嵌合型猪圆环病毒（PCV1—2）及PCV2 ORF2真核表达质粒对商品猪的免疫保护

应用本实验室构建的嵌合型猪圆环病毒（PCV1—2）及真核表达质粒pcDNA3．1／V5-His-ORF2作为免疫原免疫母源抗体ELISA效价在0．07～0．60不等的商品猪,9头猪随机分为4组,1组（3头）肌肉注射免疫10^3.5 TCID50的PCV1-2／头,2组（2头）肌肉注射真核表达质粒200μg／头,3组（2头）肌肉注射空载体（pcDNA3．1）200μg／头,4组（2头）不免疫作为攻毒对照组。于免疫后42d,PCV1—2组及真核表达质粒组产生了PCV2抗体。免疫后42d所有组攻毒PCV2和PRRSV,剂量分别为2×10^4.5 TCID50／头和10^6 TCID50／头。攻毒后21d,攻毒对照组猪淋巴结比免疫组显著肿大,免疫组猪血清、淋巴结中PCV2病毒栽量低于对照组,攻毒对照组猪淋巴结中PCV2抗原含量高于免疫组。这些结果表明,嵌合型PCV1-2及真核表达质粒肌肉注射免疫商品猪后,对PCV2感染能产生保护性免疫应答,有可能成为候选疫苗。     

猪圆环病毒2型ORF2/猪白介素2嵌合表达质粒在猪体内诱导的保护性免疫反应

为探讨猪圆环病毒2型(PCV2)ORF2/猪白介素-2(PoIL-2)嵌合重组表达质粒(rpcDNA3.1/PCV2-linker-PoIL-2)在猪体内的免疫效果和免疫保护效果进而研制高效PCV2核酸疫苗,将35只10日龄健康仔猪平均分成7组分别以rpcDNA3.1/PCV2-linker-PoIL-2重组表达质粒或PCV2ORF2基因原核表达的rCap蛋白进行免疫及免疫保护试验。共进行4次肌肉注射免疫,每次间隔2周;于第4次免疫后3周通过口腔和鼻腔途径感染PCV2细胞强毒。分别于第4次免疫后和攻毒后不同时间通过检测免疫猪血清抗体水平和外周血T淋巴细胞增殖活性、辅助性T细胞(Th)和细胞毒性T细胞(Tc)亚群的百分含量、排毒率和病毒血症阳性维持时间等指标以评价其免疫和免疫保护效果。结果表明,各免疫组猪均产生了抗PCV2特异性ELISA免疫抗体,但rCap蛋白免疫组猪抗体水平较低;rpcDNA3.1/PCV2-linker-PoIL-2质粒对猪体的免疫和免疫保护效果显著优于rpcDNA3.1/ORF2质粒;在rpcDNA3.1/PCV2-linker-PoIL-2质粒或rpcDNA3.1/ORF2质粒中添加rCap蛋白对重组质粒免疫及免疫保护效果无明显影响。因此,选取rpcDNA3.1/PCV2-linker-PoIL-2质粒为下一步研制PCV2核酸疫苗的主要成分。     

PCV2ORF2基因与猪IL-18基因共表达DNA疫苗的免疫原性

为研制新型的PCV2基因疫苗,本研究将PCV2ORF2和猪IL-18基因插入真核表达质粒pIRES中,构建共表达capsid (Cap)蛋白和猪IL-18的质粒pIRES-OFR2/IL18和表达Cap蛋白的质粒pIRES-OFR2.将质粒pIRES-OFR2/IL18和pIRES-OFR2通过腿部肌肉多点注射8周龄昆明小鼠,间隔3周再免疫1次.加强免疫3周后用PCV2 Wuzhi株进行攻毒.pIRES-OFR2/IL18免疫昆明小鼠后能促进外周血T淋巴细胞增殖和血清中特异性抗体水平的增加,能明显增强PCV2 DNA疫苗对强毒的攻击保护,pIRES-ORF2/IL18免疫组要优于pIRES-ORF2免疫组及其它对照组,差异显著.表明猪IL-18基因与PCV2ORF2共表达可增强猪体对DNA疫苗的免疫应答,提高对PCV2强毒的抵抗力.     

PCV2感染对仔猪NO-NOS系统的动态影响

将19头PCV2和PRRSV血清抗体阴性的普通断奶仔猪分为对照组(4头)和攻毒组(15头),攻毒组每头仔猪滴鼻接种PCV2病毒悬液4mL(5×105 TCID50/mL)并用KLH刺激,分别在攻毒后14、21、35d各扑杀5头仔猪采集血清和脾脏、淋巴结、胸腺(对照组4头在攻毒当天扑杀)。检测各种组织中NO、TNOS和iNOS的含量。结果显示,攻毒猪各组织中NO含量均在攻毒后14d显著升高(P<0.05),然后缓慢下降;血清和3种免疫器官中NOS(包括TNOS和iNOS)活性的变化趋势比较一致,均表现出先升高然后再逐渐降低的特点。此外,iNOS活性变化与其相应的组织中NO含量呈显著正相关(P<0.01)。结果表明,PCV2在感染早期(1～14d)可激活仔猪多种组织中NO-NOS系统,中后期逐渐恢复,这种变化与PCV2所致的组织损伤密切相关,因此,NO-NOS系统可能是PCV2相关疾病发生发展的重要信号。     

猪圆环病毒2型核酸疫苗的小鼠免疫保护试验

为了研究猪圆环病毒2型(PCV2)核酸疫苗在小鼠攻毒试验中的免疫保护效果,以PCV-2 GXWZ-1株为模板,扩增出ORF2基因及其截短基因8个片段(A(ORF2)、B(51-100aa)、C(101-150aa)、D(181-235aa)、E(151-200aa)、F(51-150aa)、G(101-235aa)、H(51-235aa)),将其插入到pcDNA3.0载体中,构建出真核表达质粒,并将其转染至PK-15细胞,用间接免疫荧光试验检测其瞬时表达情况。将试验小鼠随机分成9组,其中免疫组7组,阴阳性对照各1组,将纯化的真核表达质粒对小鼠进行组合免疫;二免后,用经处理过的PCV-2 GXWZ-2株阳性病料悬液腹腔注射免疫组和非免疫对照组小鼠,阴性对照组用生理盐水腹腔注射;其后进行体重记录、病理切片制作及PCR检测。结果表明:共有6个真核表达质粒成功在PK-15细胞中表达。在攻毒后的3周内,阳性对照组小鼠PCR诊断均为阳性;免疫组中,部分组小鼠在攻毒后第1周或在第2周为阳性,到第3周时各免疫组小鼠全部为阴性;阴性对照组始终为阴性。免疫组在病理保护学方面明显优于非免疫对照组,非免疫对照组的体重增长速率略低于免疫组和阴性对照组。由此可见猪圆环病毒2型核酸疫苗在小鼠攻毒试验中有明显的保护作用。     

猪圆环病毒2型(PCV2)疫苗对PCV2与猪繁殖和呼吸综合征病毒共感染猪群免疫力评价

本试验旨在探讨猪圆环病毒2型(PCV2)疫苗对PCV2、猪繁殖与呼吸综合征病毒(PRRSV)感染猪生产性能及免疫保护力的影响。将287头仔猪随机分为2组,一组于出生后14 d和28 d免疫107.0TCID50PCV2疫苗各1 mL,另一组不予免疫。记录各组临床症状变化、断奶体重、保育后体重,并按时采血检测PCV2抗体。结果显示,在PCV2、PRRSV感染猪场,免疫组发病2头,死亡0头;对照组发病18头,死亡12头,免疫组发病率和死亡率明显低于对照组。二者在保育阶段增重变化差异显著(P<0.05),PCV2疫苗免疫后能够较快地产生保护力。     

PCV2a/2b灭活疫苗与PCV2b亚单位疫苗免疫效力比较试验

比较猪圆环病毒(PCV)2a、PCV2b基因型全病毒灭活疫苗和PCV2b亚单位疫苗对PCV2b基因型强毒株免疫攻毒保护效力。取3种类型PCV2疫苗,分2次免疫PCV2抗原和特异性抗体阴性的BALB/c小鼠和14日龄仔猪,间隔21d,分别于免疫后14、21和35 d进行PCV2特异性ELISA抗体测定。使用临床分离鉴定的PCV2b基因型强毒株进行攻毒,通过攻毒前后临床观察、体温变化、平均相对日增重及PCV2核酸载量检测等对疫苗免疫效果进行评价。结果:不同类型PCV2疫苗免疫BALB/c小鼠后14 d能检测到PCV2特异性抗体,在35 d时抗体水平持续升高,其中PCV2b亚单位疫苗产生抗体水平高于全病毒灭活疫苗;不同类型疫苗均能刺激仔猪产生较好的免疫应答,其中PCV2b亚单位疫苗刺激机体免疫应答强于灭活疫苗;仔猪免疫攻毒试验表明,3种类型疫苗均可刺激机体产生抵御PCV2强毒攻击的特异性免疫应答,免疫组体温变化、相对日增重及PCV2核酸载量检测均与攻毒对照组间存在显著性差异,3种类型疫苗之间差异不显著。试验表明:3种类型PCV2疫苗免疫小鼠和仔猪后均能诱导产生抵御PCV2b基因型强毒攻击的特异性免疫应答反应,有效抵抗PCV2感染和提高猪的生长性能。     

猪圆环病毒2型和副猪嗜血杆菌5型二联灭活疫苗研制与免疫效力研究

猪圆环病毒2型(PCV2)和副猪嗜血杆菌(HPs)混合感染十分普遍,造成我国养猪业严重经济损失。本研究将杆状病毒表达的重组PCV2 Cap蛋白(BCap)和PCV2灭活病毒液(i PCV2),分别与HPs血清5型(HPs5)灭活菌液混合,加入水性佐剂混合制备成BCap/HPs5与i PCV2/HPs5二联灭活疫苗,进行猪体免疫保护试验。仔猪免疫保护试验结果为:两种疫苗免疫后均可诱导猪体产生PCV2和HPs抗体。免疫后35天用PCV2攻击,两免疫组猪均无明显临床症状,相对日增重(RDWG)与空白对照组相似,但高于攻毒对照组(P0.05);攻毒后14天,病毒血症明显低于攻毒对照组(P0.05);攻毒后28天剖解,腹股沟淋巴结PCV2载量明显低于攻毒对照组(P0.05),病理学变化明显轻于攻毒对照组。免疫后35天用HPs攻击,对照组猪均出现明显临床症状,但两免疫组猪无明显临床症状;攻毒后14天剖检,两免疫组病理学变化方面相似,但明显轻于攻毒对照组。结果表明,BCap/HPs5与i PCV2/HPs5两种二联苗免疫仔猪后均能诱导机体产生免疫应答,产生免疫保护作用,具有较好应用前景。     

猪繁殖与呼吸综合征病毒基因工程双标记疫苗rHN4-△25+NP49株最小免疫剂量的测定

将35头40日龄猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪瘟病毒(Classical swine fever virus,CSFV)、猪圆环病毒(Porcine circovirus type 2,PCV2)抗原及抗体均为阴性的健康仔猪,随机分成7组,每组5头,命名为XJ1~XJ7组。XJ1~XJ6组分别免疫PRRSV基因工程双标记疫苗rHN4-△25+NP49株,接种剂量分别为102TCID50/头、103TCID50/头、104TCID50/头、105TCID50/头、106TCID50/头和107TCID50/头,XJ7组为对照组,按1mL/头剂量接种DMEM。各试验组在免疫后28 d,用3×104.0TCID50/头剂量感染HP-PRRSV HuN4第5代强毒,疫苗免疫和攻毒后观察和检测各组试验猪的免疫效果。结果表明:攻毒前XJ2-XJ6组PRRSV ELISA抗体在免疫后14 d全部转为阳性,XJ1组在免疫后21 d转为阳性,但维持在较低水平;XJ2-XJ6组针对标记基因NP49的特异性抗体在免疫后28 d转阳性。攻毒后对照组全部发病,且死亡2头,XJ1有4头发病,XJ2-XJ6组无发病和死亡,本研究结果表明PRRSV基因工程双标记疫苗rHN4-△25+NP49株对仔猪提供免疫保护的最小免疫剂量为103TCID50/头。     

猪SERPINC1基因对PCV2复制的效应及其转录调控

旨在研究猪SERPINC1基因对PCV2复制的影响,并探究SERPINC1基因的转录调控。本研究以15头纯种的莱芜猪(LW)和15头大约克夏-长白杂交的商品猪(YL)为试验动物。每个品种分为2组:接毒组(10头)和对照组(5头)。对接毒组的猪肌肉注射3 mL 6.3×10~(-3) TCID_(50)的PCV2-SD毒株,对照组的猪肌肉注射3 mL的磷酸盐缓冲液。在前期转录组测序的基础上,分析SERPINC1基因对PCV2复制的效应,并分析检测SERPINC1基因启动子和转录活性。结果表明,过表达SERPINC1能显著抑制猪肺泡巨噬细胞(PAM)中PCV2的复制。分别克隆了LW和YL猪SERPINC1基因转录起始位点上游3 854 bp的序列,并进行启动子活性的分析,发现PCV2接毒后LW猪SERPINC1基因的启动子活性显著升高(P0.05),而YL猪的启动子活性无显著变化。在SERPINC1基因的5′调控区找到4个调控该基因转录的关键区段,并对上述关键调控区中的4个多态性位点对SERPINC1基因启动子活性的影响进行了分析。综上表明,这4个多态位点均不影响SERPINC1基因的启动子活性。该研究结果为找到与猪抗PCV2有关的基因及分子标记奠定了基础。     

Effect of porcine circovirus type 2 (PCV2) vaccination on PCV2-viremic piglets after experimental PCV2 challenge

The objective of this study was to evaluate the effect of porcine circovirus type 2 (PCV2) vaccines on PCV2-viremic and -seropositive piglets born from naturally PCV2-infected sows against postnatal PCV2 challenge. The experimental design was aimed at mimicking commercial swine rearing conditions to evaluate the response of the PCV2 vaccine on PCV2-viremic and -seropositive piglets after experimental PCV2 challenge. PCV2a (or 2b)-viremic piglets received a PCV2 vaccine at 21 days of age followed by a PCV2b (or 2a) challenge at 49 days of age (28 days post vaccination). The PCV2 vaccines elicited a high level of humoral (as measured by immunoperoxidase monolayer assay and neutralizing antibody titers) and cellular (as measured by the frequency of PCV2-specific interferon-γ-secreting cells) immune response in the PCV2-viremic piglets after vaccination even in the presence of maternally derived antibodies (MDA). The initial infection of PCV2 in the pigs was not affected by PCV2 vaccination, however the challenging PCV2 was reduced by PCV2 vaccination on PCV2-viremic pigs. The results from this study demonstrate that the PCV2 vaccine used in this study is effective at reducing PCV2 viremia and lymphoid PCV2 DNA, even for PCV2-viremic pigs with passively acquired MDA at the time of vaccination.     

Detection of porcine Circovirus type 2 (PCV2) variants PCV2-1 and PCV2-2 in Brazilian pig population

In the present study whole genome of six Brazilian isolates of PCV2 were sequenced, analyzed and compared with 35 other sequences (24 from other countries and 17 from Brazil). The phylogenetic analysis showed that mostly Brazilian variants of PCV2 were grouped as PCV2-1. Two isolates among the six analyzed here could not be grouped with any other PCV2-2 analyzed in this study. One of these isolates was from an aborted fetus with myocarditis and the other from a PMWS affected pig. The results pointed here showed that both groups of PCV2 are present in Brazilian pig population without any clear geographical correlation.     

猪圆环病毒Ⅱ型新疫苗可预防猪圆环病毒Ⅱ型相关疾病

美国堪萨斯州州立大学科学家完成了一项研究,结果表明新研制的猪圆环病毒Ⅱ型疫苗能有效预防猪圆环病毒Ⅱ型。病毒学家、诊断医学和病理学     

Porcine circovirus type 2 (PCV2) vaccination reduces PCV2 in a PCV2 and Salmonella enterica serovar Choleraesuis coinfection model

We previously reported that prior porcine circovirus type 2 (PCV2) infection potentiates the severity of clinical signs, lung lesions, and fecal shedding and tissue dissemination of Salmonella enterica serovar Choleraesuis in infected pigs. Here, we evaluated whether PCV2 vaccination is effective in reducing fecal shedding and tissue dissemination of S. Choleraesuis and improving clinical signs associated with PCV2 and S. Choleraesuis infection in 15 Cesarean-derived, colostrum-deprived pigs randomly assigned to 3 groups (n = 5/group). The vaccinated and co-infected (VAC-COINF) group received 2 ml of a commercial PCV2 vaccine at age 3 weeks. The VAC-COINF and co-infected (COINF) groups were inoculated intranasally with PCV2 and S. Choleraesuis at 5 and 7 weeks of age, respectively. The CONTROL group pigs received a similar volume of PBS for sham-vaccination and sham-inoculation. PCV2 vaccination clearly reduced PCV2 DNA load in the serum and postmortem tissue samples and decreased PCV2 antigen levels in tissue samples of the VAC-COINF group. After S. Choleraesuis infection, the incidence of several clinical signs increased in the VAC-COINF group compared to that in the COINF group. The microscopic lung lesions and weight gain, fecal shedding and tissue dissemination of S. Choleraesuis except in the spleen were not significantly different in the VAC-COINF and COINF groups. Thus, PCV2 vaccination reduced PCV2 in the S. Choleraesuis and PCV2 coinfection model and the effects on S. Choleraesuis were minimal.     

Porcine reproductive and respiratory syndrome virus (PRRSV) influences infection dynamics of porcine circovirus type 2 (PCV2) subtypes PCV2a and PCV2b by prolonging PCV2 viremia and shedding

The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on porcine circovirus type 2 (PCV2) subtypes a (PCV2a) or b (PCV2b) viremia and shedding characteristics in oral, nasal and fecal samples in experimentally infected pigs. Twenty-three, 2- to 6-week-old pigs were randomly divided into five groups: negative control (n=3), PCV2a-I (n=5), PCV2a-PRRSV-CoI (n=5), PCV2b-I (n=5), and PCV2b-PRRSV-CoI (n=5). Blood, oral, nasal and fecal swabs were collected in regular intervals from day post inoculation (dpi) 0 until dpi 70 and tested by quantitative real-time PCR for the presence and amount of PCV2 DNA and by ELISA for the presence of PCV2-specific antibodies. The results indicate that there were significantly (P<0.05) higher loads of PCV2a and PCV2b DNA in serum, oral swabs, nasal swabs and fecal swabs and a higher prevalence of detectable PCV2 antigen in tissues of pigs concurrently infected with PCV2 and PRRSV compared to pigs singularly infected with PCV2 further confirming that PRRSV enhances replication of PCV2. Moreover, PRRSV infection significantly prolonged the presence of PCV2 DNA in serum and increased the amount of PCV2 DNA in oral and nasal secretions and fecal excretions in the later stages of infection between dpi 28 and 70. Shedding patterns were similar between groups infected with PCV2a and PCV2b, indicating that there was no subtype-specific interaction with the PRRSV isolate used in this study. The results from this study highlight the interaction between PRRSV and PCV2 and the importance of controlling PRRSV infection in order to reduce PCV2 virus loads in pig populations.     

猪圆环病毒Ⅱ型免疫方案比较

希腊首都雅典成为讨论养猪业最大麻烦制造者一猪圆环病毒Ⅱ型(PCV2)防控措施的地点,约400名从事猪兽医工作的兽医师与会倾听了有关PCV2的科学研究和实际应用的进展,并在此背景下论述各自的防治经验.     

猪圆环病毒病与猪圆环病毒Ⅱ型疫苗免疫策略的回顾

猪圆环病毒Ⅱ型(PCV2)能够影响猪场中各阶段猪只的生产性能。因此,C.Charreyre、F.Joisel和Henry Too认为,重要的是要保护胚胎、胎儿、仔猪和育肥猪、后备母猪和后备公猪、经产母猪和成年公猪。饲养管理和疫苗接种是控制猪场彻底免受PCV2不利影响的两个关键点。     

猪圆环病毒3型与猪圆环病毒2型二重PCR检测方法的建立

为建立同时检测PCV3和PCV2的二重PCR方法,分别以PCV3和PCV2基因组的保守区域设计2对特异性引物,片段大小分别为649,295 bp。将反应条件进行优化,构建PCV3和PCV2的二重PCR检测方法。检测结果表明该方法能特异性扩增PCV3和PCV2,且无法扩增其他猪常见病毒;PCV3和PCV2的最低检出限分别为508,412 copies/μL。临床样品检测结果显示,PCV3和PCV2阳性率分别为2.1%(3/145),62.1%(90/145),PCR产物经测序证实为PCV3。本研究建立的二重PCR方法具有速度快、特异性强和灵敏度等优点,可以用于PCV3和PCV2的检测和流行病学调查。     

Reproduction in porcine circovirus type 2 (PCV2) seropositive gilts inseminated with PCV2b spiked semen

Background

Since 1999, field evidence of transplacental infection by porcine circovirus type 2 (PCV2) and reproductive failure has been reported in pigs. The objective of this study was to evaluate the clinical and pathological consequences of PCV2 infection in conventional PCV2-seropositive gilts by insemination with PCV2b-spiked semen.

Results

Six PCV2 seropositive gilts were inseminated with PCV2b-supplemented semen (infected) and three animals with semen and cell culture medium (controls). Only three out of the six infected animals were pregnant by ultrasonography on day 29 after insemination, while two out of the three controls were pregnant. One control gilt aborted on day 23 after insemination but not due to PVC2. Viraemia was demonstrated in four out of six infected and in one control gilt that became infected with PCV2a. Anti-PCV2 antibody titres showed dynamic variations in the infected group throughout the study. Among infected gilts, the animal with the lowest anti-PCV2 titre (1/100) at the beginning of the experiment and another that reached a similar low value during the experiment showed evident seroconversion over time and had also PCV2 positive foetuses. One placenta displayed mild focal necrosis of the chorionic epithelium positively stained by immunohistochemistry for PCV2 antigen.

Conclusions

PCV2-seropositive gilts can be infected with PCV2 after intrauterine exposure and low maternal antibody titre may increase the probability of a foetal infection.