2016——2018年我国部分猪群塞尼卡病毒回顾性监测

塞尼卡病毒(Senecavirus A,SVA)是最近新出现的可引起猪水泡样病变的单股小RNA病毒。为了解SVA的流行状况,采集对2016—2018年从我国辽宁等省份猪群的164份病料和2018年从我国福建等7省份35个屠宰场的458份组织混合样品和95份血清样品,用荧光实时定量RT-PCR方法进行SVA检测和分析。结果显示:在2016年的48份样品中,从3个省份检测出7份阳性样品,阳性率为14.6%。2017年的32份病料中,从5个省份检测出7份阳性样品,占比为21.9%;2018年的84份病料中,从3个省份检测出19份阳性样品,占比为22.6%。屠宰场组织样品中,从6个省份检出55份SVA阳性样品,阳性率为12.0%;血清样品中,从1个省份检出7份阳性样品,阳性率为7.4%。结果表明,我国猪群SVA感染年份至少可追溯到2016年;SVA在我国流行面相对较广,且猪群中存在隐性带毒,因此须重视猪群的SVA监视和监测。     

增强猪传染性胸膜肺炎灭活苗对小鼠保护力的研究

为了提高猪传染性胸膜肺炎灭活苗的保护力,本研究利用基因重组技术表达了猪传染性胸膜肺炎放线杆菌6种主要致病因子蛋白:rApxⅠ、rApxⅡ、rApxⅢ、rApxⅣ、rApfa和rOMP。设立试验Ⅰ组(灭活苗),试验Ⅱ组(灭活苗、rApxⅠ、rApxⅡ、rApxⅢ和rOMP),试验Ⅲ组(灭活苗、rApxⅠ、rApxⅡ、rApxⅢ和rApxⅣ),试验Ⅳ组(灭活苗、rApxⅠ、rApxⅡ、rApxⅢ和rApfa)和空白对照组(PBS),每组26只BALB/c小鼠,分3次免疫,每次间隔2周,每周检测血清抗体水平,在免疫后第2周、第4周和第5周,MTT法检测脾脏T细胞增殖水平及ELISA法测定IFN-γ分泌水平。在第3次免疫后的第7 d,将每组剩余20只小鼠平均分成2组,分别以APP1型菌(5×10~9 CFU)和APP7型菌(1×10~(11) CFU)进行攻毒保护试验。结果表明,向灭活苗中添加重组亚单位成份可以提高机体的细胞免疫和体液免疫水平,并能提高灭活苗的交叉保护率;试验Ⅱ组的ApxⅠ和OMP抗体水平以及细胞免疫水平均显著高于其它各组(p<0.05),对于APP1和APP7型菌的攻毒也提供了明显优于其它各组的保护结果,试验Ⅲ、Ⅳ组的细胞、体液免疫水平及保护率较试验Ⅰ组也有所提高。结果显示出ApxⅠ、OMP抗体水平和细胞免疫水平与攻毒保护率之间存在着正相关性,向灭活苗中加入重组蛋白成份可以提高疫苗的交叉保护,试验Ⅱ组通过激活体液免疫和细胞免疫,对两个血清型APP的攻击提供了很好的保护作用,从而为猪传染性胸膜肺炎新型疫苗的研制提供参考。     

间接ELISA检测猪伪狂犬病血清抗体

用猪肾传代细胞IBRS－2增殖猪伪狂犬病病毒（PRV）鄂A株，病毒培养上清液经硫酸铵沉淀、聚乙二醇（Mr20000）浓缩后作为包被抗原。用纯化的猪血清IgG免疫家兔，HRP村记撮的兔抗猪IgG，制备出高效价的酶标抗体，酶标抗体工作浓度为1：50000；经各种条件的选择，建立了检测猪伪狂犬病血清抗体的间接ELISA。所建立的间接ELISA抗原包被浓度为39.2mg/L，血清最佳稀释度为1：20，与猪细小病毒、猪、O型口蹄疫、猪衣原体标准阳性血清呈阴性反应，与标准阴性血清和临床未感染PRV的猪血清呈阴性反应；与猪伪狂犬病标准阳性血清、免疫猪血清和临床发病猪血清呈明显的阳性反应；与美国进口的PRV抗体检测ELISA诊断试剂盒检测结果比较，45份猪血清的阴、阳性检出符合率均为100％。表明建立的间接ELISA具有敏感性高、特异性强、重复性好的优点，可用于猪伪狂犬病血清抗体的定性和定量检测。     

猪繁殖-呼吸综合征活疫苗对仔猪的安全性试验

本试验用猪繁殖-呼吸综合征（PRRS）活疫苗和国内分离的PRRS强毒CH—1a株接种PRRS阴性的断奶仔猪，分别在接种后的3、7、14d各剖杀1头，取各脏器分别做冰冻切片和病理切片观察。用间接免疫荧光法检测各脏器PRRS病毒的分布。结果表明，PRRS活疫苗在免疫初期，抗原主要分布在脾脏、淋巴结，其次是肾脏和肺脏，少见于肝脏和心脏，第14d时在脾、淋巴结和肾脏有一定量的抗原，而肺脏相比则数量很少，肝脏和心脏未检到PRRS病毒抗原的存在，表明接种PRRS活疫苗随着时间的推移抗原分布呈下降趋势。而强毒抗原分布以脾脏最多，依次是肾脏、肺脏、淋巴结、肝脏、心脏，接种后第14d仍能在各脏器检到PRRS病毒抗原。病理组织学检测结果表明，活疫苗产生以下颌淋巴结、脾脏增生为特征的免疫应答，组织损伤轻微，对肺的病变较少，且仔猪生长良好。强毒则引起以大面积的肺泡隔增宽为特点的间质性肺炎和微循环障碍的病理变化，淋巴小结、脾脏滤泡发生崩解与周围界限不清，个别淋巴细胞核浓缩，组织损伤严重。本试验表明弱毒疫苗对仔猪是安全的。     

PCV2衣壳蛋白羧基端基因在T4噬菌体表面的展示及ELISA方法的建立

用PCR扩增猪圆环病毒Ⅱ型广东分离株的衣壳蛋白羧基端基因,将PCR产物连接到pR质粒,转化DH5α细胞,筛选阳性克隆进行PCR鉴定并测序后,转化E2菌,将重组E2菌与缺陷型噬菌体T4-Z1同源重组后,得到重组噬菌体,经SDS-PAGE和Western blotting分析,表明衣壳蛋白片段在噬菌体表面正确展示,表达的融...     

Study on Anti-viral Effect of Rhubarb on Porcine Epidemic Diarrhea Virus in vitro

The inhibitory effects of rhubarb on cell lesion, which was caused by porcine epidemic diarrhea virus (PEDV), and its mechanism had been studied in this research.The maximum safe concentration of rhubarb solution applied to Vero cells was determined through morphological observation, and then the PEDV infected Vero cell model in vitro was established, by which the inhibitory effect of rhubarb on virus was studied.The experimental method had also been established based on this cell model.The result showed that the maximum safe concentration was 3.28 mg/mL.Both morphological observation and MTT assay were indicated that rhubarb could not only block the attachment of the virus to Vero cell and inhibit the synthesis and reproduction of the virus, but also could directly inactivate the virus.This research showed that rhubarb had an inhibitory effect on PEDV, and provided a theoretical basis for the prevention and treatment of clinical porcine epidemic diarrhea.     

猪β防御素研究进展

β防御素是猪体内分泌的一类抗菌肽，广泛分布于各个组织中，在抵抗病原入侵和免疫调节中发挥着重要作用。猪β防御素对细菌、真菌和病毒都有很强的杀灭作用，并且具有分子量小、热稳定性好、无残留等优点，是理想的抗生素替代品。研究表明，猪β防御素的表达与个体抗病力正相关，增加内源性防御素表达或直接摄入外源性防御素均能增强机体免疫力并促进生长。脂肪酸、氨基酸、维生素、益生菌、病原微生物等外源刺激皆可影响β防御素的表达。随着防御素相关研究的深入，将有望解决养猪生产中长期使用抗生素而导致的耐药性、药物残留及环境污染等问题。文章介绍了猪β防御素的结构特征、表达特异性及生物学功能，主要综述了外源刺激对β防御素表达的影响及其作用机制，初步说明外源β防御素作为饲料添加剂在仔猪生产中的效果，旨在为猪β防御素的深入研究及其在生猪健康养殖中的应用提供参考。     

Use of Porcine Meal in Plant‐based Practical Diets for Pacific White Shrimp,Litopenaeus vannamei

Two growth trials were conducted to evaluate and confirm the efficacy of a porcine meal (PM) with high protein content (>90%) as an alternative feed ingredient in commercial‐type feed formulation for Pacific white shrimp, Litopenaeus vannamei. Six experimental diets were formulated for the two growth trials. The first five diets contained increasing levels (0, 1, 2, 4, and 6%) of PM as a replacement for soybean meal in a plant‐based diet with low inclusion level (6%) of fish meal (FM). The last experimental diet was produced utilizing 4.2% PM to completely replace FM. In Trial 1, shrimp (1.5 g initial mean weight, 20 shrimp/tank, n = 4) were offered test diets for 6 wk in a semirecirculation system. At the end of Trial 1, shrimp fed with the diet containing 6% PM exhibited significantly enhanced weight gain (WG), feed conversion ratio (FCR), and survival compared to those fed with the diet devoid of FM. As survival was poor across all treatments and different densities could mask growth results the trial was repeated. In Trial 2, shrimp (0.85 g, 15 shrimp/tank, n = 4) were offered diets for 6 wk. Dietary supplementation of PM at 6% significantly improved WG, FCR, and apparent net protein retention in contrast with the treatment devoid of FM, confirming the same trends in Trial 1. No significant difference was detected in protein, lipid, moisture, and mineral profiles of whole‐body shrimp as well as survival across all the treatments. Results of this study indicate that PM is a good high protein source in shrimp feeds, which can be included up to 6% in the low FM‐based diet without compromising the growth of shrimp.     

Identification and Molecular Characteristics Analysis of Porcine Epidemic Diarrhea Virus Variants

To ascertain a diarrhea case in a pig farm in Shandong province,the pathological changes of dead piglets were observed and nested RT-PCR test was carried out on 7 diarrhea samples for porcine epidemic diarrhea virus (PEDV).Pathological examination revealed that intestine was detected as enlargement,hyperemia and edema.After histological examination,the typical microscopic lesions of intestine were disappearance of epithelial cells,villus shrinkage and shortening.The result of nested RT-PCR showed that all of the 7 samples could amplify a specific target band of PEDV.Molecular characteristics of two field strains showed that they had an amino acid homology of 92.3% to 92.4% with vaccine CV777 and 96.6% to 98.6% with other previous field strains which sequences were downloaded from GenBank.Phylogenetic tree analysis further revealed that all of PEDV strains could be mainly divided into two clusters of G1 and G2. G2 consisted of the field strains of our study and other field strains from USA,China and so on,which had the same sequence characteristics of two insertions and one deletion,while G1 consisted of all vaccines of CV777 and several older field strains from China and Korea.These results indicated that our field strains were the dominant strains in recent epidemic diarrhea occurrence.Moreover,its molecular characteristics might be a characterization of differentiating the field and vaccine strains.     

基于S基因序列分析新型猪流行性腹泻病毒的遗传演化

最近，笔者实验室在青藏高原地区发现两种新亚型藏猪源猪流行性腹泻病毒（PEDV），为进一步调查新型PEDV是否在四川腹泻猪群中存在或流行，对实验室2018-2019年保存的116份猪腹泻粪便或肠组织样本进行PEDV的检测及其纤突蛋白基因（spike）分子特征研究。结果表明：腹泻样本的PEDV检出率为42.2%（49/116，95% CI=33.1%~51.8%），并获得了13条完整的S基因序列，全长为4 149~4 170 bp，序列相似性为94.2%~99.9%，其中SWUN-H3-CH-SCYA-2019的S基因与藏猪源新G1亚群PEDV的序列相似性高达97.0%~98.6%。遗传演化研究结果表明13株PEDV S基因划分为G1和G2大群，其中SWUN-H3-CH-SCYA-2019位于藏猪源新G1亚群；SWUN-19-CH-SCZY-2018、SWUN-4-CH-SCXC-2018、SWUN-1-CH-SCNJ-2019和SWUN-3CH-CH-SCZG-2019位于G2亚群中一个独立的分支，且与藏猪源新G2亚群毒株有着较近的亲缘关系。为了进一步研究13株PEDV的演化过程，以贝叶斯进化分析软件包（BEAST）进行分歧时间估算，结果表明SWUN-H3-CH-SCYA-2019的分歧时间约为2012.3年，早于藏猪源新G1亚群其余毒株的最早分歧时间（2015.7年）；SWUN-4-CH-SCXC-2018、SWUN-19-CH-SCZY-2018和SWUN-3CH-CH-SCZG-2019的分歧时间约为2014.2年，早于G2亚群的藏猪源毒株2014.7年，所有藏猪源PEDV的分歧时间均晚于四川毒株。本研究在四川地区首次发现了藏猪源PEDV，并且从毒株的分歧时间推断青藏高原的藏猪源PEDV来源于四川，为新型PEDV分子遗传进化的监测提供了依据。