune-enhancing activity, anti-viral activity in vitroTPPPS molecular weight and monosaccharide composition of each component were significantly different, indicating that there may be significant differences in the biological activity of TPPPS1-3. In order to further understand the biological activity of TPPPS, the biological activity of TPPPS1, TPPPS2 and TPPPS3 were detected in vitro. The results show that TPPPS2 and TPPPS3 significantly promote the proliferation of spleen lymphocytes, and the effect of TPPPS3 stronger than TPPPS2; TPPPS3 significantly enhanced the concentration of cytokine interleukin-2(IL-2) and tumor necrosis factor(TNF), and TPPPS2 only can significantly improve the concentration of IL-2. TPPPS1 induced significantly NO production, indicating that TPPPS1 has the most significant antioxidant activity. 50 μg/m L TPPPS3 has the most significant suppression effect on proliferation of ALV-B, and the role of period is in the adsorption process of viruses. This study establishes foundation for the using of TPPPS1-3 reasonable proportion to promote effective immunomodulators and the analysis of in vitro antiviral mechanism.2. Antiviral activity mechanism analysis of TPPPS in vitro2.1 The establishment of receptor-virus ELISA methodThe mechanism of TPPPS suppression effect was researched on the process of virus adsorption. In this study, according two proteins, virus glycoprotein gp85 and host receptor protein tv-b, playing an important role in the ALV-B adsorption process, we established two ELISA methods. Gp85 and tv-b protein was prepared by prokaryotic expression method, and both polyclonal antibodies of two proteins were also prepared. Two receptors-virus ELISA method was the establishment with these two proteins and antibodies(Method I: gp85—tvb—tv-b antibody—HRP-secondary antibody; Method II: tv-b—gp85—gp85 antibody—HRPsecondary antibody). The each conditions of ELISA method was determined with square test.The results show that the optimal concentration of gp85, tv-b, tv-b antibody, and the negative and positive of the critical values in the process I respectively ??are 16 μg/m L, 1:80, 32μg/m L, 0.178, the optimal concentrationin in process II are 32 μg/m L, 1:80, 32 μg/m L, 0.196.The blocking solution and TMB chromogenic time is respectively 5% skim milk and 15 min.This test successfully established two ELISA methods for the analysis of antiviral mechanism of TPPPS in vitro.2.2 Analysis of the antiviral mechanism of TPPPS in vitroUsing the established two receptors-virus ELISA method, the gp85 protein(or tv-b protein) coated with 200 μL different concentrations(1, 10, 100, 1000, 104, 105μg/m L) in ELISA plate, and then observed the results of changes in subsequent trials. The results show that TPPPS can significantly reduce the P/N value in the ELISA method I, and the process II is not significant change, suggesting that TPPPS3 can bind virus gp85 protein and unable to bind tv-b receptor protein. The binding interfere the binding between virus and tv-b receptor protein, which is one of the mechanisms TPPPS inhibits virus amplification. This study lay the foundation for further understand the role of TPPPS in ALV-B adsorption process and the inhibition mechanism of TPPPS to ALV-B in vivo.3. Immunomodulation of TPPPS on immune suppression chicken infected ALV-B3.1 Build immune suppression modelsThe infection of ALV can cause 1-2% mortality in chickens(occasionally up to 20% or more), and most chickens’ manifestation is primarily decreased performance and immunosuppression. In this study, 50-day-old chickens infected with ALV-B, and changes of infected chickens in various physiological indices were respectively monitored in 1-9 weeks after infection. The test indicators include: morbidity and mortality, body weight and immune organ index, anti-ALV-B-specific antibody levels, IFN-γ and IL-2 levels, T lymphocyte proliferation and CD4+ and CD8+ lymphocyte subsets. The detection results of the above indicators showed that chickens are not found to be died, but its growth condition and immune organ was significantly worse than normal chickens, and immune response,lymphocyte activity, CD4+ and CD4+ / CD8+ was significantly decline. The results showed that the immune system of infected chickens is significantly suppressed. The immunosuppression model provides the basis for analysis changes in immune response of chickens infected ALV-B.3.2 Immunomodulatory of TPPPS on immunosuppressive model chicksIt is reported that TPPPS has immune recovery effect to immune suppression mice caused by cyclophosphamide. In this study, TPPPS was subcutaneous injected in immunosuppressive model chicks to analyze immunomodulatory of polysaccharides on chickens. Newcastle disease virus(NDV) vaccine was immunized in one week old chicks.The immune parameters were detected for analyze immune recovery role of TPPPS on model chickens infected with ALV-B. The results showed that in TPPPS group chicks, body weight and immune organ index, peripheral blood lymphocyte proliferation, CD4 lymphocyte subsets and CD4+/CD8+ ratio, IL-2 and IFN-γ, and ALV-B antibody positive rate is increased significantly, and effect of the dose of 400 mg/kg(per kilogram of chicken) TPPPS is the most significant. In addition, the anti-NDV antibody titers of 400 mg/kg TPPPS group were significantly higher than other experimental groups. These results indicate that TPPPS can be used as an immune enhancer and play role in reducing the immune suppression caused by ALV-B. This research provides a new ideas and approaches for the prevention and control of avian leukemia. |
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