国产与进口猪伪狂犬病gE基因缺失苗免疫对比试验

为比较国产与进口猪伪狂犬病娅基因缺失疫苗免疫效果,并制定合理的免疫程序,笔者选用国产和进口的猪伪狂犬病gE基因缺失弱毒疫苗,用3种不同的免疫程序,对240头母猪和1200头仔猪进行免疫试验。试验期间,定时随机采集免疫猪的血液,用ELISA试剂盒进行抗体检测。结果表明,母猪和仔猪无论免疫国产或进口猪伪狂犬病gE基因缺失弱毒疫苗,免疫效果均良好。母猪免疫抗体合格率达100％,仔猪49日龄前免疫抗体合格率达100％,免疫效果差异不显著。但免疫猪群在75日龄后抗体逐渐降低,120日龄后基本消失。为维持猪体免疫力,不给野毒入侵的机会,建议仔猪首免后,在适当时间进行二次加强免疫。     

猪伪狂犬病病毒gE基因缺失苗免疫试验

为了掌握猪伪狂犬病gE基因缺失疫苗免疫效果,并制定合理的免疫程序,选用国产和进口的猪伪狂犬病gE基因缺失弱毒疫苗,用4种不同的免疫程序,对250头母猪和1000头仔猪进行了免疫试验。试验期间,按比例定时采集免疫猪的血液,用ELISA试剂盒进行抗体检测,证明猪伪狂犬gE基因缺失疫苗,无论国产苗或进口苗都可以产生良好的免疫效果;无论跟胎免疫、1年2次普免,每隔4个月定时免疫,效果均良好。种猪免疫抗体合格率达100%,仔猪49日龄前抗体合格率达100%,75日龄后抗体逐渐降低,120日龄后基本消失。为了使猪体免疫力更强,不给野毒入侵的机会,建议仔猪在首免后,适当时间进行二次加强免疫。而只给公猪、母猪春秋两季免疫,不给仔猪免疫组,仔猪在35日龄后抗体降为阴性,不能抵抗野毒的侵袭,这种免疫方法不宜推广。     

伪狂犬病免疫净化方案的评价

从一伪狂犬病阴性场选肉仔猪77头,均在2日龄鼻内接种伪狂犬病gE基因缺失弱毒疫苗1头份,免疫前采血,以后每间隔两周采血1次直至18周龄,检测并分析每份血样中的gE和gB抗体。另从一伪狂犬病阴性场选后备母猪50头,于77、147、175日龄肌注伪狂犬病gE基因缺失弱毒疫苗,于40、60、76、91、114、143、161、173、191、226日龄采血,检测并分析每份血样中gB抗体。结果表明,肉仔猪各周龄伪狂犬病野毒抗体(gE)均为阴性,疫苗母源抗体(gB)随日龄的增加而下降,但在114日龄有5头猪gB抗体阳转;免疫伪狂犬病gE基因缺失弱毒疫苗的后备母猪,首免后抗体上升,二免后抗体明显上升、并保持高水平,第3次免疫后抗体较二免后上升不明显。     

两种猪伪狂犬病毒基因缺失疫苗产前免疫母猪对仔猪抗体水平的影响

试验选择广东某规模化猪场怀孕母猪30头及其所产仔猪30头用进口猪伪狂犬病病毒基因缺失疫苗(B苗)和国产猪伪狂犬病病毒基因缺失疫苗(A苗)进行免疫试验.结果表明:各组母猪产前10 天免疫接种后20～30 d的母猪抗体水平接近或达到峰值,其所产仔猪10～60 d获得的母体抗体恰巧达到最佳.根据母猪产仔时抗体水平及其后代母源抗体维持时间的关系,建议母猪产前20～30 d为猪伪狂犬病病毒加强免疫接种的适宜时间,仔猪60日龄为适宜的首免时间;使用B苗和A苗免疫对母猪产前免疫是必要的,有利于控制带毒母猪猪伪狂犬病病毒野毒的排放,降低感染其他猪只的机会,提高仔猪保护率.     

猪伪狂犬病母源抗体衰减规律及适时首免试验

试验采用伪狂犬病gB抗体ELISA试剂盒,分别检测3日龄和1、2、3、4、5、6、7、8、9、10周龄未免疫猪伪狂犬病母源抗体和相应日龄免疫后3周的伪狂犬病gB抗体,以gB抗体阳性率和S/N值分析猪伪狂犬病母源抗体衰减规律和最佳首免时间。结果表明,母猪分娩前1个月免疫伪狂犬病gE基因缺失弱毒苗,所产仔猪能获得高水平的母源抗体;随着仔猪日龄增加,抗体水平逐渐降低,能维持到10周龄。仔猪在5周龄前免疫伪狂犬病疫苗,母源抗体干扰主动免疫,5周龄后免疫抗体水平均有上升;该场仔猪伪狂犬病最佳首免时间为35~42日龄。     

猪伪狂犬病流行病学的调查

猪伪狂犬病是导致母猪流产,产死胎、木乃伊胎或弱胎的重要疫病之一,尤其会导致新生仔猪出现大量死亡,15日龄内的仔猪死亡率达100%,少数耐过的猪也因为发育不良而成为僵猪,因此,该病一直是制约养猪业发展的重要疫病之一.笔者通过对部分规模猪场猪伪狂犬病的流行病学调查发现,伪狂犬病gE基因缺失疫苗(Bartha-K61株)已免猪场的gB免疫抗体平均合格率可达90.16%,而在已免猪场中还存在gE抗体阳性,平均阳性率为11.76%;通过对已免猪场与未免猪场以及免疫前后的对比和分析,提出单纯依靠疫苗注射并不能完全实现猪伪狂犬病野毒感染率的降低,建立科学的免疫程序,加强防疫管理,实施科学净化才是有效防控猪伪狂犬病野毒感染的重要措施.     

猪伪狂犬病阳性场gE与gB抗体监测及风险评估

采集衡阳某猪场119份血清样品进行伪狂犬病gE-ELISA（酶联免疫吸附试验）与gBELISA抗体检测。gE抗体检测结果表明,该猪场所检测猪群除公猪外都有伪狂犬病野毒阳性,是伪狂犬病阳性场,总阳性率为47%。公猪伪狂犬病野毒抗体阳性率为0,说明公猪暂时还没有感染伪狂犬病野毒,属于阴性猪;母猪群伪狂犬病野毒（gE）阳性率达50%~60%;30~40日龄及60日龄阶段猪群gE抗体阳性率分别为30%和55%,加上可疑的数量达到40%~65%,这阶段野毒抗体阳性估计主要受母源gE抗体的影响,且与母猪阳性背景基本一致,但也不能排除个别仔猪阳性抗体来自病毒感染;90~120日龄阶段gE阳性率不降反升,达到100%,说明90~120日龄阶段猪群是在保育转至肥育舍后被伪狂犬病野毒再次感染,gE抗体S/N值显著下降,抗体水平较高。gB抗体结果表明,该猪场伪狂犬病gB抗体阳性率为98%,公猪伪狂犬病gB抗体阳性率为100%,且公猪的gE抗体阴性,说明公猪gB抗体来源于疫苗免疫;母猪群伪狂犬病gB抗体水平高且整齐,可能是野毒感染和疫苗免疫综合作用的结果;30~40日龄阶段gB抗体水平整齐,这是由于母猪群gB抗体水平高且均匀整齐,其仔猪母源抗体相应较好;60日龄阶段gB抗体水平不整齐,这与母源抗体消退有关;90~120日龄阶段gB抗体水平显著提升,这可能是后期野毒感染所致,因为其gE抗体不降反升。综合该猪场gE和gB抗体检测结果分析表明,该猪场伪狂犬病在种猪群得到了有效控制,生产比较稳定,但种猪带毒且散毒,肥育猪的感染压力加大,造成病毒不断循环,肥育猪伪狂犬病野毒感染的控制成了稳定伪狂犬病阳性场的关键。目前,猪伪狂犬病阳性场应定时监测gE和gB抗体,根据猪场血清学检测结果,结合临床实际情况实时调整免疫程序,以便稳定生产和预防伪狂犬病的暴发。     

山西猪伪狂犬病免疫与野毒感染情况调研

山西2013年以来,即使使用gE基因缺失活疫苗免疫的猪场,也有母猪发生流产、仔猪疑似伪狂犬病病死情况,给养猪生产造成一定的经济损失。2016年、2017年、2018年项目组对山西不同地市的11个规模型种猪场调研证实它们使用的是正规厂家生产的猪伪狂犬病gE基因缺失疫苗,大部分猪场母猪采取普免,仔猪采取3次免疫;共检测11个猪场母猪血样388头份,野毒感染阳性率为57.94%,最高阳性率为83.33%;仔猪血样727头份,阳性率为34.66%,最高为70.00%,只有一个猪场仔猪没有检出野毒感染抗体。另外,从疑似伪狂犬病病死猪病料检测出猪伪狂犬病病毒阳性19份。初步掌握了山西猪伪狂犬病免疫与感染情况,为有效控制该病流行提供依据。     

种猪场伪狂犬病净化技术探讨与应用

选择徐州六马种猪科技有限公司种猪场作为伪狂犬病净化创建场,通过开展对种公猪、生产母猪、后备种猪、待售种猪伪狂犬病野毒感染抗体(gE抗体)和疫苗免疫抗体(gB抗体)检测,进行猪伪狂犬病野毒感染情况的本底调查,了解各阶段猪群健康状态、免疫情况、免疫抗体水平和野毒感染状况。根据猪伪狂犬病毒野毒感染状况,采用不同的净化方案,进行猪伪狂犬病净化。通过净化,该场种公猪、生产母猪、后备种猪及待售种猪连续2次以上抽检结果均为伪狂犬病gE抗体阴性且gB抗体合格率70%以上,达到猪伪狂犬病净化创建场标准;同时,猪群的健康状况、母猪生产性能和仔猪成活率均有明显提高,为今后猪场伪狂犬病净化工作的开展提供技术参考。     

猪伪狂犬病净化技术的研究

在某600头母猪的伪狂犬病阳性猪场,应用猪伪狂犬病gE基因缺失弱毒苗对全场所有猪只严格按免疫程序进行强化免疫,8个月后对本场种猪群猪伪狂犬病开展流行病学调查,当抽样检测gE抗体阳性率低于5%时,对全群种猪进行检测并淘汰gE阳性猪只,对免疫后的后备母猪进行逐头检测,gE阴性猪只并入生产群。通过反复检测、淘汰,3年后,该场猪伪狂犬病野毒阳性率由2008年的11.53%下降为0,达到净化标准。     

猪伪狂犬病病毒gE蛋白在野毒诊断中的应用进展

With mass immunization application of pseudorabies virus （PRV） gE gene deleted attenuated vaccine in China, it played an important role in prevention and control of porcine pseudorabies, meanwhile it also needed to establish a differential diagnosis technology for gE gene deleted vaccine matching to eliminate virus affected swine and reduce immune pigs to be killed, and gradually realized the purification of PRV. With the gradual progress of molecular biology and immunology, molecular biological and serological diagnosis methods based on PRV gE protein continuously developed and improved. In this paper, recent study on various molecular and serological diagnosis methods of gE protein based on the current situation and development prospect were reviewed, in order to provide reasonable scientific basis for diagnosis and prevention of PRV in China.     

广西某集约化猪场猪伪狂犬病的监测及净化

为了解某集约化猪场伪狂犬病病毒（PRV）的感染情况,清除感染猪只,净化伪狂犬病,提高猪群的健康水平.采用PRV gE抗体ELISA检测试剂盒,对该场免疫了猪伪狂犬病基因缺失疫苗的猪群进行3次野毒感染检测;应用荧光PCR方法,对临床发病的仔猪进行PRV病原检测.经过动态监测、加强综合防控和逐步淘汰措施,该猪场的PRV野毒感染率从35.29％逐步降低至0％,猪场自繁的仔猪存活率高,无PRV感染,净化效果良好.     

Cloning and Sequence Analysis of gE and gI Genes of Pseudorabies Virus NP Isolate

According to published gE and gI gene sequences of pseudorabies virus (PRV) in GenBank, we designed two pairs of primers for PCR amplification of gE and gI genes of PRV NP isolate, after PCR products recycling, cloning and sequencing, the sequencing results were consistent with expectations of PRV gE and gI genes.Homology comparison analysis results revealed that compared with the domestic PRV strains, the homologies of gE and gI amino acids of PRV NP isolate were 95.7% to 99.8% and 89.9% to 99.5%, respectively.Phyogenetic tree analysis and amino acid sequence alignment results found that the gE amino acid sequence site changes of PRV NP isolate were the same with the PRV strains which were isolated from domestic in 2012, thus we could speculate that PRV NP isolate had mutanted.This study had laid the foundation for the epidemiological investigation and analysis of PRV, also provided the scientific basis for the development of scientific, effective and new pseudorabies vaccine.     

猪伪狂犬病病毒NP株gE、gI基因的克隆及序列分析

根据GenBank中已发表的猪伪狂犬病病毒(PRV) gE、gI基因的序列设计了2对引物,对PRV NP株的gE、gI基因进行了PCR扩增、回收、克隆、测序,测序结果与预期的PRV gE、gI基因片段相符。同源性比对分析结果显示,PRV NP株gE、gI基因推导的氨基酸序列与国内分离的PRV毒株的同源性分别为95.7%~99.8%、89.9%~99.5%。遗传进化树分析和氨基酸序列比对结果发现PRV NP株的gE氨基酸序列发生变化的位点与2012年国内分离到的PRV流行株相同,从而推测NP株为PRV变异毒株,本研究为PRV的流行病学调查分析奠定了基础,也为开发科学、有效的新型猪伪狂犬病(PR)疫苗提供科学依据。     

长沙市对伪狂犬病野毒感染的调查

为了解长沙市规模猪场伪狂犬病流行情况,对市内47个规模猪场送检的1534猪血清样品和22个已免疫伪狂犬基因缺失苗规模场送检的1300份血清样品,用ELISA方法对猪伪狂犬病野毒感染抗体（gpI抗体,下同）和免疫抗体（gB抗体,下同）进行检测。结果表明,长沙市规模猪场猪伪狂犬病gpI抗体阳性率为20.9%,场阳性率为44.7%;22个免疫猪伪狂犬基因缺失苗规模猪场猪伪狂犬病gpI抗体阳性率下降1.8%,场阳性数下降4.6%。结果表明,通过利用猪伪狂犬病基因缺失疫苗科学免疫,同时配合伪狂犬野毒抗体检测技术可以控制和净化规模猪场猪伪狂犬病流行。     

Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses

The successful eradication of pseudorabies in U.S. domestic swine was accomplished through the use of glycoprotein E (gE) deleted modified live virus vaccines and an accompanying gE differential enzyme-linked immunosorbent assay (ELISA). Yet, pseudorabies virus (PRV) was established in feral swine in the United States, becoming a potential reservoir of PRV for infection of domestic swine and other native wildlife. A critical need for the current PRV surveillance program in the United States is the rapid detection of PRV infection. For this reason, a set of 2 real-time polymerase chain reaction (PCR) assays by using TaqMan chemistry was developed and evaluated for their capability in the detection and differentiation of field and vaccine strains of PRV. PCR primers and probes were designed for gB and gE genes of PRV, respectively. The newly developed PRV-specific real-time PCR assays could detect all wild-type PRV isolates from diagnostic submissions and differentiate them from vaccine strains. The analytical sensitivity of the assays was approximately 0.1 plaque-forming units per reaction. The assays were highly specific for PRV, because no positive results were obtained from testing other common swine viral pathogens and other animal herpesviruses. The results of testing samples from domestic and feral swine and from bovine showed that the real-time PCR assays are more sensitive than gel-based PCR. These results demonstrated the potential application of the developed real-time PCR assays as a differential test for rapid and specific detection of PRV in domestic and feral swine, as well as nonporcine species that can be infected with PRV and serve as carriers.     

近期部分规模化猪场猪伪狂犬病野毒抗体监测情况调查

猪伪狂犬病仍然是我国猪群的重要威胁性传染病，通常造成母猪繁殖障碍以及仔猪的神经症状和高死亡率，同时伪狂犬病病毒也是猪呼吸系统疾病综合征的重要原发性病原。用gE-ELISA野毒鉴别诊断方法共检测了来自8个省、市46个猪场1940份血清样品，其中阳性样品657份，样品总阳性率33.87%，阳性猪场25个。在检测的各场中，阳性率最高猪场达到75.76%(125/165)。而部分进行了科学的疫苗免疫和实施了严格的生物安全措施的猪场始终保持猪伪狂犬病阴性(0/135)。利用基因缺失疫苗科学免疫配合伪狂犬抗体鉴别诊断技术是控制和净化伪狂犬病的重要手段。     

Survival and immunization of raccoons after exposure to pseudorabies (Aujeszky's disease) virus gene-deleted vaccines

In a controlled experiment, 16 wild-trapped raccoons were exposed to 1 of 2 genetically modified live pseudorabies virus (PRV) vaccines used in swine. One vaccine had genes deleted for thymidine kinase (TK(-)) and glycoprotein G (gG(-)); the other had an additional deletion for glycoprotein E (gE(-)). These vaccines were administered orally and intranasally at four dose levels: 10(3), 10(4), 10(5), and 10(6) TCID(50). The 21 days survival rate was 37.5% for the gG(-)TK(-) vaccine; all of the survivors developed antibodies to PRV. All animals receiving the gG(-)gE(-)TK(-) vaccine survived; 75% (all except the lowest dose) developed anti-PRV antibodies. Survivors were challenged intranasally with a 3.2x10(3) TCID(50) dose of the virulent wildtype PRV Shope strain. Two of the remaining three gG(-)TK(-) vaccinated raccoons survived the challenge; for the gG(-)gE(-)TK(-) vaccine, the survival rate was 50% (4/8). The raccoons with higher vaccine-induced antibody titers were more likely to survive the challenge with the virulent PRV; there was a 100% mortality rate for raccoons lacking detectable anti-PRV antibodies. This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent virus.     

伪狂犬病病毒gE/gI基因在昆虫细胞中的表达及鉴定

为获得具有生物活性的伪狂犬病病毒(Pseudorabies virus,PRV)gE/gI蛋白,建立PRV抗体快速检测方法。将含有PRV gE、gI基因的质粒pFastBacdual-GP67-gE/gI转化至宿主菌,经位点特异性重组和蓝白斑筛选后获得重组杆粒rBacmid-gE/gI,转染Sf9细胞,获得重组杆状病毒。采用悬浮的Sf9细胞进行发酵并纯化产物,SDS-PAGE和Western blot分析镍柱纯化后的重组蛋白。结果显示,重组杆粒在4800 bp处克隆出预期大小的条带,表示重组杆粒构建成功。SDS-PAGE和Western blot表明,在50 ku和65 ku处出现预期大小的条带,能与PRV标准阳性血清特异性反应,不与PRV gE/gI缺失疫苗免疫血清反应。结果表明gE/gI重组蛋白具有良好的反应原性,为PRV抗体快速检测及区分PRV野毒感染和疫苗免疫奠定了基础。