某规模化猪场猪瘟疫苗免疫抗体检测与分析

采用猪瘟抗体检测试剂盒对某规模化猪场种猪和仔猪猪瘟疫苗免疫的抗体水平进行检测、分析。结果表明,不论是种猪还是仔猪,要有足够的免疫剂量才能达到较好的免疫效果。     

实时荧光定量RT-PCR法比较猪瘟疫苗病毒含量

为有效检测福建省使用猪瘟兔化弱毒疫苗病毒含量,给养殖户选择高效疫苗提供科学指导。本研究收集福建省内使用的10个不同厂家生产的23个批次猪瘟疫苗,应用实时荧光定量RT-PCR方法检测疫苗中病毒核酸的含量,以其中的标准品对照,比较相应的兔体反应量。结果表明：抽检23个猪瘟疫苗样品合格率仅为69.56%,传代细胞苗的病毒含量相对较高,脾淋苗病毒含量优于细胞苗;不同厂家生产的同类型疫苗病毒含量差异大,同一厂家生产的同类疫苗存在批间差异。实时荧光定量RT-PCR检测猪瘟疫苗病毒含量是一种便捷、高效的方法,为今后临床猪瘟疫苗的使用提供有效指导依据。     

适于猪场污水中快速生长富油微藻的筛选

微藻的大规模培养以及生产生物燃料在很大程度上依赖于所使用的微藻藻株性能.为了从自然水域中筛选、分离能够异养生长并同步处理养猪废水和实现油脂积累的高油脂产率微藻,本研究采用平板划线、单胞分离和毛细管分离3种方法,对51个采样点的样品进行分离、纯化,获得了118株藻,71株能够进行异养生长,其中33株能够在猪场污水中生长,且17株藻生长良好.根据形态特征对分离得到的部分藻株初步鉴定为小球藻(Chlorella sp.)和栅藻(Scenedesmus sp.).比较这17株藻在猪场污水中的生长速率和油脂含量,藻株13-6、19-4、20-6和34-2的比生长速率分别为0.147、0.162、0.177和0.154 d-1,较其他藻株生长更快;19-4、24-1和34-2的油脂含量分别为19.7％、22.9％和28.8％,高于其他藻株.从中选取生长快且油脂含量高的藻株19-4和34-2经18S rDNA鉴定为Chlorella sorokinlana和Chlorella sp.,分别命名为C.sorokinlana 19-4 (GenBank登录号:KU948990)和Chlorella sp.34-2 (GenBank登录号:KU948991).将微藻培养体系扩大至30 L反应器,利用稀释猪场污水培养C.sorokinlana 19-4和Chlorella sp.34-2,最大比生长速率分别达到0.153和0.149 d-1;油脂含量分别达到18.73％和29.27％;最大生物量浓度分别达到0.78和1.12 g/L.C.sorokinlana 19-4对废水培养基中总氮、总磷的最高去除率分别高达70.56％和90.98％,34-2则分别为60.24％和85.07％.脂肪酸组分分析表明,Chlorella sp.34-2主要含有Cl6∶0、C18∶2n6c及C18∶3n3,其脂肪酸组分含量符合生物柴油生产的原料要求标准.结果说明这两株微藻在净化废水和生产生物柴油中具有潜在的应用前景.利用猪场污水养殖微藻,既可以节约大量营养盐成本,促进微藻生物柴油产业的推进,又可以净化污水,促进水资源再利用,实现能源与环境的双赢.     

RNA可视化原位杂交技术对感染细胞中猪瘟病毒RNA定位与分布

【目的】为研究CSFV RNA在体外感染细胞中的分布及定位,建立了一种准确、敏感的RNA可视化原位杂交技术。【方法】本研究通过比对GenBank公布的CSFV、BVDV和BDV全序列,避开BVDV和BDV的同源区,设计了CSFV RNA及内参基因β-actin的特异探针。以CSFV中等致病力毒株（HeBHH1/95）为参考毒株,在PK15细胞中培养病毒,加入RNA可视化原位杂交特异探针和相应试剂,采用荧光共聚焦显微镜进行成像观察。通过综合分析观测结果、荧光强度、重复性等因素,采用正交试验优化了对原位杂交过程中具有重要影响的蛋白酶K浓度和甲醛固定时间,建立了CSFV RNA可视化原位杂交技术,并与FAT方法比较该技术灵敏度;用我国目前流行的CSFV 1.1、2.1、2.2、2.3基因亚型及BVDV、PPV、PRV和PCV-2病毒进行特异性试验。最终,以CSFV强致病力毒株（SM）接种PK15细胞,病毒感染后0.5、1、3、6、8、10、14、18、24、36、48、72、96h(hours post inoculation,hpi)取样,每个时间点2个重复,采用CSFV RNA可视化原位杂交技术进行检测。为佐证病毒蛋白在细胞中的定位及分布,同时采用FAT方法对SM株E2蛋白在PK15细胞中的表达情况进行动态研究。【结果】采用该技术在荧光共聚焦显微镜下可观察到CSFV RNA在细胞中的定位;当蛋白酶K浓度为1:1 000、甲醛固定时间为30min时为最优反应条件;灵敏度试验表明该技术对病毒的检测极限为10-8/200μL,比FAT高3.5个数量级;特异性试验结果显示该探针能与CSFV 1.1、2.1、2.2、2.3亚型结合,与BVDV、PPV、PCV-2、PRV无交叉反应。采用该技术对CSFV RNA感染后在靶细胞中的定位与分布研究结果显示:0.5hpi在胞核和胞浆均能检测到RNA,0.5-6hpi RNA主要分布于胞核内并在核内富集;10hpi胞浆内RNA逐渐增多,胞核内RNA逐渐减少,24hpi RNA主要集中在胞浆内细胞核周围;36hpi核外RNA大量聚集增多,72hpi达到峰值;96hpi RNA总量有所下降。而FAT结果显示：8hpi在少数细胞的胞浆内检测到病毒E2蛋白,10-24hpi蛋白表达数量一直较少;36hpi蛋白表达量不断增多,72hpi到最大值;96hpi荧光信号由强变弱。病毒蛋白在细胞浆内的聚集数量与细胞浆中RNA含量成正相关。【结论】首次建立了CSFV RNA可视化原位杂交检测技术,并对CSFV强致病力毒株在细胞中的RNA定位分布进行了研究,发现病毒RNA吸附和进入靶细胞的时间早于0.5hpi,观察到CSFV RNA有在细胞核内的生活史。     

猪支原体肺炎控制和净化评价方法研究进展

简述了猪支原体肺炎各种防控措施的常规评价指标,包括临床症状和发病率的降低、肺部肉变的评分、血清抗体滴度的变化及肺炎支原体病原监测等,介绍了体液免疫和黏膜免疫评价指标分别在猪支原体肺炎灭活疫苗和弱毒活疫苗免疫效力评价中的作用,并对气溶胶中猪肺炎支原体的检测成为猪支原体肺炎防控和净化评价的主要方法之一的可能性进行了论述。     

Multilocus sequence typing as a tool for studying the molecular epidemiology and population structure of Brachyspira hyodysenteriae

The purpose of this study was to develop and apply a multilocus sequence typing (MLST) scheme to study the molecular epidemiology of Brachyspira hyodysenteriae, the aetiological agent of swine dysentery. Sequences of seven conserved genomic loci were examined in 111 B. hyodysenteriae strains. Fifty-eight of these previously had been analysed by multilocus enzyme electrophoresis (MLEE), and for some the results of pulsed field gel electrophoresis (PFGE), restriction endonuclease analysis (REA) and/or serotyping also were available. The discriminatory power of these methods was compared. The strains were divided into 67 sequence types (STs) and 46 amino acid types (AATs) by MLST. The Index of Association value was significantly different from zero, indication that the population was clonal. Eleven clonal complexes (Cc) comprising between 2 and 10 STs were recognised. A population snapshot based on AATs placed 77.5% of the isolates from 30 of the AATs into one major cluster. The founder type AAT9 included 13 strains from nine STs that were isolated in Australia, Sweden, Germany and Belgium, including one from a mallard. The MLST results were generally comparable to those produced by MLEE. The MLST system had a similar discriminatory power to PFGE, but was more discriminatory than REA, MLEE or serotyping. MLST data provided evidence for likely transmission of strains between farms, but also for the occurrence of temporal “micro-evolution” of strains on individual farms. Overall, the MLST system proved to be a useful new tool for investigating the molecular epidemiology and diversity of B. hyodysenteriae.     

High HEV presence in four different wild boar populations in East and West Germany

Swine Hepatitis E virus (HEV) can be transmitted from pigs to humans causing hepatitis. A high prevalence of HEV in wild boar populations is reported for several European countries, but actual data for Germany are missing. During the hunting season from October to December 2007 liver, bile and blood samples were collected from wild boars in four different German regions. The samples were tested for HEV RNA by quantitative PCR (qPCR) and anti-HEV IgG antibodies by two different ELISAs and a Line immunoassay. A seroprevalence of 29.9% using ELISA and 26.2% in the Line immunoassay was determined. The seroprevalence rate varied greatly within the analyzed regions. However, qPCR analysis revealed a higher prevalence of 68.2% positive animals with regional differences. Surprisingly, also adult wild sows and wild boars were highly HEV positive by qPCR. Compared to liver and serum samples, bile samples showed a higher rate of positive qPCR results. Sequencing and phylogenetic analysis of a 969 nt fragment within ORF 2 revealed that all isolates clustered within genotype 3 but differed in the subtype depending on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one population HEV isolates were closely related, but social groups of animals in close proximity might be infected with different subtypes. Two full-length genomes of subtypes 3i and 3e from two different geographic regions were generated. The wild boar is discussed as one of the main sources of human autochthonous infections in Germany.     

Serotype distribution and production of muramidase-released protein, extracellular factor and suilysin by field strains of Streptococcus suis isolated in the United States

Streptococcus suis is an important swine pathogen and a zoonotic agent. Differences in virulence have been noted among the 33 described serotypes, serotype 2 being considered the most virulent. In this study, we aimed at assessing the serotype distribution and the production of virulence-associated markers by strains recovered from diseased pigs in the United States (U.S.). Results showed that among the 100 strains evaluated, serotype 3 (20% of the isolates) and serotype 2 (17%) were the most prevalent. We then investigated the presence in these isolates of the genes sly, epf and mrp, encoding the virulence-associated markers suilysin (SLY), extracellular factor (EF) and muramidase-released (MRP) protein, respectively. The effective production of the markers by the strains was also verified. Results showed that the presence of the gene did not always correlate with actual expression of the respective protein. In the case of MRP, this was due, in most cases, to frameshift mutations at the 5′ end of the gene resulting in premature stop codons. The most prevalent phenotypes among U.S. strains were MRP⁺EF⁻SLY⁻ (40%) and MRP⁻EF⁻SLY⁺ (35%). Serotype distribution greatly differed from that reported in several European countries, as did the production of virulence markers, particularly for serotype 2. On the other hand, our results for the U.S. S. suis isolates are similar to those reported for Canadian strains, suggesting a common status in North America.     

不同杂交组合商品猪的肥育性能与胴体品质研究

本研究选用二元长大（L×Y）、三元杜长大（D×LY）、四元皮杜长大（PD×LY）三个杂交组合猪群为实验动物,进行生长发育性能、胴体品质性状测定,旨在为优质肉高效配套杂交组合筛选提供理论依据。研究表明：PD×LY日增重、料肉比及屠宰率最优,日增重、屠宰率与L×Y差异显著（P〈0．05）;PD×LY眼肌面积、后腿比例分别为48．09cm2、34．23％,为三组合中最高,并与L×Y差异显著（P〈0．05）,与D×LY组差异不显著（P〉0．05）。     

吉林省养猪业现状和发展策略

阐述了吉林省的养猪优势,饲料资源丰富、人均国土资源占有量大、畜禽品种资源丰富等等。提出了吉林省养猪业的发展策略、发展目标及发展思路。规划了养猪产业的区域布局。明确了吉林省养猪业的主攻方向和发展重点,应加强种源基地建设、建立遗传资源场、对限制养猪业发展的关键技术进行联合攻关、建立牧业小区和家庭牧场、大力发展各种类型的养猪合作社和养猪协会,建立完善的技术服务体系。