I群禽腺病毒的分离鉴定与同源性分析
为了解鸡群中I群禽腺病毒(FAdV-I)的流行现状,从山东、黑龙江、吉林等地部分大规模养鸡场采集病料,进行病毒分离、PCR鉴定及同源性分析。结果显示:从76份病料中分离到10株FAdV-I,分离率为13%;10株FAdV-I中,1株为血清4型,6株为血清8b型,1株为血清9型,2株为血清11型。结果表明,山东、黑龙江、吉林等地均存在一定的FAdV-I流行,主要流行血清型已由4型转变为8b型,因此需要评估当前的4型灭活苗能否有效防控这些地区的FAdV-I流行。Isolation and Identificationand Homology Analysis on Group I Fowl AdenovirusIn order to recognize the prevalence ofgroup I fowl adenovirus(FAdV-I)in poultry,samples were collected from several large-scalechicken farms in Shandong,Heilongjiang and Jilin provinces for FAdV isolation,PCR assay and homology analysis. Resultsshowed that 10 FAdV-I strains were isolated from 76 samples with the positiverate reaching 13%. Among the 10 FAdV-I strains,1 of them belonged to serotype 4,6 of them belonged to serotype 8b,1 of them belonged to serotype 9,and 2 of them belonged to serotype 11. Theresults indicated that certain FAdV-I infection existed in Shandong,Heilongjiang and Jilin provinces,and the main serotypes had been transferredfrom serotype 4 to serotype 8b,thus the current serotype 4 inactivated vaccines need to be evaluatedto determine if they could control the spread of FAdV-I effectively. 全文下载链接:http://kns.cnki.net/kcms/detail/37.1246.S.20190222.0911.032.html?uid=WEEvREcwSlJHSldTTEYzU3EydDRPZTFBeXhDa29ZZ2ljVUtvTXF2T3dtND0=$9A4hF_YAuvQ5obgVAqNKPCYcEjKensW4IQMovwHtwkF4VYPoHbKxJw!!
2019-03-04
H5N1和H7N9亚型双价灭活流感疫苗对鹌鹑及鸽子的免疫原性
为评价重组流感病毒(H5+H7)二价灭活疫苗(H5N1 Re-8株+H7N9 H7-Re1株)对鹌鹑和鸽子的安全性及免疫效果,2017年12月—2018年6月,采用哈尔滨维科生物技术开发公司生产的重组流感病毒(H5+H7)二价灭活疫苗,对鹌鹑和肉鸽,在25日龄首免,46日龄二免(鹌鹑和肉鸽的剂量分别为0.3 mL和1 mL),观察禽群临床表现,定期采集血清测定抗体效价。结果显示:鹌鹑首免后21 d,H5和H7亚型HI抗体效价均达到高峰,分别为(7.7±1.3)log2和(8.5±1.3)log2,抗体合格率大于70%的持续期约为35 d;二免后7 d,H5和H7亚型抗体效价均达到高峰,效价分别为(9.7±0.9)log2和(10.3±0.9)log2,H5和H7亚型抗体合格率大于70%的持续期分别约为147 d和175 d。鸽子首免后21 d,H5和H7抗体效价也均到达高峰,分别为(9.8±0.8)log2和(10.4±1.1)log2,两种亚型抗体合格率大于70%的持续期均超过181 d;加强免疫后7 d,H5和H7抗体效价也均达到高峰,效价分别为(10.2±1.0)log2和(10.7±0.9)log2,189 d后效价分别为(5.4±1.3)log2和(6.1±1.5)log2,合格率分别为96.7%和100%。临床观察,未发现该疫苗有严重免疫不良反应。结果表明,(H5+H7)二价灭活疫苗对鹌鹑和鸽子安全有效。生产中推荐:鹌鹑25日龄首免,46日龄和6月龄各加强免疫1次,剂量为0.3 mL;鸽子25日龄首免,以后每隔6个月加强免1次,剂量为1 mL。Immunogenicity Study onBivalent Inactivated Vaccines of H5N1 and H7N9Subtype Influenza in Quailsand PigeonsIn order to evaluate the safety and immuneeffects of the bivalent inactivated vaccine(H5N1 Re-8 + H7N9 H7-Re1 strain)of recombinant influenza virus(H5+H7)in quails and pigeons,the bivalent inactivated vaccines produced by HarbinWeike Biotechnology Development Company were immunized in quails and youngpigeons from December 2017 to June 2018. The dose for a quail/pigeon was 0.3/1mL in first immunization at 25 days of age and enhanced immunization at 46 daysof age respectively. The clinical manifestations of poultry flocks wereobserved and the titers of antibodies were determined by collecting serumsregularly. The results showed that the titers of anti-HI antibodies of H5 and H7subtypes reached peak values 21 days after first immunization in quails with(7.7±1.3)log2 and(8.5±1.3)log2 respectively. The duration of antibody eligibility exceeding 70%was about 35 days. The titers of anti-HI antibodies of H5 and H7 subtypesreached peak values 7 days after enhanced immunization with(9.7±0.9)log2 and(10.3±0.9)log2,respectively. The effect duration ofantibodies against H5 and H7 subtypes exceeding 70% were about 147 and 175 daysrespectively. The titers of H5 and H7 antibodies reached their peaks 21 daysafter first immunization in young pigeons with(9.8±0.8)log2 and(10.4±1.1)log2 respectively. The period when thequalification rates of the two subtypes of antibodies exceeded 70% was morethan 181 days. The titers of H5 and H7 antibodies reached their peaks 7 daysafter enhanced immunization with(10.2±1.0)log2 and(10.7±0.9)log2,respectively. After 189 days,the titers were(5.4±1.3)log2 and(6.1±1.5)log2 respectively,and the qualification rates were 96.7% and 100% respectively. Noserious side effects were found in clinical observation. The results showedthat(H5+H7)bivalent inactivated vaccine was safe and effectivefor quails and pigeons. Therefore,it was recommended that quails should be immunized at25 days of age,and they should receive enhanced immunes at46 days and 180 days at the dose of 0.3 mL. The pigeons should receive fistinoculation at 25 days of age,and enhanced immunizations every 6 months with the dose of 1 mL. 全文下载链接:http://kns.cnki.net/kcms/detail/37.1246.S.20190222.0911.030.html?uid=WEEvREcwSlJHSldTTEYzU3EydDRPZTFBeXhDa29ZZ2ljVUtvTXF2T3dtND0=$9A4hF_YAuvQ5obgVAqNKPCYcEjKensW4IQMovwHtwkF4VYPoHbKxJw!!
2019-03-04
3种食源性细菌免疫磁珠联合ATP发光检测技术研究
沙门氏菌、单核细胞增生李斯特菌和金黄色葡萄球菌是食品中常见的致病菌,在国内外常引起食源性疾病。为解决食源性疾病中的微生物检测问题,本研究将免疫磁珠技术和ATP发光技术联合用于食品微生物检测,选择沙门氏菌、单核细胞增生李斯特菌病和金黄色葡萄球菌为检测对象,建立快速检测这3种常见食源性病原菌的富集及检测新方法。该方法在显著缩短预增菌时间的条件下,通过免疫磁珠的富集作用获得与常规增菌时间同样的效果,样品中的微生物浓度增加了10倍,大大提高了样本检出率,缩短了检测周期。结果表明,本研究建立的免疫磁珠富集后联合ATP发光技术具有快速、敏感、特异和低廉的优点,可用于检测食品中沙门氏菌、单核细胞增生李斯特菌以及金黄色葡萄球菌,具有较好的推广应用前景。 Development of a Technology of Immunomagnetic BeadsCombined with ATP Chemiluminescence for Detection of three Kinds of FoodborneBacteriaAt present,Salmonella,Listeria monocytogenes and Staphylococcus aureus arecommon pathogenic bacteria in food,usually causing foodborne diseases. In order to solvethe problems of microbial detection,the immunomagnetic beads technology and ATPluminescence technology were combined,and a new method was established for rapid detectionof the three foodborne pathogens. Results showed that,under the condition of significantlyshortening the pre-enrichment time of bacteria,the same effects as the conventional enrichment timewas obtained by the immunomagnetic beads,and the microbial concentration of the samples wasincreased by 10 times,which greatly improved the sample detection rate and shortened thedetection cycle. In conclusion,the established method in this study was rapid,sensitive,specific and low-cost in detection of Salmonella,Listeria monocytogenes and Staphylococcusaureus in food,and it would have application prospects. 全文下载链接:http://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&filename=ZGDW201901021&dbname=CJFDTEMP
2019-03-01
弓形虫GT1虫株GRA15蛋白的表达及其反应原性分析
为了分析弓形虫GT1虫株GRA15(GRA15GT1)蛋白的反应原性,通过PCR扩增编码GRA15GT1 52~635氨基酸肽段的基因片段,构建pGEX-6P-1-GRA15GT1载体,转化BL21菌诱导表达;通过SDS-PAGE和Western blot方法进行表达验证及反应原性分析。结果显示:SDS-PAGE及以GST标签抗体为一抗进行Western blot,均有目的条带,比理论值稍大;以猪弓形虫阳性血清为一抗的Western blot条带与GST抗体孵育后的条带大小一致。上述结果表明,GRA15GT1蛋白具有较好的反应原性,这为下一步分段表达GRA15GT1蛋白,研究其在弓形虫血清学分型中的应用奠定了基础。Expression and Immunoreactivity Exploration of GRA15Proteinof Toxoplasma gondii GT1 StrainIn order to analyze the immunoreactivity of GRA15protein of Toxoplasma gondii GT1 strain(GRA15GT1),the gene encoding a 584-residue peptide(from aa52 to aa635)was amplified by PCR,then the pGEX-6P-1-GRA15GT1 vector wasconstructed and transformed into BL21 strain of E. coli. After inducibleexpression,the purified GRA15 protein were obtainedand verified by SDS-PAGE and Western blot. Results showed that,the target strip was slightly larger thanits estimated size when the GST-tag mouse monoclonal antibody was used asprimary antibodies,as well as the swine anti-T. gondii positive serum. The resultsindicated GRA15GT1 protein had good immunoreactivity,which would lay a foundation for the next stepof GRA15GT1 protein expression and its application in serological typing ofToxoplasma gondii.全文下载链接:http://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&filename=ZGDW201901020&dbname=CJFDTEMP
2019-03-01
袋鼠疱疹病毒1型实时荧光PCR方法的建立
为实现袋鼠临床样本中疱疹病毒1型(Macropodid herpesvirus 1,MaHV-1)的出入境快速检测,基于MaHV-1的gB基因序列设计特异引物和TaqMan探针,建立了一种MaHV-1荧光PCR检测方法。试验结果显示,该方法只对MaHV-1 gB基因呈现特异性扩增,与禽传染性喉气管炎病毒、伪狂犬病毒和牛传染性鼻气管炎病毒不发生交叉反应,对阳性标准质粒对照(pCR-MaHV-1-gB)的最低检测限为8个拷贝数/反应。该方法的组内和组间试验的Ct值变异系数介于0.17%~0.96%之间,具有良好的重现性。试验结果表明,本研究建立的实时荧光PCR方法可用于袋鼠MaHV-1的病原学检测。Development of a Real-time PCR for Detection ofMacropodid Herpesvirus 1For rapidly detecting macropodid herpesvirus 1(MaHV-1)in clinic samples from kangaroos at entry-exit ports,a real-time PCR was developed by designingprimers and probes based on the sequence of gB gene. The results showed themethod could only amplify the MaHV-1 gB gene and no cross-reaction with otherkinds of virus was observed,including infectious bovine bronchitis virus,avian laryngotracheitis virus andpseudorabies virus. The detection limit was 8 copies/reaction for the positivestandard plasmid control(pCR-MaHV-1-gB). The coefficients of variation(CV)of intra-assay and inter-assay were between 0.17 % to 0.96 %,showing good repeatability. In conclusion,the established method was applicable forpathogenic detection of MaHV-1 from the kangaroos. 全文下载链接:http://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&filename=ZGDW201901019&dbname=CJFDTEMP
2019-03-01
牛支原体和牛病毒性腹泻病毒二重二温式PCR检测方法的建立
为建立一种牛支原体(Mycoplasma bovis,MB)和牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)的快速鉴别诊断方法,针对MB的uvrC基因和BVDV的5'端非编码区(5'-UTR)保守基因序列,分别设计两对特异引物,并将三温式PCR扩增程序简化为二个温度梯度,建立了鉴别MB和BVDV的二重二温式PCR方法。该方法能同时扩增MB和BVDV,扩增产物大小分别为412和170 bp。特异性试验结果显示,该方法对参试的所有毒株只扩增MB和BVDV基因组,对其它牛病原体无扩增;敏感性试验结果显示,该方法最低能同时检测到104拷贝的两种目的核酸;干扰性试验结果显示,该方法能同时检测两个模板不同浓度的组合,试验结果不受模板影响。综上,本研究所建立的二重二温式PCR方法特异、敏感、快速、简便,可应用于MB和BVDV临床鉴别诊断和流行病学调查。 Development of Two-temperature Duplex PCRfor Detection of Mycoplasma bovis and Bovine ViralDiarrhea VirusIn order to establish a rapididentification and detection method for Mycoplasma bovis(MB)and bovine viral diarrhea virus(BVDV),two pairs of specific primers were designed based onthe conserved sequences of MB uvrC gene and BVDV 5'-UTR,and a new modified two-temperature duplexPCR was developed from three-temperature conventional PCR. According to theresults,the developed assay could amplify the genesof both MB and BVDV simultaneously,and the PCR products were 412 bp for MB and 170 bp forBVDV,respectively. The specificity test resultsshowed no cross-reaction with other bovine pathogens was observed. Thesensitivity test results showed that the detection limit was 104 copies ofnucleic acids of two target genes. The interference test results showed the combinationof different concentrations of the two templates could be detected by themethod,and the experimental results were notaffected by the template concentrations. In conclusion,the developed two-temperature duplex PCRassay was specific,sensitive,rapid and simple,and it could be applied in differential diagnosis for clinical samplesand epidemiological investigation. 全文下载链接:http://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&filename=ZGDW201901018&dbname=CJFDTEMP
2019-03-01
猪流行性腹泻病毒RT-LAMP可视化检测方法的建立
为建立一种适用于现场快速检测的猪流行性腹泻病毒(PEDV)LAMP技术,基于羟基萘酚蓝(HNB)的可视化显色特点,根据PEDV M基因编码区序列,设计合成1套引物,通过反应物浓度和反应条件优化,建立了可闭管检测的PEDV RT-LAMP检测方法。特异性和灵敏度试验结果显示,建立的RT-LAMP检测技术快速、灵敏、特异,可于1 h内检出0.2 mL 0.1 TCID50/mL的病毒RNA,与实时荧光RT-PCR检测方法灵敏度一致,与猪瘟病毒、猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型以及猪链球菌2型核酸不发生交叉反应。利用该方法对187份送检的粪拭子及病死猪组织样品进行应用检测,检出阳性样品9份,与荧光定量RT-PCR方法检测结果一致。试验结果表明,所建立的方法快速、特异,重复性满足要求,适用于送检样品的PEDV快速检测。 Development of HNB-based Visual RT-LAMP Method forDetectionof Porcine Epidemic Diarrhea VirusIn order to establish a RT-LAMP method forrapid detection of porcine epidemic diarrhea virus(PEDV),based on color reaction of HNB and according to M genesequence of PEDV,a set of primers were designed andsynthesized. By optimizing the reaction concentration and conditions,the RT-LAMP method was developed.Specificity and sensitivity results showed the established method was fast,sensitive and specific. It could detectPEDV-RNA extracted from 0.2 mL virus suspension(0.1 TCID50/mL),which was consistent with the real-time RT-PCR method.No cross reactions were observed with the nucleic acids of classical swinefever virus(CSFV),pseudorabies virus(PRV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus type 2(PCV-2) and Streptococcus suis type 2. Using the RT-LAMPmethod,a total of 9 positive samples were detectedfrom 187 samples of fecal swabs and dead pigs submitted,which was also consistent with the resultsof real-time RT-PCR. As a conclusion,the established method was rapid,specific and repeatable,and it was suitable for rapid detection of PEDV in submitted tissuesamples.全文下载链接:http://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&filename=ZGDW201901016&dbname=CJFDTEMP
2019-03-01
农业农村部召开全国非洲猪瘟防控工作视频会强调 坚决贯彻落实中央决策部署 毫不松懈抓好非洲猪瘟防控和生猪生产
进一步强化饲用血液制品生产过程管控——农业农村部畜牧兽医局有关负责人就防范饲料传播非洲猪瘟病毒风险答记者问
以质量兴牧促进畜牧业转型升级
农业农村部办公厅关于切实加强重大动物疫病强制免疫疫苗监管工作的通知
2019年第二期国家官方兽医师资培训班在青岛举办
非洲猪瘟防控国际研讨会在京举办
动物源食品中呋喃西林及其代谢物氨基脲研究进展
农业农村部办公厅关于近期两期规模养殖企业非洲猪瘟疫情违法违规查处情况的通报
成立农业农村部动物病原微生物实验室生物安全评审专家委员会
一株类禽型H1N1猪流感病毒的进化分析与分子特征