摘要
本研究从规模化猪场分离到一株猪源病毒,通过细胞培养、电镜观察、乳鼠血清中和实验,以及送中国农业科学院兰州兽医研究所口蹄疫国家参考实验室鉴定为口蹄疫亚洲Ⅰ型病毒。对病变细胞进行超速离心,电镜负染可见大小约25-30nm的病毒粒子;血清中和实验,FMDV AsiaⅠ标准血清组乳鼠存活,其余FMDV标准血清组、对照组乳鼠死亡。
根据GenBank中的FMDV AsiaⅠ的全基因序列,在VP1基因两端设计一对引物,扩增出1012bp的VP1衣壳蛋白的完整片段。通过对病毒VP1基因的测序显示:本次分离株VP1 cDNA全长633bp,GC含量为59.56%。该毒株与中国报道株的同源性在82.0%—99.2%之间,与国外报道序列的同源性在82.9%—98.4%之间。同源性比较显示本次分离毒株不属于2001年报道口蹄疫亚洲Ⅰ型的4个基因型。结果显示我国口蹄疫亚洲Ⅰ型病毒为两个不同基因性的流行,即06年以前以Gansu 1、Yunnan株为代表的GenotypesⅣ,还有06年后报道的和本次分离株的新基因型。比较显示本次分离株在149突变为脯氨酸(Pro)和154位突变为天冬氨酸(Asp),该两处的氨基酸突变在其他报道序列中未见报道。
本试验选择母猪70头,小猪70头,随机均分为7组,每组母猪10头,小猪10头,分别编为1—7组(1—3组采用口蹄疫AsiaⅠ-0二价疫苗,4—6组采用口蹄疫AsiaⅠ单价疫苗,7组为正常免疫0型疫苗组),分别按正常免疫剂量、1.5倍剂量、正常免疫剂量免疫后15日加强一次共三个剂量组进行肌肉注射免疫。各组均正常饲养,于免疫后0、7、15、30、60、90、120日时耳静脉采血并分离血清。采用国际兽疫局推荐的液相阻断酶联免疫反应(LPB-ELISA)检测口蹄疫抗体效价。注射剂量比较表明,母猪采用1.5倍剂量、仔猪采用加强免疫其免疫抗体滴度水平高于另外两种方法;不同疫苗产生抗体滴度比较表明,采用口蹄疫AsiaⅠ型单价灭活疫苗免疫后所产生的抗体滴度高于使用口蹄疫AsiaⅠ-0型双价灭活疫苗试验组;口蹄疫0型抗体滴度的比较表明,采用口蹄疫AsiaⅠ-0型双价灭活疫苗进行免疫时,0型抗体和采用单价苗免疫结果差异不显著;对免疫后抗体滴度达到保护的猪只数量进行统计,结果显示采用AsiaⅠ-0型二价疫苗组免疫后抗体滴度能达到保护的母猪有70%、小猪有60%;AsiaⅠ单价苗免疫后抗体滴度能达到保护的母猪有83%、小猪有73%;
对试验猪抗体监测结果显示,猪群在免疫后15日左右出现最高抗体滴度,随后逐渐减弱。同时通过加强免疫组监测结果显示,抗体滴度log2值在低于4.0以下时进行免疫,可以提高抗体滴度。根据试验结果,推荐猪群在首次免疫采用AsiaⅠ单价疫苗,母猪采用1.5倍剂量免疫,首次免疫后120天可以加强一次。小猪在免疫后抗体滴度低于母猪,推荐小猪采用正常剂量免疫,免疫后15日时加强一次免疫,首次免疫后120天时加强一次。
This study separated a virus from swine farms,which was identified as the foot and mouth disease virus Asia I by using cell culture,electron microscope,serology neutralization technology and sending to Lanzhou Institute of Veterinary,national foot and mouth disease reference laboratory for identification.By ultracentrifugation,virion with a diameter of about 25-30nm can be observed in the electron microscope.The suckling mice treated with FMDV Asia I standard blood serum were live and others were dead.
A pair of primers was designed according to the complete gene sequence of FMDV Asia I in GenBank to amplify capsid protein VP1 gene and the gene of 1012bp was amplified.The gene sequencing showed cDNA of VP1 of this strain was in size of 633nt and having a GC contents of 59.56%.The VP1 gene homology of this strain versus other strains reported in China was 82.0%-99.2%,and that versus strains reported abroad was 82.9%-98.4%.Sequence homology comparison showed that this strain does not belong to the four genotypes reported at 2001.China's FMDV Asia I mainly have two different prevailing genotypes,which are GenotypesⅣbefore 2006 such as Gansu 1,Yunnan,and the new genotype reported after 2006 including this isolation.The comparison showed the VP1 amino acid sequence of this strain has two mutations in the 149(Pro)and 154(Asp) respectively which was not reported in other papers.
In this research,seventy sows and seventy piglets were randomly divided into seven groups,each group having 10 sows and 10 piglets(group1-3:inoculated FMDV Asia I-O association vaccine;group4-6:inoculated FMDV Asia I vaccines;group 7:inoculated FMDV O vaccines),the former two treatments were each inoculated for 3 different immunization doses which was normal dose,1.5 times of normal dose and repeated inoculation at 15 days after normal dose.Each group was fed equally,and serums were separated from ears' intravenous blood at the time of inoculating,7d、15d、30d、60d、90d、120d after vaccination.The antibody efficacy was examined by means of liquid-phase blocking enzy-linked immunosorbent assay(LPB-ELISA),which is recommended by the OIE.In view of vaccination doses,the result showed that sows treated with 1.5 times of normal dose and piglets inoculated repeatedly at 15 days after normal dose having more antibody efficacy;in view of different vaccines,the result showed that the antibody efficacy induced by FMDV Asia I vaccine was higher than that of FMDV Asia I-O association vaccines;in view of the antibody efficacy of FMDV O,the result showed the difference of antibody efficacy was not significant between FMDV O group and FMDV Asia I-O group.The statistics on the number of pigs that were immuno-protected showed that inoculated with FMDV Asia I-O association vaccine,70% of sows and 60%of piglets were protected;inoculated with FMDV Asia I vaccine,83%of sows and 73%of piglets,were protected.
Antibody monitoring results indicated that around 15 days after immunization swine show a maximum antibody titer,which then drop gradually;besides repeated immunization can enhance antibody efficacy when antibody is less than 4.0.According to results of the trial,FMDV Asia I vaccine is recommended for the first immunization;1.5 times of normal dose for first immunization and an enhancement on the 120th day for sows, normal dose for first immunization and enhancements on 15th and 120th days respectively for piglets are also recommended.
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