| Hydropericardium-hepatitis syndrome is a kind of poultry infectious disease caused by fowl adenovirus serotype-4（FAd V-4）,characterized mainly by pericardial effusion and multifocal liver necrosis with high morbidity and mortality.At present,the main methods to control the disease in the world are dominated by application of inactivated and attenuated vaccines.In recent years,the development of novel anti-FAd V-4 vaccines has become a research hotspot.Lactic acid bacteria（LAB）is recognized as a safety microorganism,and the development of subunit vaccine based on LAB carrier has been paid more and more attention.In the preliminary research work of our laboratory,E.faecalis MDXEF-1 strain derived from chicken was used as the host bacteria,and the Fiber2 protein of FAd V-4was expressed on the surface of the recombinant bacteria,which was testified to induce a strong specific immune responses after oral immunizations.However,how to further improve the growth rate of recombinant E.faecalis and how to avoid degradation in gastric after oral administration are needed to be further explored.In this experiment,the above problems were solved based on preliminary study,which were briefly described as follows:Firstly,phthaloylated inulin nanoparticles（PINs）were prepared by chemical modification,and PINs was characterized by 1H nuclear magnetic resonance spectra（1H-NMR）,dynamic light scattering（DLS）,electrophoretic light scattering（ELS）and scanning electron microscopy（SEM）.The results showed that the partial substitution of PINs-phthalic acid was 37.04%,the average particle size was 338nm,the surface charge was-28.70 m V,and smooth and stable spherical shape of narnoparticles were observed under SEM.Based on the above results,fluorescein isothiocyanate（FITC）labeled PINs（FITC-Pins）were prepared.FITC-PINs were observed to be internalized into the recombinant E.faecalis by FITC and the co-culture of PINS with recombinant E.faecalis was found to promote the proliferation of recombinant E.faecalis,and results of Western blot confirmed that the co-culture of PINS with recombinant E.faecalis could significantly promote expression of Fiber2 protein in recombinant E.faecalis.In order to determine the survival and release characteristics of recombinant E.faecalis loaded with inulin nanoparticles in simulated stomach and intestinal fluid,digestion experiments in simulated gastric and intestinal fluid were carried out in vitro.The results showed that the encapsulation rate of sodium alginate in each group was above 97%.The free bacteria could not be detected after exposure to simulated gastric fluid for 2 h,but the number of viable bacteria encapsulated by sodium alginate was all above 80%.The viable rate of recombinant E.faecalis microencapsulated and loaded with nanoparticles after exposure to simulated gastric juice was significantly higher than that in other groups（p<0.05）.There was no significant difference（p>0.05）in the release rate of each group in simulated intestinal fluid after encapsulation by sodium alginate,and all completed the release within 30 min.In order to evaluate the immune responses induced by microencapsulated recombinant E.faecalis loaded with inulin nanoparticles after oral immunization of chickens,200 chickeds with 3-day-old were randomly divided into 7 groups,including positive infection control group,negative control group and blank control group with sodium alginate microcapsule.Oral immunization was performed for three consecutive days at 7-9 days,21-23 days and 35-37 days,and once a day.The jejunum lavage fluid and serum were prepared in each group at the ages of 7,21,35 and 49 days,and the levels of serum Ig G antibodies and s Ig A antibodies in the jejunum lavage fluid were detected by indirect ELISA.On 12 th day after the third immunization,the serum of chickens was collected from each group and the level of neutralizing antibody was calculated by detecting cytopathic effect（CPE）of chicken liver cancer cells（LMH）in vitro.Lymphocyte suspension was prepared from peripheral blood of chickens in each group,and the proliferation of peripheral blood lymphocytes（PBL）was detected by CCK8 kit.Spleens of chickens in each group were sampled,and the m RNA expression levels of IL-2,IL-4,IL-6,IL-10 and IFN-γin spleen tissues were detected by q RT-PCR.The results showed that E.faecalis without encapsulation,Micro-E/Fiber2 encapsulated by sodium alginate,Micro-E/Fiber2 encapsulated by sodium alginate and inulin culture（Micro-E/Fiber2-inulin）and Micro-E/Fiber2-PINs encapsulated by sodium alginate loaded with inulin nanoparticles,could all induce chickens to produce specific Ig G and s Ig A against Fiber2 protein,and the antibody level in Micro-E/Fiber2-PINs group was significantly higher than that in other groups（p<0.05）.After three times of immunization,the proliferation ability of peripheral blood lymphocytes and the relative expression of IL-2,IL-4,IL-6,IL-10 and IFN-γin spleen tissues of chickens immunized with recombinant E.faecalis protein were significantly higher than those in PBS group and blank microcapsule group,and the increase of the above immune responses in Micro-E/Fiber2-PINs group was significantly different from other groups（p<0.05）.To evaluate the immune protective effects,chickens in each group except Negative control group were infected with FAd V-4/GS01 through thoracic injestion at the dose of 105.2 ELD50 per chicken at12 days after the tertiary immunization.The immune protection was evaluated by detecting the index changes of enzymes related to liver function,viral load in liver,pathological changes of liver,survival rate,weight gain rate and lesion score of clinical symptom of chickens in each group.The results showed that compared with the positive control group,the activities of aspartate aminotransferase（AST）,alanine aminotransferase（ALT）and lactate dehydrogenase（LDH）in serum and the viral load in liver of chickens in Micro-E/Fiber2-PINs group were significantly lower than those in other groups（p<0.05）.The levels of albumin（ALB）and total protein（TP）were significantly higher than those in other groups（p<0.05）.The pathological changes in heart,liver,spleen and kidney of chickens in Micro-E/Fiber2-PINs group were less severe than those in infected control group and other three immunized groups,the survival rate of chickens in Micro-E/Fiber2-PINs group was 100%,and the body weight gain and pathological score were significantly different from those in other groups.Taken together,phthaloylated inulin nanoparticles can promote the proliferation of recombinant E.faecalis,and the recombinant bacteria loaded with inulin nanoparticles and encapsulated by sodium alginate microcapsules can tolerate gastric acid environment,thus induce efficient immune responses and provide immune protection after intestinal release. |
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