| Porcine Rotavirus (PRV) belongs to the family reoviridae. It is one of the major pathogens of viral diarrhea in piglets and causes significant losses in animal husbandry in the worldwide. The young animals are the most easily infected. Its infection is over 80%, and its mortality is 50-100% in 1~10 day piglets. The main symptoms is severe diarrhea , and partial infected piglets were dead due to severe loss of water, imbalance of acid alkali and secondary infection. Now there is no effective medicine to cure RV, thus rotavirus vaccines are considered as the most effective way to prevent disease infection. WHO appealed scientists in countries to develop effective rotavirus vaccine many times, and took it as the one of the most priority developed proceedings.This experiments made a porcine rotavirus oil emulsion inactivated vaccine using formaldehyde solution and BEI respectively and tested the immunogenicity by immunizing rabbits through intramuscular injection.The porcine rotavirus JL94 isolates proliferated in the MA-104 cell and identified by electric mirror, RT-PCR, sequencing and immunofluorescence. It was the typical rotavirus. When the titer of the P58 virus solution reached 108.25TCID50/ml to prepare the basic virus. The virus was checked on bacteria and mycoplasma, and the result was negative. It was expansion of training and prepared the productive virus as the antigen solution which was used to prepare the vaccine.The antigen solution was inactivated by formaldehyde solution and BEI respectively. The oil adjuvant added into the inactivated antigen solution, and then the rabbits were inoculated with the vaccine through intramuscular injection. The rabbits divided into three groups, 2~3 individuals/group, and was named the formaldehyde group, BEI once immunity group and BEI secondary immunity group. The rabbits were immunized with the inactivated vaccine and had no feel bad and death. The injection part have no pathology change, and the rabbit had no badness reaction. It was testified that the vaccine had nicer security. The rabbit sera were collected before immunization and every week after the first immunization. Collected the blood serum and detected the IgG using the neutralization test and ELISA method. The results of ELISA test show that the ELISA antibody level of formaldehyde group had no significantly raise after first immunity. The antibody had apparently raise after 1w when secondary immunity with a peak titer 1:9600, and the level began to go down. Titers of ELISA was maintained 1:1200 in 14w~17w. The ELISA antibody titers of BEI once immunity group and BEI secondary immunity group had apparently raise after immunity with peak titer of 1:9600. The ELISA antibody titers of the once immunity group can maintain 1:1000 and the secondary immunity group was 1:4000 after 21w. The results of the neutralization test show that the neutralizing antibody titers of formaldehyde group had no apparently raise after first immunity. The antibody had apparently raise after secondary immunity with a peak titer 1:969, and the level began to go down. The neutralizing antibody titers was maintained 1:255 after 15w. The neutralizing antibody titers of BEI once immunity group and BEI secondary immunity group was approximately same. The antibody had apparently raise after 3w with a peak titer 1:1800, and the level began to go down after 14w. Titers of IgG antibody of the once immunity group can maintain 1:119 and the secondary immunity group was 1:230 after 21w.The results of the two test methods can testify that contrast the group immunized with formaldehyde-inactived vaccine, the group immunized with BEI-inactived vaccine induced significantly humoral immune responses, and the antibodies maintained a high level for along time. Compared one time immunity with secondary immunity, we can conclude it is just need once immunity. So we can testify that the BEI-inactivated porcine rotavirus vaccine had good immunogenicity and could significantly improve the anti-rotavirus antibodies titers. |
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