Given that probiotics always have host-homologous and strain-specific effects on the hosts, lactic acid bacteria extracted and identified from porcine specimens can be potentially developed as probiotics for pig production. We aimed to identify lactic acid bacteria that are potentially probiotic, have good capacity of inhibiting pathogenic bacteria in intestine and are promising to be used as substitutes for antibiotics in pig production. Potential probiotic strains were extracted from 15 fecal specimens collected from 15 apparently healthy pigs, and were identified via 16S rDNA sequencing. The antimicrobial activity, tolerance to acid and bile salts, Caco-2 cell adhesiveness and susceptibility to antibiotics of the isolates were evaluated in vitro, and oral toxicity of the isolates were evaluated in mice. One Lactiplantibacillus plantarum (BJR2), two Lacticaseibacillus casei (HJD and TH2), one Lacticaseibacillus rhamnosus (MRS1), and two Enterococcus faecium (S-3 and S-4-H) were extracted from healthy pigs and underwent 16S rDNA sequencing identification. L. plantarum BJR2 and L. casei HJD exhibited broad-spectrum and higher antimicrobial activity against indicator enteric pathogens, including Salmonella choleraesuis CVCC 2139, Escherichia coli (O147:K89) CVCC 199, Escherichia coli (O141:K99) CVCC 223 and Escherichia coli (O139) CVCC 1496, among 6 tested strains. In addition, both L. plantarum BJR2 and L. casei HJD exhibited good tolerance to low pH (pH 2.5 and pH 3.5) and 0.30% bile salts, had relatively strong Caco-2 adhesiveness and carried no transferable resistant genes against antibiotics encoded by plasmid. In safety trials, these two isolates had no α or β-hemolysis activity, and were proved safe through oral toxicity tests in mice. It is concluded that L. plantarum BJR2 and L. casei HJD are potential probiotic candidate strains and their probiotic effects need to be further studied in pigs.

The experiment was to investigate the preventive effect of anti Escherichia coli IgY and IgY nanoparticle-in-microsphere in the prevention of Escherichia coli-induced diarrhea. Mice were fed with IgY continuously for 8 d, and the trend of their weight, changes in blood routine, organ index, phagocytic index of macrophages and phagocytic percentage were recorded. Established Escherichia coli diarrhea model of mice. Mice were randomly divided into IgY group, nanoparticles microspheres group and control group, then given drugs in three ways of oral administration, intraperitoneal injection, intraperitoneal injection + oral administration. The control group used PBS to replace IgY. The results showed that in the IgY feeding experiment, the average daily weight gain of mice in IgY group was higher than that of control group, with an increase of 11.25%. The liver index, spleen index and macrophage phagocytosis percentage of the IgY group were significantly higher than those of control group (P<0.05), and the macrophage phagocytosis index of IgY group was extremely higher than that of control group (P<0.01). In the diarrhea prevention test, both IgY and IgY nanoparticle-in-microsphere had a certain protective effect. The mortality rates of mice in IgY group under oral administration, intraperitoneal injection, intraperitoneal injection + oral administration were 16.67%, 33.00% and 0, the corresponding mortality rates for IgY nanosphere-microsphere group were 83.33%, 0 and 16.67%. The study indicates that IgY and IgY-NiSMs have obvious prevention effects in diarrhea model of mice.

为确诊某猪场猪发病死亡原因,采集死亡猪病变组织,用常规方法分离鉴定细菌并进行致病性和耐药性检测.结果显示:从病死猪肺脏分离到1株奇异变形杆菌GX-PM1,与GenBank收录的奇异变形杆菌AYQ-1株同源性高达99.9％;小鼠致病性试验鉴定为强毒株:药敏实验检测结果显示,该菌株对阿米卡星、头孢氨苄为极敏,对青霉素G、头孢拉定、左氟沙星等为高敏,对阿莫西林、恩诺沙星等药物效果为中敏,对四环素、红霉素、复方新诺明、磺胺异恶唑等8种药物完全耐药;对耐药基因进行PCR扩增发现在该菌株中存在四环素类、氯霉素类、磺胺类耐药基因;GX-PM1株毒力基因型为ureC(-)、atfA(+)、pmfA(+)、zapA(+)、mrpA(-)、atfC(+)、ucaA(+)、rsbA(-);生物膜实验研究表明,分离株具有生物被膜形成能力,且为强黏附型(+++).本研究表明此疫情由奇异变形杆菌引起,可用头孢类和氨基糖苷类药物进行治疗.

为了解近年广西地区猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)基因组特征和分子遗传多样性,本研究设计11对特异性引物,对中国广西南宁猪腹泻病例中检测到的1株PEDV GXNN进行了全基因组测定,同时进行相似性比对、遗传进化、基因变异及S基因重组分析.结果显示,广西变异株GXNN与其他PEDV分离株具有相似的基因组特征,基因组全长28 035 bp,与不同参考株核苷酸相似性为96.4％～98.7％,S、OR F3、M 和 N基因核苷酸相似性分别为 93.7％～98.9％、90.9％～99.4％、97.4％～99.7％和 95.6％～99.2％;氨基酸相似性分别为 92.9％～99.5％、91.3％～99.1％、97.4％～99.1％和96.4％～99.5％.GXNN与近年国内大多数分离株亲缘关系较近,处于同一分支,为GII-b亚型,而与经典疫苗毒株、国内早期流行株、国外流行株及广西CH-GX-2015-750A分离株亲缘关系都较远,为不同亚型.与目前常用5个疫苗毒株相比,S基因变异较大,在118、844和905位插入氨基酸Q,在COE(Core neutral-izing epitope)区和主要抗原表位区发生 4 个独特的氨基酸位点突变,其他区域发生14个位点突变;ORF3、M和N基因分别在126位、4D4区和PN-D4区发生126 T/A、199 A/V和103 T/A突变.重组分析发现在S基因的高变区826～3 142 nt存在1个潜在的重组区域.本研究成功获得1株PEDV全基因组序列,对其进行遗传变异分析,为PEDV分子流行病学研究和新型疫苗研发提供参考依据.

Feline parvovirus (FPV), a type of parvovirus prevalent worldwide, can cause foetal death and acute enteritis in adult cats with severe leukopenia, and yet there are no effective drugs to prevent or treat FPV. Here, the immune effects of two FPV vaccines on horses were compared. IgG was extracted from FPV-immunized horse sera. Equine F(ab′)2 fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. The results showed that the inactivated FPV oil vaccine was more effective than the inactivated FPV propolis vaccine in helping healthy horses to produce hyper-immune serum. Four methods were tested, among which the optimized octanoic acid-ammonium sulphate precipitation method was proved to be the best process for extracting IgG. The optimal condition for preparing F(ab′)2 by pepsin digestion was 30 °C for 3.5 h, and the content, purity and recovery of F(ab′)2 were 8.64 mg/mL, 90.36% and 93.24%, respectively. Our equine immunoglobulin F(ab′)2 fragments effectively neutralized activity in vitro against FPV, alleviated the clinical symptoms of FPV-infected cats, reduced the viral loads in the intestine and had prophylactic effects in FPV-infected cats. These results indicate that the F(ab′)2 fragment prepared from inactivated FPV-immunized horses may be used as a prophylactic agent for diseases caused by FPV.

旨在构建检测猪瘟病毒(CSFV)的双功能单链抗体.通过连接肽将抗人红细胞膜H抗原单链抗体基因2E8-ScFv和抗猪瘟病毒单链抗体基因CSFV-ScFv拼接成融合基因,大肠杆菌密码子优化及人工合成后,构建原核表达载体pCzn1-2E8-CSFV,转化BL21 (Plyss)进行重组蛋白IPTG的诱导表达,利用SDS-PAGE对表达产物进行鉴定,通过间接免疫荧光(IFA)、ELISA和血凝试验,验证重组蛋白双功能活性.结果 表明,重组蛋白主要以包涵体形式在大肠杆菌中表达,分子质量约为55 kDa.IFA和ELISA结果表明,纯化复性后的重组蛋白与猪瘟病毒具有良好的结合活性;红细胞凝集试验表明,重组蛋白能与人红细胞特异性结合,同时也能与猪瘟病毒发生特异性结合,具有双功能特性.本研究成功表达了抗人红细胞膜H抗原/抗猪瘟病毒双功能单链抗体,为进一步构建猪瘟病毒红细胞凝集试验快速检测方法奠定基础.

本试验旨在从竹鼠肠道内容物中分离筛选易培养、具有高纤维素酶活性的菌株.称取竹鼠肠道内容物1.0 g,以刚果红平板进行纤维素降解菌的初筛,再分别以羧甲基纤维素钠(CMC-Na)、微晶纤维素、D-水杨苷和滤纸为碳源,采用3,5-二硝基水杨酸(DNS)法测定菌株内切葡聚糖酶(Cen)、外切葡聚糖酶(Cex)、β-葡萄糖苷酶(BG)和滤纸酶(FPA)活性.采用生理生化试验和16S rDNA PCR方法进行菌株种属鉴定.结果显示:从竹鼠肠道内容物中分离获得4株产纤维素酶的需氧菌(GL-4、GL-5、GL-6和GL-8),其中GL-4的Cen和FPA活性最高,分别为49.32和25.13 U/mL;GL-8的Cex活性最高,为30.59 U/mL;GL-5的BG活性最高,为35.08 U/mL.对纤维素酶总活性最高的GL-4进行了培养条件优化,其在37℃、pH 8.0条件下培养24 h时,FPA活性最高.对产纤维素酶活性较高的3株菌(GL-4、GL-5和GL-8)进行种属鉴定,显示3株菌均为枯草芽孢杆菌.综上可知,本试验从竹鼠肠道内容物中分离到3株需氧枯草芽孢杆菌,这3株菌均具有较高的产酶能力;对菌株GL-4进行培养条件优化,得出其产酶最适条件为发酵时间24 h、发酵培养基pH 8和发酵温度37℃,可作为高粗纤维含量非粮型饲料开发利用的候选菌株.

检测广西巴马小型猪内源性反转录病毒(PERV)整合到HEK293细胞基因组(HEK293-PERV-BM)后,宿主细胞的细胞周期、细胞凋亡及cyclin D1、CDK4、c-Myc、p53、p16和k Ras等相关基因表达的改变,推测相关作用机制.检测发现P10代巴马小型猪内源性反转录病毒感染HEK293细胞模型(HEK293-PERV-BM)的细胞周期主要被阻滞在S期和G2/M期,细胞凋亡受到抑制,P25代细胞周期主要被阻滞在S期,细胞凋亡明显受到诱导.通过实时荧光定量PCR和Western blot检测发现细胞周期相关基因的表达与细胞感染模型传代次数(P1～P35)有密切关系,其中cyclin D1、c-Myc和k-Ras基因表现为先降低后升高,而CDK4和p16基因则恰好相反,同时,p53基因表达的改变不明显,据此可以推测P10代细胞周期的改变可能由Rb调控通路诱导发生,P25代细胞周期的改变可能由Rb调控通路与p53调控通路协同诱导发生.上述结果提示PERV的感染可能存在潜在的危险.

[目的]了解猪博卡病毒(Porcine bocavirus,PBoV)在广西猪群中的流行状况及遗传学特征,为有效防控PBoV感染提供科学依据.[方法]采用PCR对采自广西境内养殖场的388份猪源样品进行PBoV检测,挑选3份阳性样品(1份为PBoVG1阳性样品,2份为PBoVG3阳性样品)进行全基因扩增,测序获得的序列使用LaserGene进行处理及多重比对分析,应用MEGA 5.2中的ClustalW进行遗传进化分析,通过邻接法(NJ)构建系统发育进化树,并以SimPlot 3.5.1进行潜在基因重组事件分析.[结果]猪内脏组织(肺脏、脾脏和淋巴结的混合样品)、扁桃体、脑组织和公猪精液的PBoV阳性率分别为25.19％、8.33％、2.94％和1.54％.在所有检测样品中,PBoVG3基因群的阳性率最高,为9.02％,PBoVG2和PBoVG1基因群的阳性率均为1.55％;不同基因群的PBoV存在混合感染现象,其中,PBoVG1+PBoVG3二重感染率为1.03％,PBoVG2+PBoVG3二重感染率为0.77％.从PBoV阳性样品中扩增获得3条PBoV全基因序列(毒株),命名为GXBH2014、GXBH2015和GXLC2014,对应的GenBanK登录号为MN747332、MN747333和MN747339.其中,GXBH2014株序列全长5055 bp,GXBH2015株序列全长5070 bp,GXLC2014株序列全长4313 bp.GXBH2014株和GXBH2015株与PBoVG3基因群参考毒株SH20F各编码基因的推导氨基酸序列同源性较高,为70.9％～98.5％;GXLC2014株与PBoVG1基因群参考毒株SX各编码基因的推导氨基酸序列同源性最高,为98.0％～99.1％.基于PBoV全基因编码区(CDS)全长序列构建的系统发育进化树也显示,GXBH2014株和GXBH2015株同处于PBoVG3基因群分支上,GXLC2014株处于PBoVG1基因群分支上.基因重组分析结果显示,在GXBH2014株的全基因序列中检测到1个潜在的重组断点,位于NP1基因的第391位核苷酸;而在GXBH2015株和GXLC2014株的全基因序列中均未检测到潜在的重组断点.[结论]广西猪群中普遍存在PBoV感染,且PBoVG1、PBoVG2和PBoVG3基因群毒株同时存在.其中,GXBH2014株可能是IA159-2株(KF025386)与MN154-1株(KF025384)基因重组进化的产物.

为掌握广西外观正常猪群中博卡病毒(PBoV)的感染情况,本研究采用PCR方法对采自广西境内的274份1～2月龄保育猪血清、咽拭子和肛拭子样品进行检测.结果显示:3种样品的阳性率分别为1.9％(2/106)、83.3％(75/90)和82.1％(64/78),不同PBoV基因群的检出率分别为G3(47.4％,130/274)>G2(32.8％,90/274)>G1(20.1％,55/274),说明PBoVG3是广西PBoV流行的主要基因群;不同基因群的混合感染情况普遍存在,G2+G3二重感染率最高,为17.9％(49/274);从PBoV阳性样品中扩增获得8条PBoV全基因序列;8个毒株分别处于PBoVG1、PBoVG2和PBoVG3三个进化分支,PBoVG2和PBoVG3的6个毒株均包括与磷酸酯酶A2(PLA2)活性相关的保守氨基酸序列HDXXY和YXGXF.本研究结果为PBoV的进一步防控提供了数据信息和科学依据.

为了制备抗猪瘟病毒(CsFV)单链抗体(scFv)并鉴定其生物学活性,试验以编码特异性抗CSFV scFv基因序列为基础,按照大肠杆菌密码子偏爱性优化并合成scFv基因,将其克隆入pCzn1表达载体,转化大肠杆菌BL21 (DE3) PlysS感受态细胞,经IPTG诱导表达后进行Ni-IDA亲和层析纯化、SDS-PAGE检测和Western-blot鉴定,通过间接免疫荧光(IFA)和间接ELISA验证蛋白抗原的活性.结果 表明:密码子优化合成的抗CSFV scFv基因全长768 bp,在大肠杆菌中主要以包涵体形式表达,其分子质量约28.0 ku,Western-blot检测显示纯化后的目的蛋白能被抗His标签单克隆抗体特异性识别,IFA和ELISA检测证实纯化复性后的蛋白可与CSFV发生特异性结合反应,且存在明显的浓度依赖性.说明应用原核表达系统可高效制备抗CSFV scFv.

为了解猫杯状病毒(feline calicivirus,FCV)在广西地区的流行情况与致病性,本研究采集55份疑似FCV拭子,按常规方法分离鉴定得到3株FCV,以VP1基因进行同源性分析,在77.5％～85％之间,这3株分离株(2019)与广西株GX01(2013)、吉林株CH-JL4(2015)、上海株SH(2014)及英国株F65(1999)、哈尔滨株HRB-SS(2014)处于同一进化分支,具有相同的遗传起源,但与目前临床上使用的国内疫苗株255、国外疫苗株F9处于不同的进化分支,存在疫苗免疫失败的风险.为了解这3株FCV的毒力,进行动物致病性试验.结果 显示,模拟自然感染的试验猫,FCV潜伏期最长为7d,5～7 d为排毒的高峰期,FCV存在短暂病毒血症的情况.器官病变集中在肺部,会出现明显的淤血、出血现象.这3株的感染率均为100％,死亡率分别为50％、50％、75％,表明这3株FCV毒株均有致病性.综合剖检和HE染色结果分析,致病力最强的是GXN3株.

从南宁某宠物医院疑似感染猫杯状病毒(Feline calicivirus,FCV)猫鼻咽拭子中,利用F81细胞分离1株FCV后,RT-PCR和电镜鉴定,测定TCID50和理化性质.同时利用RT-PCR分段扩增病毒全基因序列,进行同源性及遗传进化分析.结果 显示,FCV GX2019分离株的TCID50为1×10-867/0.1 mL,基因组全长为7 687 bp,与FCV CH-JL4(长春株)毒株核苷酸序列相似性最高,为85.3％,属于同一分支.氨基酸分析表明,与国内FCV疫苗株255、国外FCV疫苗株F9、FCV跛行综合征株2280及FCV GX01分离株氨基酸变异率分别为46.7％、26.7％、53.3％、40.0％,其中B细胞抗原识别表位中氨基酸位点发生了改变.

检测不同代次广西巴马小型猪内源性反转录病毒(PERV)感染HEK293细胞模型(HEK293-PERV-BM)中,HERV-W各基因mRNA和syncytin-1蛋白表达水平,以及HERV-W各基因变异情况,评价PERV的整合对HERV-W的影响.结果 显示,与HEK293细胞相比,PERV整合后,不同代次HEK293-PERV-BM中HERV-Wgag、pol、env和syncytin-1的mRNA相对表达量出现不同程度的改变,且改变趋势相似,即P10或P20后开始升高,至P30最高;选取相对表达量差别较大代次,检测其syncytin-1蛋白,各代次间syncytin-1蛋白表达量的改变较mRNA的差别小.此外,PERV整合后,在P1～P55中HERV-W结构基因仅发生个别位置的点突变,未见发生固定位置的点突变或基因片段的缺失、插入及重组.本试验为系统评价PERV-BM在异种器官移植中的安全性提供参考.

Bama miniature pig (BM) as the inbreed pigs is considered to be a potential donor for xenotransplantation. However, porcine endogenous retrovirus (PERV) integrated in pig's genome, is likely to infect human cells in vitro. PERV infection from BM remains unknown. This study aimed to investigate PERV infection in BM in comparison with that of PK15 cells and further to identify the integration sites of PERV from BM (PERV-BM) in human genome. Two full-length PERV-BMs were cloned from 15 isolates of PERV from Bama miniature pigs and sequence analysis indicated that PERV-BM had limited infection to human cells. Subsequently, PBMC from the BM and PK15 cells were co-cultured with HEK293 cells, respectively. Using nested RT-PCR assay and TEM observation, one PERV-BM was observed to infect HEK293 cells with lower transcriptional level than PERV from PK15 cells (PERV-PK) did, while the others showed no sign of transmission of PERV to human cells. Furthermore, 4 and 157 integration sites were identified from PERV-BM-infected HEK293 cells (HEK293-PERV-BM) and PERV-PK-infected HEK293 cells (HEK293-PERV-PK), respectively, which displayed variations of PERV integration. For further verification of the PERV-BM integration, the expression level of cyclin D1, c-myc, p53 and p16 genes of HEK293-PERV-BM decreased significantly while the CDK4 genes were upregulated dramatically in comparison with those of the HEK293-PERV-PK. It is the first time that PERV-BM has been confirmed to be low infection to human cells and highly integration into the PK15 cells. Therefore, BM is a potential xenograft donor due to less PERV transmission to human beings. (C) 2019 PVJ. All rights reserved

Akabane virus (AKAV) is an important insect-borne virus belonging to the genus Orthobunyavirus, the Peribunyaviridae family. An AKAV defined as GXDH 01 here, was isolated for the first time from blood from a sentinel goat in China in 2016, and its full-length open reading frames (ORFs) were sequenced in this study. Sequence analysis suggested that the isolate GXDH 01 probably had undergone a reassortment incident and acquired L segments from other strain originating from an attenuated vaccine, such as OBE-1. This study aims to provide more understanding as to the origin and epidemiology of AKAV in China.

旨在利用杆状病毒-昆虫细胞表达系统表达融合VP22短肽的重组猪细小病毒(PPV)衣壳蛋白VP2,分析其免疫原性.以PPV N株为模板,通过重叠延伸PCR技术分别将VP22融合到VP2基因的N端或C端,构建重组杆状病毒rBV-VP2、rBV-VP22-VP2和rBV-VP2-VP22,通过感染昆虫细胞进行重组蛋白的表达,并在小鼠上初步验证其免疫原性.间接免疫荧光和Western blot分析结果表明表达产物均能与鼠抗PPV抗体发生特异性反应;透射电镜观察到rBV-VP2、rBV-VP22-VP2的表达产物可装配形成直径约22～24 nm的病毒样粒子,VP2-VP22没有观察到病毒样粒子.PPV ELISA抗体检测结果显示,3种表达产物均能有效诱导小鼠产生PPV特异性抗体,其中VP2、VP22-VP2组产生抗体水平与PPV灭活苗相当;细胞因子检测结果显示,3种表达产物均能有效刺激细胞因子IL-2和IFN-γ的分泌,其中VP22-VP2组IL-2和IFN-γ的含量要显著高于VP2和PPV灭活苗组及重组蛋白VP2-VP22组,表明VP2 N端融合VP22蛋白的免疫效果要优于其C端,VP22-VP2虽不能提高VP22蛋白的体液免疫效果,但具有增强小鼠机体细胞免疫反应的能力.

为了建立竹鼠细小病毒PCR检测方法,试验通过对竹鼠细小病毒及其同属各病毒基因序列进行比对分析,设计了针对竹鼠细小病毒NS1基因片段的一对特异性引物,经优化建立了竹鼠细小病毒PCR检测方法.结果表明:在56℃退火温度条件下,能得到理想的目的片段.经琼脂糖凝胶电泳,该方法能特异性地扩增竹鼠细小病毒,不能扩增竹鼠圆环病毒、竹鼠内源性反转录病毒、竹鼠乳酸脱氢酶增高病毒;检验敏感性高,灵敏度可达125 pg/μL.

为了解广西牛片形吸虫病的流行情况,在广西8个地级市不同规模牛场及散养户共采集了1 571份牛粪,采用水洗沉淀法进行检测,同时屠宰时随机抽查50头牛,观察组织及病理变化.调查结果表明,广西牛片形吸虫感染较为严重,平均感染率达69.3％(1 089/1 571),其中南宁71.9％ (41/57)、柳州46.3％ (139/300)、梧州72.3％ (88/122)、北海37.5％ (6/16)、崇左87.8％ (652/742)、河池69.1％ (65/94)、百色39.6％(87/220)和贵港55％(11/20),各地感染情况均有不同.牛感染片形吸虫主要病变器官为肝脏、胆囊、淋巴结,而胆囊是片形吸虫病寄生的主要地方.本调查为指导广西防治牛片形吸虫病,促进肉牛产业发展提供参考依据.

以巴马小型猪内源性反转录病毒(porcine endogenous retrovirus,PERV)感染HEK293细胞模型(PERV-BM-HEK293)为研究对象,分析PERV整合对HEK293细胞活性的影响,同时建立7种与人细胞增殖和周期调控密切相关基因的实时荧光定量PCR(q-PCR)检测方法,并对细胞感染模型中7个基因的mRNA表达情况进行分析.细胞活力分析结果显示,随着培养代次及培养时间增加,相对于母源HEK293细胞PERV感染模型的细胞活力下降;q-PCR检测方法建立结果显示,所构建的cyclinD1、CDK1、CDK4、k-ras、c-myc、p53、p16基因检测方法检测各基因在1.0×101～1.0×109 copies/μL反应范围内有很好的线性关系,扩增产物熔解曲线只出现特异性单峰,各组内变异系数在0.31％～2.01％之间,组间变异系数在0.46％～2.19％之间,重复性好,可稳定地用于相关基因mRNA的定量检测.用建立的q-PCR检测方法对PERV感染模型进行细胞周期调控相关基因mRNA表达分析,结果显示,与母源细胞相比,模型细胞cyclinD1、CDK1、CDK4、k-ras、p53和p16基因的mRNA表达无明显变化,原癌基因c-myc基因表达下调,提示PERV感染可能抑制c-myc基因表达.该研究结果可为评价巴马小型猪来源PERV在人-猪异种移植中生物安全性提供参考.