桂林市年出栏生猪600万头以上,饲养总量与规模长期位于广西前三行列,养猪业在农业中地位越来越重要. 在集约化养猪生产循环中,仔猪阶段是提高生产效率最为关键的环节. 据统计,仔猪阶段(仔猪出生后～2周)因各种原因造成的死亡率占整个仔猪阶段(出生至断奶后1～2周)的65％左右,而仔猪阶段的死亡率占猪一生死亡率的70％[1] ,由此可见,仔猪阶段对疫病的预防与控制好坏对养殖效益起到关键作用.

为建立一种快速检测猪流行性腹泻病毒(Porcine Epidemic Diarrhea Virus,PEDV)病原的方法,根据GenBenk上的猪流行性腹泻病毒N基因序列,设计合成一对引物,建立了检测PEDV的一步法RT-PCR方法,并对其特异性、灵敏性及重复性进行了研究.结果表明:建立的一步法RT-PCR方法具有较好的特异性、灵敏性及重复性,最低可检测出约1 pg PEDV的RNA.该方法为P E DV的病原检测及其分子流行病学调查提供了有效的诊断方法,可用于猪流行性腹泻的早期确诊和病毒鉴定.

[Objective] To provide a scientific basis for better prevention and control of PEDV,the infection of PEDV and its current characteristics of genetic evolution in Guangxi were studied.[Method]86 PEDV field strains were isolated from 25 farms in Guangxi during 2012-2016.N gene was amplified from the 86 PEDV field strains by RT-PCR,cloned,sequenced and compared with the other reference strains in GenBank.[Result] Sequence analyses of the N genes were performed,and the results showed that 86 strains had nucleotide homology of 94.2 ％-100.0 ％ and 94.1％-100.0 ％ homology with reference strains.Phylogenetic analysis of N gene indicated that Guangxi strain could be divided into two groups.Cluster 2 consisted of the Korean strains DR13,Japanese strain 83P-5 and six strains from our study (GP35-8,LC037-22,LC107-5,LC107-5,LC107-19,LC107-16).Other81 strains and Chinese strain DX,LJB-03,HuN,JS-2004-2 belonged to cluster 3 while cluster 1 and cluster 4 were composed of other reference strains.[Conclusion] PEDV was the main pathogen that caused diarrhea of neonatal piglets in Guangxi.The PEDV-N genes were divided into two series,and the N genes of the current PEDV were still relatively conservative.

概述ELISA技术原理、特点和基本类型及其在口蹄疫、猪蓝耳病、猪瘟、猪伪狂犬病、小反刍兽疫等5种家畜疫病诊断应用中涉及的检测标准、试剂种类、影响因素和常见问题,为兽医实验室人员准确掌握ELISA技术提供有益参考.

[目的]通过原核表达及纯化获得A型口蹄疫病毒(FMDV)结构蛋白VP1,为建立A型FMDV的ELISA诊断方法及开发安全、高效、广谱的新型基因工程疫苗提供技术支持.[方法]以含A型FMDV VP1基因的重组质粒pMD18-T-A-VP1为模板,通过特异性引物扩增A型FMDV的VP1基因,构建表达质粒pET-32a-VP1和pGEX-6p-1-VP1,然后转入感受态细胞E.coli BL21 (DE3)中诱导表达融合蛋白.[结果]诱导表达获得的VP1融合蛋白主要以包涵体形式存在,分别经His· Bind和GST· Bind柱层析纯化,SDS-PAGE分析结果表明融合蛋白纯度较高;Western blotting检测分析发现,VP1融合蛋白能与豚鼠抗A型FMDV阳性血清发生特异性结合,但不与豚鼠抗O型和Asia1型FMDV阳性血清反应.[结论]经原核表达及纯化获得的A型FMDV VP1融合蛋白具有良好的特异性和抗原性,可用于易感动物的免疫及血清抗体筛查.

【目的】通过原核表达并纯化获得O型口蹄疫病毒（FMDV）VP1基因及其多表位基因的重组蛋白,为建立O型FMDV抗体ELISA检测试剂盒及制备动物高免血清提供技术支持。【方法】以O型FMDV VP1全基因重组质粒p MD18-T-T-VP1及串联的VP1多表位基因重组质粒p MD18-T-O-VP1为模板,通过特异性引物扩增并回收目的基因,构建重组质粒p ET-32a-VP1和p GEX-6p-1-VP1,然后转入大肠杆菌BL211（DE3）中诱导表达,并以SDS-PAGE和Western blotting对融合蛋白进行分析鉴定。【结果】O型FMDV的VP1基因及其多表位基因在大肠杆菌BL21（DE3）中得到正确表达,表达的两种融合蛋白主要以包涵体形式存在,纯度较高,且均能与猪抗O型FMDV阳性血清发生特异性结合,具有良好的反应原性。【结论】表达获得的融合蛋白具有良好的反应原性,可作为包被抗原应用于O型FMDV抗体检测试剂盒研发。

为了深入了解VP1蛋白的结构和功能,并建立A型口蹄疫(FMD)血清抗体检测方法,试验采用纯化的重组蛋白pET-32a-VP1免疫Balb/c小鼠,以纯化的重组蛋白pGEX-6p-1-VP1作为检测抗原包被酶标板检测抗体效价.将免疫小鼠的脾细胞与骨髓瘤细胞SP2/0融合,其杂交瘤细胞用间接ELISA法进行筛选,获得4株能稳定分泌抗口蹄疫病毒(FMDV)结构蛋白VP1的特异性杂交瘤细胞株,分别命名为1D10、3D12、4E12和5G7.结果表明:这4株单克隆抗体均为IgM亚型和K轻链,单克隆抗体间可能识别同一个表位,或表位重叠性大.4株单克隆抗体均能被A型FMDV阳性血清所阻断,具有特异性,且以单克隆抗体4E12产生的抗体效价最高、稳定性强、亲和力最大,并能与重组蛋白FMDV-A-VP1特异性结合.说明单克隆抗体4E12可作为单克隆抗体竞争ELISA法的竞争抗体用于检测A型FMDV血清抗体.

以构建好的O型 FMDV VP1基因的重组质粒PMD18-T-VP1为模板，通过PCR扩增出VP1基因主要抗原位点VP1（61～180 bp）的核苷酸及VP1（421～639 bp）的核苷酸，采用重组PCR方法将扩增所得的2个基因片段用linker连接，结果构建出1个含384 bp的重组质粒，将该重组质粒定向插入原核表达载体pGEX-6P-1，成功构建了pGEX-6P-1-VP1（384 bp）串联表达载体。该串联表达载体的构建为进一步研制 FM D V的诊断抗原及多肽疫苗奠定了基础。

Competitive ELISA was developed for detecting the antibodies against the foot-and-mouth disease virus (FMDV)with the purified recombinant pGEX-6P-1-VP1 protein as the antigen and the rabbit-anti-VP1 serum as the competitive antibody.The positive threshold of the competitive ELISA was calculated using statistical methods.The results showed that (a)the dose of the coating antigen protein was 1ug/ml;(b)the working concentration of the detected serum sample was 1 ∶ 40dilution,and the enzyme-labeled goat anti-rabbit lgG was 1 ∶ 2000;(c)the working titer of specific rabbit serum to N protein was 1 ∶500;and,(d)comparing to the liquid-phase blocking ELISA,the sensitivity and specificity of the competitive ELISA were 91.3% and 95.1%,respectively,and the coincident rate between the two methods was 96.7%.It appeared that this newly developed diagnostic method could be applied for the detection of the serum antibodies against serotype O FMDV with high sensibility,specificity and repeatability.

[目的]对猪嵴病毒(PKV) VP1基因进行测序与同源性分析,确认其可能来源,为今后的生物学特性分析及仔猪腹泻防治工作提供科学依据.[方法]采用RT-PCR对GXPKV-1毒株VP1基因进行克隆,运用DNASTAR软件包中Megalign程序对测序获得的VP1基因进行核苷酸序列及其推导氨基酸序列同源性分析.[结果]GXPKV-1毒株ⅥP1基因全长762 bp,共编码254个氨基酸,与GenBank已公布的13株参考毒株VP1基因的核苷酸同源性为74.1％～85.4％,推导氨基酸同源性为81.1％～93.3％.根据VP1基因推导氨基酸序列进行系统发育进化分析,发现GXPKV-1毒株与Gansu-2012、JS1419等国内参考毒株同属于同一亚群,而与瑞士分离株Swine-S-1-2007、泰国分离株THA-2008、美国分离株H24-2012-USA及四川分离株CHN-SC-2011-02等的亲缘关系较远.[结论]猪嵴病毒GXPKV-1毒株起源于国内流行毒株的传播,在新的环境下虽然其VP1基因核苷酸发生变异,但由于同义翻译,推导氨基酸的同源性仍然较高,说明碱基突变并未引起蛋白结构的改变.

本研究从2012～2014年收集广西地区猪群发生疑似仔猪腹泻的仔猪肠系膜淋巴结或肠样粪便样品,通过RT-PCR方法对收集到的1 600份样品进行猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)和猪库布病毒(KobuV)检测和流行病学调查.结果显示:2012年TGEV、PEDV、KobuV流行率分别为1.2％、19.7％、38.2％,2013年分别为20.6％、85.7％、22.7％,2014年分别为0、4.1％、3.0％.不同时间有不同的主导病毒,提示我们兽医工作者应根据病原的变化,及时调整防控措施和对策.

新城疫是由新城疫病毒引起的鸡急性高度接触性传染病，常呈败血症经过。主要症状是呼吸困难，下痢、神经紊乱、粘膜和浆膜出血［1］。目前该病在我国部分地区仍有散发和流行，并表现出宿主范围扩大、病原基因型多样化、免疫带毒现象普遍等流行病学特征，对养禽业生产危害巨大［2］。我县长期落实相关监测方案，将鸡新城疫免疫作为重点工作来抓，免疫密度均保持在95％以上。由于我县地处高寒山区，家禽养殖基本以散养为主，饲养环境参差不齐，与相邻的湖南等地家禽交易频繁，给我县的防疫工作带来了很大困难。为了摸清我县的新城疫免疫效果，为制订下一步的高效免疫方案提供科学依据，我县对2012年至2014年的鸡新城疫免疫血清进行了监测分析。

为了解广西狂犬病病毒(RV)感染与分布情况,本研究对采集自广西境内9个市31个县(区)1 007份犬脑组织、1 324份犬唾液拭子及564只野生蝙蝠进行RV检测,结果显示有6个县的犬脑组织检测出RV,阳性率为0.65％(1/153)～5.38％(5/93),县(区)阳性率为27.27％(6/22),广西划分的5个地理区域均有分布.外观健康犬和疑似发病犬脑组织平均阳性率分别为1％(10/1 005)和100％(2/2).犬唾液拭子和野生蝙蝠均未检测到RV.结果表明广西家犬的RV感染率存在一定的地域性差异,平均感染率较低,但个别县(区)连续几年阳性率均处于较高水平,应加强对犬只的管理和疫苗免疫.

【Objective】Epidemic survey was carried out to find out the prevalence of bovine tuberculosis in Guangxi.【Method】The tuberculin skin test (TST) and interferon gamma (IFN-g) ELISA assay were used to detect the bovine tuber-culosis in Guangxi from 2008 to 2011. Totally 5478 cows and 1580 buffaloes had been quarantined for 4 years. 【Result】Dur-ing 2008-2011, 63 dairy cows (accounted for 1.15% of the total ) and 17 buffaloes(accounted for 1.08% of the total)were detected as positive in Nanning, Liuzhou, Beihai, Qinzhou and Guilin. Overall, the positive rate of tuberculosis in cow and buffalo decreased year by year. Through self-made box of interferon gamma assay and BovigamTMkit, it was found that the positive rate of bovine tuberculosis detected by interferon gamma assay which had high accuracy was lower than that by TST.【Conclusion】For better detection and purification of bovine tuberculosis, the herd should be quarantined by tuberculin skin test, and then tested by interferon gamma assay.

2011年12月27日接到荔浦县荔城镇沙街村吴家厂一养殖户报告,该场150多头10㎏～12㎏体重的仔猪发生严重咳嗽与腹泻,已于当日早上死亡2头,无法确诊,请求前往帮助诊治。

猪瘟是一种由猪瘟病毒引起的高度接触性传染病,是目前影响我国养猪业最严重的传染病之一[1,2].近几年养猪业的不断发展,猪瘟在不少地方仍不断发生和流行,特别是由于非典型猪瘟和温和性猪瘟的出现,猪瘟的流行病学、临床表现、病理变化方面发生了很大变化,给猪瘟的诊断和防治工作带来了新的难题[3,5].

A H9N2 subtype strain of swine influenza virus(SIV) was isolated from swine in Guangxi by SPF chicken embryo.The SIV strain had a stable haemagglutiain(HA) values for 27 after there generations,and can be inhibited by H9 positive serum.The HA and NA genetic amplification showed that this strain had the height homology with the subtype H9N2 SIV,which named A/Swine/Guangxi/01/2009.