| Classical swine fever(CSF)is an acute,febrile and septic infectious disease caused by classical swine fever virus.CSF is one of the category I animal epidemic diseases and an OIE notifiable epidemic diseases,which largely affects the sustainable development of swine industry.Currently,much progress has been made for the control of CSFV by the application of CSFV vaccine.However,the occurrence and prevalence of CSFV are still often found in the immunized pig herds,which are likely related to many reasons including unreasonable immunization procedures,poor vaccine quality,and especially the lack of monitoring and tracking of the immune effect of the immunized pigs.Therefore,surveying and monitoring the antibody level of swine fever in immuned herds,determining the immune efficacy of swine fever vaccine and the infection status of swine fever virus,and monitoring the genetic variation trend of epidemic CSFV strains will provide an effective theoretical basis for the effective prevention and control of the occurrence and epidemic of swine fever.In this study,we employed the immunology,molecular biology,virology and other approaches to study the antibody level,infection status of CSFV and genetic variation of CSFV in pig herds immunized with CSFV vaccine in given regions of China from 2015 to 2017,and monitors and analyzes the immune effect of SFV gene engineering subunit vaccine.Additionally,the emerging infection of atypical pestivirus in piglets recently in China is preliminarily studied,and the main results were as follows:1.Forty-eight thousand seven hundred and thirty-one serum samples were collected from 2009 pig farms immunized with CSF vaccine of 26 provincial,municipal and autonomous regional and tested by ELISA.The results showed that during 2015-2017,the average positive rate of swine fever antibody was68.30%~82.94% depending on the regions and the growth stages.,.The antibody level in sows(reserve sows,boars and sows)was the highest with a positive rate of79.92%~88.93% and an average blocking rate of 62.48%~71.55%.The antibody level of the pigs in the weaner stage was the worst with the positive rate of only48.19%~57.41% and the average blocking rate of 41.34%~46.84%.During 2015 to2017,the number of samples with an average blocking rate between 50% and 90%was the most,ranging from 57.14% to 62.12%.Samples were collected in different months or years were shown no difference on antibody level.2.Three thousand and sixty~nine samples collected were processed and used for the detection of the potential CSFV nucleotide sequence using RT-PCR.The results showed that the detection rate of CSFV was 16.40% in 2016,followed by 13.30% in2015,and then 12.80% in 2017.Comparison of the CSFV detection rate among different quarters showed that the second quarter had the highest CSFV infection rate(14.80%~24.40%).The mixed infection of swine fever virus with other three viruses including Porcine respiratory and reproduction syndrome virus,pseudorabies virus and porcine circovirus were also investigated.Results showed that the proportion of mixed infection of CSFV with other viruses was 0.98%~9.24%.Among them,the mixed infection with PRRSV was the highest with coinfection rate uo to6.70%~9.24%.The mixed infection with pseudorabies was the lowest,ranging 0.98%to1.20%.3.Sequencing and phylogenetic analysis of the 18 strains of CSFV isolated from Shandong and Henan provinces was performed.The results show that the nucleotide homology for E2 between the 18 strains and the domestic and foreign reference strains ranged from 79.7% to 99.0%,the nucleotide homology with the standard strain Shimen strain E2 ranged from 81.0% to 94.7%.Among them,the lowest nucleotide homology between SD11 strain and vaccine strain E2 was 79.9%,and the highestnucleotide homology between SD05 and strain and vaccine strain E2 was 99.2%.The amino acid sequence homology of E2 gene of 18 strains was 50.2% ~ 98.5% with reference strain,52.1% ~ 86.9% with standard strain Shimen and 52.1% ~ 97.7% with vaccine strain C,respectively.Among them,SD10 strain had the lowest amino acid sequence homology with vaccine strain,and HN02 strain had the highest amino acid sequence homology with vaccine strain.The 18 isolates of epidemic strains mainly belonged to subgroups 1.1 and 2.1,with subgroups 2.1 as the dominant;2.1b became the dominant epidemic strain,accounting for 72.22%.These results demonstrated the the genetic diversity of the epidemic CSFV strains in China.4.Molecular biology and bioinformatics approaches were used to study the epidemic diseases characterized with newborn piglet tremor as the main clinical characteristics in large~scale pig farms in Henan province in recent years.Results uncover that the gene sequences of atypical pestivirus amplified from the infected pigs.Comparing the nucleotide sequence of the amplified fragment with those of the available atypical pestivirus in GenBank found that Xuchang 2018 strain had a89.5%~94.2% of sequence identity with the reported APPV isolates.Among them,the highest homology was 97.6% between Xuchang 2018 and Jiangxi~7.Hematoxylin and eosin staining showed that a large number of vacuoles were observed in the white matter in the cerebellum of infected pigs.The content of myelin in the cerebellum were significantly reduced in the brain of infected animals with the local glial cells around the blood vessels.To our best knowledge,the report demonstrated the existence of APPV infection in pigs in Henan Province,which provides a basis for the prevention and control of this disease in the future.5.The antibody change elicited by the subunit vaccine was studied by comparing the genetic engineering CSFV subunit vaccine with the traditional CSFV live attenuated vaccine.400 piglets were randomly divided into experimental group(swine fever subunit vaccine)and control group(traditional vaccine).200 piglets in each group were randomly selected and sampled at 30 days,60 days,90 days,120 days and 155 days respectively.10 blood samples were collected randomly in each group.Piglets in the experimental group were injected with a CSFV subunit vaccine into their neck muscles at 30 days of age,while piglets in the control group were injected with a live attenuated vaccine of classical swine fever at 30 days of age and60 days of age,respectively.The results showed that the antibody level of the experimental group reached the highest titer at 90 days after immunization with the blocking rate reaching 88.23% before decline.On day 155,the antibody titer still remained at a higher level with a relative long maintenance time and less affected by maternal antibodies and PRRS. |
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