三个厂家猪瘟活疫苗免疫效果比较试验

在同一条件下对3个厂家生产的猪瘟细胞源活疫苗进行了免疫效果评价试验,并与猪瘟传代细胞苗免疫效果进行比较。结果发现3个厂家生产的猪瘟细胞源活疫苗存在一定差异,2个厂家的免疫效果较好,1个厂家的免疫效果较差。猪瘟传代细胞苗免疫效果优于猪瘟细胞源活疫苗,猪瘟传代细胞苗免疫1次,抗体合格率高,持续时间长,猪瘟细胞源活疫苗要免疫两次才能获得比较好的效果。同时,我们发现高致病性猪蓝耳病活疫苗（JXA1-R株）对猪瘟抗体产生有一定影响。     

Sow performance in an endemically porcine reproductive and respiratory syndrome (PRRS)-infected farm after sow vaccination with an attenuated PRRS vaccine

The objective of this field study was to evaluate in an endemically porcine reproductive and respiratory syndrome (PRRS) virus-infected farm the reproductive performance of sows after their vaccination with a PRRS attenuated vaccine. In a farrow-to-finish pig farm with history of endemic PRRS virus infection, a total of 200 gilts and sows were used. They were divided in 2 groups of 100 animals. The first group was used as untreated controls, while the animals of the second group were vaccinated against PRRS virus using the attenuated Porcilis PRRS vaccine (Intervet International, The Netherlands) based on European strain. All health and reproductive parameters were recorded from the time of vaccination up to next weaning. No adverse systemic or local reactions or side effects relative to vaccination were noted. Compared to controls, vaccinated sows showed significantly improved farrowing rate (89% versus 78%) and a tendency for fewer returns to oestrus, particularly those at irregular intervals. Fewer sows farrowed prematurely and showed post-partum dysgalactia syndrome, but more live pigs were born and weaned in each litter after vaccination. It was concluded that vaccination of sows with Porcilis PRRS attenuated vaccine in farms with endemic PRRSV infection has beneficial effects on their health and fertility.     

不同类型CpG基序对细粒棘球蚴Eg95抗原的免疫增强作用研究

为探究不同类型CpG基序对细粒棘球蚴（Echinococus granulosus）Eg95抗原的免疫增强作用,将pET-32a-3Eg95重组大肠杆菌,经IPTG诱导表达,亲和层析纯化重组蛋白pET-32a-3Eg95。将重组蛋白分别与常规佐剂Quli-A、pUC18-CpG和CpG ODN混合制备基因工程亚单位疫苗样品,免疫小鼠。通过间接ELISA方法检测抗体水平,通过实时荧光定量PCR方法测定体外刺激试验后细胞因子的相对表达量,检测不同佐剂在体液免疫和细胞免疫方面对Eg95抗原免疫效果的增强作用。结果显示,诱导表达重组蛋白的大小约为56 ku,与理论值相符,用羊细粒棘球蚴阳性血清检测有特异性条带;pUC18-CpG组和CpG ODN组小鼠免疫后14、28、42 d的抗体平均水平（D_{450 nm}）分别为3.10、3.03、3.22和2.98、3.12、3.27,与Quli-A组抗体平均水平相比均差异显著（P<0.05）;CpG ODN组血清中抗体平均水平略高于pUC18-CpG组,但差异不显著（P>0.05）。重组蛋白刺激后pUC18-CpG组与CpG ODN组IFN-β、IFN-γ和TNF-α的相对表达量分别为6.88、2.35、6.28和5.03、2.85、7.07,与Quli-A组相比差异均显著（P<0.05）。因此相对于常规佐剂Quli-A,pUC18-CpG和CpG ODN在体液免疫和细胞免疫方面对Eg95抗原都有显著的免疫增强作用,CpG ODN的免疫增强作用略优于pUC18-CpG,二者均可作为免疫佐剂,增强现有包虫病基因工程疫苗抗原的免疫效果。     

Immune Enhancement Activity of Different Types of CpG Motifs on the Recombinant Eg95 Antigen of Echinococus granulosus

To study the immune enhancement activity of CpG motifs on the recombinant Eg95 antigen of Echinococcus granulosus,Escherichia coli BL21(DE3) containing pET-32a-3Eg95 was grown in LB medium,and IPTG was then added for induction.The recombinant Eg95 protein was expressed and later harvested by affinity chromatography.The obtained antigen was mixed either with the conventional adjuvant Quli-A or with different types of CpG motifs (pUC18-CpG or CpG ODN),to prepare vaccine candidates for mice immunization.The average levels of serum IgG antibody were tested by indirect ELISA and the quantification of cytokine expression were determined by Real-time quantitative PCR after in vitro stimulation.The results identified that the size of the recombinant protein was 56 ku as predicted,and the recombinant protein could be recognized by serum from Echinococcus granulosus-infected sheep.The average levels of serum IgG antibody (D_{450 nm}) of the pUC18-CpG and CpG ODN groups on 14,28 and 42 d were 3.10,3.03,3.22 and 2.98,3.12, 3.27,respectively.Antibody levels from both CpG groups were significantly higher compared to the conventional adjuvant Quli-A group (P<0.05) The average level of serum IgG antibody in CpG ODN group was a little higher than that in pUC18-CpG group(P>0.05).After stimulation by recombinant protein 3Eg95,the relative expression levels of IFN-β,IFN-γ and TNF-α of pUC18-CpG and CpG ODN groups were 6.88,2.35,6.28 and 5.03,2.85,7.07,respectively,and both were higher than that of the Quli-A group (P<0.05).The results indicated that pUC18-CpG and CpG ODN had significant immune enhancement property on Eg95 antigen in both humoral and cellular immune response,and the immune enhancement activity of CpG ODN was slightly better than that of the pUC18-CpG.Hence,pUC18-CpG and CpG ODN could be used as efficient immune adjuvants enhancer in future vaccines against Echinococcus granulosus infection.     

Microbial ecology of swine farms and PRRS vaccine vaccination strategies

The present study investigated the microbial ecology and vaccination strategies against porcine reproductive and respiratory syndrome (PRRS) in field condition. Four representative farms with a history of PRRS were included in this study. Over the almost 3-year period, the average detection rate was 68.9%, making PRRSV the first most frequently detected virus, followed by porcine circovirus type 2 (PCV2) (64.2%), pseudorabies virus (PRV) (11.03%) and classical swine fever virus (CSFV) (4.41%). Streptococcus suis (77.92%), Haemophilus parasuis (51.25%) and Escherichia coli (52.39%), Pasteurella multocida (26.77%) were isolated most frequently in association with PRRSV. Under the present microbial ecology, production performances of sows their offspring after mass vaccination with a PRRS attenuated vaccine were evaluated. In addition, large scale PRRS vaccines usage and efficacy were further performed. The results indicated that mass vaccination following our immunization program can improve health status and production performances of both sows (2ml/i.m. booster after 4 weeks, and then immunized quarterly) and their offsprings (1ml/i.m. on 14-18 days of age).     

Experimental inoculation of late term pregnant sows with a field isolate of porcine reproductive and respiratory syndrome vaccine-derived virus.

The use of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in piglets has been associated with reproductive disorders in non-vaccinated sows. Vaccine-derived virus (VDV) has been isolated from foetuses, stillborn pigs, and dead piglets, indicating that the live vaccine spread from vaccinated piglets to non-vaccinated sows, and that the virus might be implicated in the severe reproductive problems observed. In the present study, one such VDV isolate was used to experimentally infect pregnant sows in the last trimester. The chosen isolate, which had more than 99.6% identity to the attenuated vaccine virus, originated from the lungs of a stillborn pig from a swine herd with a sudden high level of stillborn pigs and increased piglet mortality in the nursing period. Intranasal inoculation of sows with the virus isolate resulted in congenital infection, foetal death, and preweaning pig mortality. As such, the present study showed that vaccine-derived PRRSV can cause disease in swine consistent with PRRS.     

非洲猪瘟疫苗研制方向及进展

非洲猪瘟(African swine fever,ASF)给全球养殖业造成了巨大的威胁和挑战.非洲猪瘟病毒(African swine fever virus,ASFV)具有比寻常病毒更加巨大的基因组、迅速的变异能力和更加复杂多变的免疫逃逸机制,目前市场上仍无有效预防疫苗.科研人员几十年来,从灭活疫苗、减毒活疫苗、亚单...     

重组白细胞介素-2提高PRRS抗体阳性猪的猪瘟疫苗免疫效果试验

观察了重组白细胞介素-2（IL-2）对健康成年猪和PRRS抗体阳性猪的猪瘟疫苗免疫效果的影响。结果显示,重组IL-2和猪瘟疫苗一起免疫健康猪,20d后间接血凝抗体滴度达到1∶64的猪的比例为87.5％,而不注射IL-2的对照组抗体滴度可以达到这一水平的比例只有25％。给经2次猪瘟疫苗免疫但抗体滴度在1∶32以下的PRRS抗体阳性猪单独注射IL-2,20d后,注射前检测不到抗体的猪都检测到了抗体,注射前抗体滴度在1∶8～1∶16之间的猪的抗体滴度提高到1∶32～1∶64。再次用猪瘟疫苗和IL-2共同免疫,可使抗体滴度提高4倍以上。而不注射的对照组抗体滴度则略有下降。说明重组IL-2可以减轻PRRS感染所引起的免疫抑制,提高猪瘟疫苗的免疫效果。     

Analysis of the Immune Efficacy of Attenuated Live Vaccine in the Pig Herds Previously Infected with Wild-type Porcine Reproductive and Respiratory Syndrome Virus

To evaluate the immune efficacy of porcine reproductive and respiratory syndrome (PRRS) attenuated live vaccine,this study selected 4 litters from two small pig farms (two litters each farm) and vaccinated with porcine reproductive and respiratory syndrome virus (PRRSV) attenuated live vaccine.Sera were collected on different days post vaccination (dpv) to detect PRRSV nucleic acid by RT-PCR,and PRRSV antibody using two different ELISA Kits,i.e.N-ELISA and G-ELISA.The results showed that PRRSV nucleic acid were positive on 0 dpv until 30 dpv in piglets and also positive on 0 dpv in the corresponding sows.All piglets were negative for PRRSV antibody on 0 dpv,but were positive on 30 dpv until 150 dpv.The positive rates detected by N-ELISA Kit were higher than those of G-ELISA Kit on 30 and 60 dpv,but lower than those of G-ELISA Kit on 120 and 150 dpv.A total of 216 sera were detected respectively by two ELISA Kits and the coincidence rate of the results was 95.83%.The P value of χ² test was more than 0.05,showing there was no significant difference between the results of two Kits.The Kappa value was 0.87,showing there was strong consistence between them.The correlation coefficient was 0.605,showing there was significantly linear correlation between them.The results indicated that the wild-type PRRSV in the previously infected pig herds could be eliminated by vaccination with attenuated live vaccine and both N-ELISA and G-ELISA Kits could be used to estimate the immune efficacy of the attenuated live vaccine effectively.     

PRRSV野毒感染猪群免疫接种弱毒活疫苗的效果分析

为评估猪繁殖与呼吸综合征（porcine reproductive and respiratory syndrome,PRRS）弱毒活疫苗的免疫效果,本研究在两个小型猪场各选取两窝仔猪,在免疫接种猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus,PRRSV）弱毒活疫苗后不同时间采集血清,应用RT-PCR检测PRRSV核酸,应用N-ELISA和G-ELISA两种试剂盒检测PRRSV抗体。结果发现,免疫前0 d可以从仔猪血清中检测到PRRSV野毒株核酸,可持续到免疫后30 d;相对应的母猪在免疫前0 d可从血清中检测到PRRSV野毒株核酸。试验仔猪在免疫前0 d未能检测到PRRSV抗体,但在免疫后30～150 d均可检测到PRRSV抗体,其中N-ELISA试剂盒的阳性检出率在免疫后30、60 d高于G-ELISA试剂盒的阳性检出率,而在免疫后120、150 d则低于G-ELISA试剂盒的阳性检出率。使用两种ELISA试剂盒共同检测216份血清样品,检测结果的总体符合率为95.83%;配对χ²检验,P>0.05,两者差异不显著;Kappa值为0.87,属于极强一致性;两者相关系数r为0.605,具有显著线性相关。表明免疫接种弱毒活疫苗可以有效清除野毒感染猪群中的野毒株,N-ELISA和G-ELISA两种ELISA试剂盒均可用于评估弱毒活疫苗的免疫效果。     

猪流感疫苗研究进展

全面综述了国内外猪流感（SI）疫苗的研究现状。从激发体液免疫、细胞免疫、交叉保护以及避免母源抗体干扰和区分野毒感染等方面分别阐述了常规疫苗、减毒疫苗、亚单位疫苗、DNA疫苗、病毒活载体疫苗的研究进展,以期找到最佳控制猪流感的疫苗及免疫方案,为进一步研究猪流感疫苗提供参考。     

猪繁殖与呼吸综合征疫苗研究进展

猪繁殖与呼吸综合征是一种引起母猪繁殖障碍和新生仔猪呼吸道症状及高死亡率的传染病。在世界包括中国在内的各养猪国家广泛流行,造成严重经济损失,受到国内外学者的高度重视,对此病的研究特别是对其疫苗的研究正日益受到关注。文章就猪繁殖与呼吸综合征病毒的灭活疫苗、弱毒疫苗和基因工程疫苗的研究进展做了详细的阐述。     

猪繁殖与呼吸综合征免疫防制新途径的研究进展

猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是一种主要表现为母猪繁殖障碍与仔猪呼吸道症状的传染病。近年来,猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)变异株不断出现,免疫逃避及持续性感染使得猪群发病率或复发率均相继增高,给养猪业带来了巨大的损失。目前所采用的胃肠道途径接种活疫苗或灭活疫苗的方法无法诱导对猪群的全面保护作用。为减少养猪业的经济损失,亟需研制新防制方法和新疫苗接种途径。作者主要从黏膜免疫的免疫部位、呼吸道保护性黏膜免疫反应诱导、黏膜免疫途径、佐剂的选择及病毒的免疫抑制反应等方面简要论述了有效防制PRRSV的黏膜免疫方法的研究进展,为进一步了解黏膜免疫抵御PRRSV突变株感染及黏膜疫苗研制等方面提供有用的信息。     

非洲猪瘟疫苗研究的困境和展望

非洲猪瘟（ASF）目前是生猪产业最重要的猪病。因为非洲猪瘟病毒（ASFV）本身的复杂性,以及和宿主相互作用的复杂性导致ASF已经被报道一百年了,还没有商业化疫苗。理想的ASF疫苗不仅要有好的免疫保护性,更重要的是其安全性,同时如果能区分野毒感染和疫苗接种,能在适合的高质量GMP车间进行稳定低价的生产,能用于不同物种就更好了。ASF灭活疫苗研制这条道路似乎不通;亚单位疫苗、DNA疫苗、病毒活载体疫苗的免疫保护能力不足;主要包含自然缺失、传代致弱、基因工程缺失的减毒活疫苗在免疫保护方面体现了非常大的优势,但是其潜在的持续感染风险,会造成猪只的副作用,包括皮肤溃烂、关节炎,导致神经系统、呼吸系统的损伤等问题非常值得警惕。复制缺陷单周期病毒能有效地解决减毒活疫苗的安全性问题,似乎是一个值得尝试的ASF疫苗研制方向,尽管存在难以确定缺失复制相关的基因,以及难以构建能高效表达缺失基因编码蛋白,且能让单周期病毒稳定大量生产的辅助细胞系。我国针对ASFV强毒的精准剔除策略,未注册非法弱毒苗造成临床严重损失,以及一些不使用疫苗但成功从国家层面净化ASF的案例,让我们认识到针对ASF基础研究的重要性。同时至少目前阶段,ASF的防控不一定要借助疫苗,更多的要做好生物安全管控和区域ASF净化。尽管ASF疫苗研制困难重重,但针对ASF理想型疫苗的研制也应该持续进行下去,未来可能作为ASF防控的一个突破点。     

Simultaneous vaccination of piglets against foot-and-mouth disease and classical swine fever

Six-week-old piglets, born of unvaccinated sows, were vaccinated against foot-and-mouth disease (FMD) with a trivalent, inactivated vaccine containing an adjuvant or vaccinated against classical swine fever (CSF) with a live attenuated vaccine or against both diseases simultaneously at two different sites. The antibody response to the FMD vaccine was not significantly influenced by the simultaneous vaccination against CSF. FMD vaccine administered simultaneously with the CSF vaccine produced a significantly higher antibody response to CSF than occurred with CSF vaccination only.     

Threonine and tryptophan supplementation enhance porcine respiratory and reproductive syndrome (PRRS) vaccine‐induced immune responses of growing pigs

The aim of the present study was to investigate influences of threonine and tryptophan supplementation (TTS) on immune response of growing pigs inoculated with modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. Twenty growing barrows (Landrace × Yorkshire) were randomly assigned to four groups according to the PRRS vaccination and TTS. Serum samples were collected from all pigs at days 0, 7, 14, 21, 28, 35, 49 post‐vaccination (day 0 defined as the day of vaccination). Pigs were euthanized and samples collected at day 49 post‐vaccination. The results showed that TTS tended to increase weight gain and average daily gain (ADG) of pigs (P < 0.1). PRRS vaccine enhanced serum PRRSV‐specific antibody, serum virus neutralizing (SVN) antibody and interferon‐γ, interleukin (IL)‐10 and IL‐1β concentrations (P < 0.05). The expression of TLR3 and TLR7 mRNA in lymph nodes were higher in TTS than in the control group after PRRS vaccine inoculation (P < 0.05). TTS diet mitigated lung damage which is induced by PRRS vaccination from microscopic evaluation. These results suggest that dietary TTS could improve growth performance of growing pigs, which may be ascribed to the improved immune response and mitigated lung damage.     

Comparative evaluation of PRRS virus infection in vaccinated and naïve pigs

The purpose of this study was to evaluate the time-course of the immune response to a field Porcine Respiratory and Reproductive Syndrome virus (PRRSV) strain in PRRS-naïve, untreated pigs, as well as in four groups of age and breed-matched pigs injected with a live attenuated PRRS vaccine, its adjuvant, an inactivated PRRS vaccine and an irrelevant, inactivated Porcine Circovirus type 2 (PCV2) vaccine, respectively. PRRSV infection was confirmed in all groups by PCR and antibody assays. The antibody response measured by ELISA took place earlier in pigs injected with the live attenuated vaccine, which also developed a much stronger serum-neutralizing antibody response to the vaccine strain. Yet, no clear protection was evidenced in terms of viremia against the field virus strain, which showed 11.1% nucleotide divergence in ORF7 from the vaccine strain. In vitro, the interferon (IFN)-γ response to PRRSV was almost absent on PVD 60 in all groups under study, whereas the prevalence of interleukin (IL)-10 responses to PRRSV was fairly high in PCV2-vaccinated animals, only. Results indicate that distinct patterns of immune response to a field PRRSV strain can be recognized in PRRS-vaccinated and naïve pigs, which probably underlies fundamental differences in the development and differentiation of PRRSV-specific immune effector cells.     

Regulating effects of porcine interleukin-6 gene and CpG motifs on immune responses to porcine trivalent vaccines in mice

In order to develop novel immunoadjuvants to boost immune response of conventional vaccines, experiments were conducted to investigate the regulating effects of porcine interleukin-6 gene and CpG motifs as the molecular adjuvants on immune responses of mice that were co-inoculated with trivalent vaccines against Swine fever, the Pasteurellosis and Erysipelas suis. Synthetic oligodeoxynuleotides containing CpG motifs were ligated into pUC18, forming recombinant pUC18-CpG plasmid. Eukaryotic plasmid expressing porcine interleukin-6 (VPIL-6) were also constructed as molecular adjuvants in an attempt to enhance levels of immune responses of mice co-administered with the trivalent vaccines in this paper. The cellular and humoral immune responses of mice were systematically analysed, and the experimental results were observed that the number of white blood cells, monocytes, granuloytes and lymphocytes significantly increased, respectively, in the mice immunized with VPIL-6, compared with those of the control; the IgG content and titre of specific antibodies to the trivalent vaccine mounted remarkably in the sera from the VPIL-6 vaccinated mice; the proliferation of lymphocytes and induced IL-2 activities were significantly increased in the vaccinated groups. The above-mentioned immune responses of mice co-inoculated with pUC18-CpG plasmid were significantly stronger than those of co-inoculated with pUC18 plasmid, suggesting that the immunostimulatory effect of oligodeoxynuleotides CpG is closely connected with the number of CpG motifs. These results suggest that the porcine IL-6 gene and CpG motifs could be employed as effective immunoadjuvants to elevate immunity to conventional vaccines.     

CpG寡核苷酸对IBDV VP2基因真核表达质粒免疫增效作用

以传染性法氏囊病病毒(IBDV)VP2蛋白基因表达质粒DNA为免疫原，以CpG的寡核苷酸(CpG-0DN)为免疫佐剂，肌肉注射于14日龄SPF鸡，1周后加强免疫1次，2次免疫后15d和21d分别测定血清ELISA抗体效价，并于免疫后21d用IBDV99儿强毒株攻毒和进行病理学观察。结果显示，(1)VP2基因重组质粒DNA与CpG共同免疫组的ELISA抗体水平明显高于VP2重组质粒免疫组；(2)IBD弱毒苗与VP2重组质粒免疫组抗体水平明显高于VP2重组质粒免疫组，且比VP2基因重组质粒DNA与CpG共同免疫组略高；(3)VP2基因重组质粒DNA与CpG共同免疫组及IBD弱毒苗与VP2重组质粒免疫组可明显降低IBDV强毒攻击后引起的急性发病率和死亡率。由此表明，CpG寡核苷酸对IBDV VP2蛋白基因真核表达质粒免疫具有明显增强作用，有很大的应用前景。     

Protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus PA/Wolgemuth/98

Protection provided by live and inactivated virus vaccination against challenge with the virulent nephropathogenic infectious bronchitis virus (NIBV) strain PA/Wolgemuth/98 was assessed. Vaccinations with combinations of live attenuated strains Massachusetts (Mass) + Connecticut (Conn) or Mass + Arkansas (Ark) were given by eyedrop to 2-wk-old specific-pathogen-free leghorn chickens. After live infectious bronchitis virus (IBV) vaccination, some chickens at 6 wk of age received an injection of either an oil emulsion vaccine containing inactivated IBV strains Mass + Ark or an autogenous vaccine prepared from NIBV PA/Wolgemuth/98. Challenge with PA/Wolgemuth/98 was given via eyedrop at 10 wk of age. Serum IBV enzyme-linked immunosorbent assay antibody geometric mean titers (GMTs) after vaccination with the combinations of live attenuated strains were low, ranging from 184 to 1,354, prior to NIBV challenge at 10 wk of age. Both inactivated vaccines induced an anamnestic response of similar magnitudes with serum GMTs of 6,232-12,241. Assessment of protection following NIBV challenge was based on several criteria virus reisolation from trachea and kidney and renal microscopic pathology and IBV-specific antigen immunohistochemistry (IHC). Live attenuated virus vaccination alone with combinations of strains Mass + Conn or Mass + Ark did not protect the respiratory tract and kidney of chickens after PA/Wolgemuth/98 challenge. Chickens given a live combination vaccination of Mass + Conn and boosted with an inactivated Mass + Ark vaccine were also susceptible to NIBV challenge on the basis of virus isolation from trachea and kidney butshowed protection on the basis of renal microscopic pathology and IHC. Live IBV-primed chickens vaccinated with an autogenous inactivated PA/Wolgemuth/98 vaccine had the highest protection against homologous virulent NIBV challenge on the basis of virus isolation.