Analysis On The Effect Of Different Combinations Of PRRS Vaccines To Induce Immune Response In Piglets

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious infectious disease characterized by reproductive disorders of sows and respiratory diseases of growing pigs caused by porcine reproductive and respiratory syndrome virus(PRRSV).In recent years,PRRSV infection is still very common in our country,causing serious economic losses to the pig industry.Vaccination is currently the most effective measure to prevent and control PRRS,which plays a positive role in controlling the occurrence and spread of PRRS.At present,the vaccines used in production practice mainly include commercial attenuated vaccines and inactivated vaccines,but there is no perfect evaluation system for their immune effects,and opinions on the use effect of PRRS vaccines vary among farms(households).Previous studies have confirmed that the combination of PRRS attenuated vaccine and inactivated vaccine can produce better immune protection,but its immune characteristics are still poorly understood.Therefore,the commercial classical PRRS attenuated vaccine(CH-1R strain)and highly pathogenic PRRS inactivated vaccine(NVDC-JXA1 strain)were used to immunize piglets in different combinations.The safety evaluation after vaccine immunization,induced humoral immunity and cellular immune response effects of the vaccines were analyzed and researched in order to lay a certain theoretical foundation and provide a feasible basis for the study of immune characteristics of different combinations of PRRS vaccines,and provide scientific guidance for the effective prevention and control of PRRS.1.Establishment of fluorescence quantitative RT-PCR detection methodAccording to the PRRSV gene sequence in GenBank,a pair of specific primers for N gene amplification was designed and amplified by RT-PCR and ligated into pMD19-T vector to construct a recombinant plasmid containing N gene fragment.Fluorescence quantitative RT-PCR amplification was performed using the SYBR Green I dye method to establish a standard curve and conduct specificity,sensitivity and reproducibility tests.The results showed that the amplification efficiency of the constructed method was 1.063,the Correlation coefficient R~2was 0.999,the slope of the standard curve was-3.18,the primer specificity was strong,and the lowest nucleic acid template was detectable.The concentration was1.0×10~2copies/?L,which was 10 times higher than the sensitivity of conventional PCR,and the coefficient of variation of the repeatability test was less than 1%.It was indicated that the established quantitative RT-PCR method has the advantages of rapidity,specificity,high sensitivity and good reproducibility,and can be used for rapid detection of PRRSV infection(including vaccine poison).2.Safety evaluation of piglets immunized with different combinations of PRRS vaccinesForty piglets with double negative PRRSV antigen and antibody at the age of 7~10 days were screened and divided into 4 groups on average(3 test groups and 1 control group D).The test group(groups A,B,and C)were immunized with the classic PRRS attenuated vaccine(CH-1R strain)for the first time at 14 days of age,Group A and B were immunized with the highly pathogenic PRRS inactivated vaccine(NVDC-JXA1 strain)and the classic PRRS attenuated vaccine(CH-1R strain)at 35 days of age respectively,the test group C was injected with normal saline,and the control group D was injected with normal saline.The clinical manifestations of the piglets immunized by the above different combinations,the average daily weight gain determination,the fluorescence quantitative RT-PCR method to monitor the PRRSV nucleic acid in the serum of the test piglets and the detection of PRRSV nucleic acid in the main tissues and organs,and observe the tissue changes for its safety evaluation.The results showed that there was no significant adverse clinical manifestation of the piglets in the experimental group after inoculation with PRRS vaccine.There was no significant difference in feeding status and mental state,and the body temperature was within the normal range.There was no significant difference in weight gain(P>0.05);the PRRSV nucleic acid duration of the classic PRRS attenuated vaccine for the 14-day-old piglets was 3weeks,and after 35 days of age(21 days after immunization),the PRRSV nucleic acid was not detected in the serum.No PRRSV nucleic acid was detected in the main tissues and organs of the piglets on the 63th day after immunization,and only slight pathological changes existed in some tissues.3.Analysis of the effect of different combinations of PRRS vaccine on humoral immune response in pigletsThe PRRSV N protein antibody and GP protein antibody detection kits were used to monitor the changes of specific antibodies in the serum of piglets at different stages after immunization with different combinations of PRRS vaccines.The results showed that the PRRSV N protein antibody in the experimental group turned positive at the 14th day,and the GP protein antibody began to turn positive on the 21st day.After the immunization,the A and B groups and the test group C and the control D were 35 to 63 days after the immunization.The mean difference of N protein antibody level between the groups was extremely significant(P<0.01),and the difference between group C and group D was extremely significant(P<0.01).At 35 days after immunization,the mean values of antibody GP protein antibody level between group A and group C were significantly different.(P<0.05);42 to 63days after immunization,the difference between group A and group B and group C was extremely significant(P<0.01),and the difference between group A and group B was 56-63days after immunization(P<0.05).4.Analysis of the effect of different combinations of PRRS vaccine on cellular immune response in pigletsThe expression of CD3,CD4 and CD8 in serum and the levels of cytokines IFN-?,TNF-a,IL-2,IL-10 and IL-12 in piglets were monitored dynamically at different stages after immunization with different combinations of PRRS vaccines were determined by ELISA.The results showed that the expression of CD3 and CD4 in the serum of the experimental group showed an increasing trend within 21 days of the first PRRS attenuated vaccine,but there was no significant change in the CD8 molecular expression test group and the control group D.After immunization on the 21st day after immunization,the expression levels of CD3 and CD4 in the serum of the experimental group began to decrease slowly and stabilized.At 35and 42 days after immunization,the expression of CD8 in serum of test group C was significantly higher than that in group A,B and control group D(P<0.05);After the PRRS vaccine was immunized,the changes of IFN-?and TNF-a content were consistent,and both of them increased first and then decreased.In the 14th day after immunization,the experimental group was significantly higher than the control group D(P<0.05).From the 21st to the 42nd day,the experimental group was significantly higher than the control group D(P<0.01);the IL-2 content showed a slowly increasing trend within 28 days after immunization,and the 14th day after immunization,the test group and control group were significant difference in group D(P<0.05);After 28 days of immunization,except group A,the test group B and C showed a slow decline trend,and the test group A was significantly higher than the other groups at 49 days after immunization(P<0.05).The content of IL-10generally showed a trend of slowly decreasing and then rising,but there were no significant difference between the test group and the control group D.The content of IL-12 increased first and then decreased slowly.Among them,within 21 days after immunization,the content of IL-12 in the test group were not significantly different from that in the control group D,but after 35 d and 56 d after immunization,the content of IL-12 in the test group were significantly different from that in the control group D(P<0.01).At 49 days after immunization,the IL-12 content in the test group were significantly different from that in the control group D(P<0.05).In summary,this study successfully established a fluorescent quantitative RT-PCR method for detecting PRRSV;It was confirmed that different combinations of PRRS vaccine had good safety in immunized piglets;The humoral immune response effect of the classical PRRS attenuated vaccine(CH-1R strain)in the first immunization and the highly pathogenic PRRS inactivated vaccine(NVDC-JXA1 strain)in secondary immunization was better than that of the single and two classical PRRS attenuated vaccines,and the PRRS attenuated vaccine(CH-1R strain)can effectively induce the cellular immune response of the body in the first immunization.