The Labrorary Research Of Triple Inactivated Vaccine Against Porcine Circovirus Disease,Haemophilus Parasuis Disease And Streptococcosis Suis Disease

Porcine Circovirus Disease（PCVD）is causing by Porcine circovirus type 2（PCV2）,which is usually infected to various stages of pigs,Haemophilus parasuis disease and Streptococcosis suis disease are the frequent disease in piglet.The co-infection of PCV2,HPS and SS2 in large-scale pig farms which has caused severe economic damage to the swine industry of China.The effective approach to prevent these diseases still depends on vaccination.It can reduce costs and bring down the stress in pigs if an effective triple inactivated vaccine is avaliable for PCV2,Haemophilus parasuis and Streptococcus suis.This study which is based on the prepared triple inactivated vaccine of three kinds of pathogens in our lab and according to the requirements of the vaccine in lab research conducted immune efficacy trial and antibody dynamic test to evaluate the immune effects of this vaccine.The main contents are as followings.1.The verification of seed virus and seed strains,and the preparation of triple inactivated vaccinePCV2 WH strain（PCV2-WH）,Haemophilus parasuis serotype 5 SH0165 strain（HPS5-SH）,serotype 4 MD0322 strain（HPS4-MD）and Streptococcus suis serotype 2 LT strain（SS2-LT）stored in our lab were validated by PCR test.After that,these four cultured pathogens were mixed with aluminum hydroxide adjuvant in a certain proportion,and the number of three batches of triple inactivated vaccines we prepared were 1501,1502 and 1503,respectively.Wherein PCV2,HPS4,HPS5,SS2 content were4×10⁶TCID₅₀/m L,2×10⁹CFU/m L,2×10⁹CFU/m L,1.5×10⁹CFU/m L for each batch.2.The immune efficacy trial of the triple inactivated vaccine for Balb/c miceThe three batches of triple inactivated vaccine named 1501,1502 and 1503seperately immunized 24 healthy female Balb/c mice with 0.2 m L per mouse and boosted immunization again 14 d later.ELISA were performed to detected blood antibody levels0、14 and 28 d later after first immunization.Four weeks after immunization,mice challenged with the 5×LD50 of HPS4,HPS5 and SS2 respectively,then we performed pathological observation of the lung.The data illustrated that 28 days after immunization,mice immunized with these batches were able to produce high levels of antibodies detected by ELISA.Wherein,after the challenge the protective efficacy rate of 1501batch were 87.5%,87.5%and 87.5%,respectively,1502 batch were 87.5%,75%and87.5%,respectively,and 1503 batch were 100%,87.5%and 87.5%,respectively.The three batches of vaccine immunized mice only had mild lung congestion,but mice in non immune group were evident in alveolar septum broadeningand intra alveolar exudate and neutrophils increasing.3.The immune efficacy trial of the triple inactivated vaccine for pigletsThirty-five piglets aged 21-24 days were randomly divided into seven groups,including:group 1-3 immunized with triple inactivated vaccine,group 4 challenged with HPS4-MD as control,group 5 challenged with HPS5-SH as control,group 6 challenged with SS2-LT as control,and group 7 as negative control.Each piglet inoculated with 1503 batch vaccine 2 m L and boosted immunization again 14d later,injected other groups of piglets with a same amount of PBS.0,21 and 35 days after immunization,antibodies in blood were detected by ELISA.5 weeks after immunization,challenging group 1 and 4,group 2and 5,group 3 and 6 were challenged with HPS4-MD(1.75×10¹⁰CFU),HPS5-SH(1.2×10¹⁰CFU)and SS2-LT（2×10⁶CFU）,seperately.The clinical symptoms were observed for 14 days after challenging,the incidence and mortality of the disease were recorded,and we performed pathological observation of lung.The information demonstrated that the protective efficiency of group 1,group 2 and group 4 are 80%,60%and 80%,respectively,the morbidity or mortality of group 3,group 5 and group 6 are60%,100%and 100%,respectively.21 days after immunization,all the immuned piglets produced antibodies of PCV2,HPS4,HPS5 and SS2 which were detected by ELISA.Meanwhile,the clinical symptoms and pathological changes of immunized challenge groups were milder than non-immunized challenge groups.4.The antibody dynamic test of the triple inactivated vaccine for pigletsOne hundred and fifty piglets aged 14days were randomly categorized into 3 groups and immunized with three batches of the triple inactivated vaccine seperately.Each piglet inoculated with 2m L vaccine and boosted immunization again 21 days later.Then 7,14,28,42,63 and 91 days after immunization,antibodies in blood which were detected by ELISA.The results showed that PCV2 antibody titers increased significantly on 28th day,then peaked on 42th.Antibodies of HPS4,HPS5 and SS2 were positive on both 28th and91th,while the antibodies for these antigens on 91th day were higher than 28th day.Overall,antibody levels of PCV2,HPS4,HPS5 and SS2 reached their peaks on 28-63,42-63,42-91 and 42-63 seperately.