鲈鱼病毒性神经坏死病的诊断与防治

福建省漳浦县某海水养鱼场饲养的10万尾鲈鱼苗(Lateolabrax japonicus)发生以自转、狂游、侧游等运动失调及大批死亡为特征的疾病。病死鱼眼睛发亮、微肿,部分鱼体表有新鲜的破溃伤口,死亡率高达65%。寄生虫、细菌学检查均为阴性。应用世界动物卫生组织(OIE)推荐的检测引物,采用RT-PCR检测方法扩增出神经坏死病毒特异性的421 bp基因片段,依此诊断为病毒性神经坏死病(Viral nerve necrosis,VNN)。采取及时捞取病死鱼,去除网箱沉渣粪便,中草药吊袋消毒,减食和氯苯尼考、黄芪多糖、益生菌拌料等防治措施,收到很好的防治效果。     

湖北省武汉市某门诊猫传染性腹膜炎病例统计分析

为了解湖北省武汉市猫传染性腹膜炎的流行情况,以华中农业大学动物医院的门诊临床病例为研究对象,运用猫传染性腹膜炎抗原快速诊断试纸条和RT-PCR检测技术,对2017年11月至2018年9月的1 036例临床病例进行猫传染性腹膜炎检测,并对病例的发病情况和特点进行统计分析。共检出猫传染性腹膜炎阳性病例61例,检出率为5.9%。从时间分布看,2018年1月的检出率最高,占13.4%,其余月份的检出率为6.0%～8.0%。从群间分布看,英国短毛猫、田园猫、美国短毛猫的患病比例较高,占总阳性病例的76.7%;雄性猫(70.0%)患病比例高于雌性猫(30.0%),1岁以下幼猫的阳性占比为72.1%。本研究对于了解猫传染性腹膜炎流行发病特点以及该病的临床防治具有一定参考价值。     

H9亚型禽流感病毒实时荧光RT-PCR检测方法的建立与应用

为建立一种快速准确检测H9亚型禽流感病毒基因的检测方法,根据GenBank中H9亚型禽流感病毒血凝素编码基因进行荧光检测引物和探针设计,建立了一种针对H9亚型禽流感病毒的荧光RT-PCR检测方法。利用该方法进行灵敏度和特异性检测,并用本方法和国家标准中的荧光RT-PCR检测方法,同时对临床样本进行检测。结果显示,仅H9亚型禽流感病毒出现了正常荧光检测曲线,而其他病毒及阴性对照均未出现;检测灵敏度为RNA终浓度10~(-4) ng/μL(1.30×10~4 copies/μL);临床样本检测结果与国标方法一致,符合率为100%。结果表明,该方法特异性强、灵敏度高,可用于H9亚型禽流感病毒检测。     

鉴别猪伪狂犬病病毒野毒与疫苗毒双重PCR检测方法的建立

依据基因库中的猪伪狂犬病病毒(PRV)基因序列,分别设计了g E和g H两对引物,以PRV闽A株、Bartha(g E-)株和Norden(Tk-)株为模板,筛选最佳反应条件,建立了检测猪伪狂犬病病毒野毒株与疫苗毒株的鉴别PCR方法。该方法能从PRV闽A株、Norden(Tk-)株中扩增出一条355 bp的条带,但Bartha(g E-)株没有扩增出该目的条带。对正常细胞、猪细小病毒(PPV)、猪圆环病毒(PCV)进行检测,结果均为阴性,没有出现交叉反应。在对单项PCR反应条件(引物浓度、Mg~(2+)浓度、退火温度等)优化的基础上,建立了鉴别猪伪狂犬病病毒野毒与疫苗毒的双重PCR检测方法,并分别用双重PCR和单项PCR检测15份临床病料,两者符合率为97.5%,表明该双重PCR检测方法有较高的敏感度,可以用于临床病料的检测。     

福建地区猪瘟病毒流行株基因分型及E2基因遗传变异分析

为了解福建地区2013—2014年猪瘟病毒(CSFV)流行毒株的分子生物学特性与变异规律,采集了福建地区多个猪场的40份疑似猪瘟样品,应用RT-PCR技术对E2基因的主要抗原编码区进行扩增及序列分析,其中31份为猪瘟阳性。序列分析结果表明,31株CSFV流行毒株之间核苷酸与氨基酸的同源性分别为78.5%~100.0%和82.2%~100.0%,与国内外分离株的核苷酸同源性为81.1%~98.9%,氨基酸同源性为79.8%~95.6%;遗传进化树分析表明,31株CSFV属于1.1和2.1亚群,其中23株为1.1亚型,8株为2.1b和2.1c亚群,没有发现2.2和2.3亚群毒株。氨基酸序列分析表明,流行株在免疫逃逸相关位点705,713,729和734位氨基酸发生变异,FJ02毒株B细胞表位关键位点771位氨基酸发生变异,表明福建地区CSFV流行毒株呈现遗传变异多样性。     

新疆部分规模化奶牛场牛病毒性腹泻调查

为了了解新疆部分规模化奶牛场BVDV的感染情况,对采集的奶牛血清、流产死胎和鼻拭子等组织病料应用Ruminant BVD/BD p80-Serum阻断ELISA实验及牛病毒性腹泻(BVD)实时荧光RTPCR的方法进行检测,BVDV血清学检测阳性率为63.8%,组织病料样品中阳性率为13.46%,结果表明BVDV在新疆规模化奶牛场中已存在感染,因此应该对发病动物进行隔离或者淘汰,避免相互感染传播,降低感染率。     

Analysis of the Immune Efficacy of Attenuated Live Vaccine in the Pig Herds Previously Infected with Wild-type Porcine Reproductive and Respiratory Syndrome Virus

To evaluate the immune efficacy of porcine reproductive and respiratory syndrome (PRRS) attenuated live vaccine,this study selected 4 litters from two small pig farms (two litters each farm) and vaccinated with porcine reproductive and respiratory syndrome virus (PRRSV) attenuated live vaccine.Sera were collected on different days post vaccination (dpv) to detect PRRSV nucleic acid by RT-PCR,and PRRSV antibody using two different ELISA Kits,i.e.N-ELISA and G-ELISA.The results showed that PRRSV nucleic acid were positive on 0 dpv until 30 dpv in piglets and also positive on 0 dpv in the corresponding sows.All piglets were negative for PRRSV antibody on 0 dpv,but were positive on 30 dpv until 150 dpv.The positive rates detected by N-ELISA Kit were higher than those of G-ELISA Kit on 30 and 60 dpv,but lower than those of G-ELISA Kit on 120 and 150 dpv.A total of 216 sera were detected respectively by two ELISA Kits and the coincidence rate of the results was 95.83%.The P value of χ² test was more than 0.05,showing there was no significant difference between the results of two Kits.The Kappa value was 0.87,showing there was strong consistence between them.The correlation coefficient was 0.605,showing there was significantly linear correlation between them.The results indicated that the wild-type PRRSV in the previously infected pig herds could be eliminated by vaccination with attenuated live vaccine and both N-ELISA and G-ELISA Kits could be used to estimate the immune efficacy of the attenuated live vaccine effectively.     

Establishment and Application of Duplex RT-PCR Assay for Detection of Porcine Epidemic Diarrhea Virus and Pseudorabies Virus

In order to establish an assay for detecting porcine epidemic diarrhea virus (PEDV) and pseudorabies virus (PRV),two pairs of primers were designed basing on the M gene of PEDV and gE gene of PRV,respectively.The total RNA of standard PEDV and PRV strains were used as templates to establish the duplex RT-PCR assay.The specificity,sensitivity,repetition and clinic detection of the established assay were tested.The result revealed that the threshold of duplex RT-PCR was 10 TCID₅₀/mL of PEDV and PRV,and no products were amplified from the cell or the nucleic acid of other 7 kinds of pathogenic viral or bacterial microorganism.The detection results for 26 clinical suspicious PEDV or PRV infected pigs were consistent with the results tested by sequencing.This study suggested that the duplex RT-PCR method was highly specific,repeatable and sensitive,and was suitable for clinic rapid differential diagnosis of PEDV and PRV.     

Establishment and Application of Single and Duplex RT-PCR Method for Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus

For the distinguishment of wild-type and vaccine viruses of classical swine fever (CSF),a rapid and simple method was established,which laid the foundation for the purification of classical swine fever virus (CSFV) in large-scale farms.It was found that T-rich insertion sequence independently that 3'-NTR in Lapinized vaccine strain according to the genome sequence comparative analysis of CSF wild virus,vaccine virus and near origin virus published in GenBank,on the basis of this,two pairs of specific primers were designed in the upper and lower ends of the insertion sequence and duplex RT-PCR primers were designed.The anneal temperature of polymerese chain reaction (PCR) was optimized,then the two single and duplex RT-PCR method for the differentiation of wild-type and vaccine viruses of CSF were established.The sensitivity and specificity results showed that the minimum amounts of nucleic acid by the single RT-PCR were 2.2 pg (one pair of primers in single RT-PCR)and 1.7 pg (another pair of primers in single RT-PCR),respectively,and that of the duplex RT-PCR were 8.2 pg (wild-type virus of CSF) and 6.7 pg (vaccine virus of CSF), respectively, and no amplification of PRV,PRRSV,JEV,BVDV,PCV2 DNA/RNA were detected by these methods.Then the methods were used to detect 146 suspicious clinical samples,and the results showed that the positive rates of the mixed infection by wild-type and vaccine viruses of CSF in breeding sows,fattening pigs,nursery pigs and suckling piglets were 6.3%,7.4%,8.3%,8.6%, respectively.The results indicated that the single and duplex RT-PCR methods had high specificity,sensitivity,and good repeatability,and it had an important reference value for the purification of CSF in large-scale farms.     

实时荧光RT-PCR鉴别小反刍兽疫病毒疫苗株和野毒株

为快速鉴别小反刍兽疫病毒的疫苗株和野毒株,本研究通过分析Gen Bank中已发布的小反刍兽疫病毒全基因组序列,设计了1对通用引物以及疫苗株和野毒株特异性探针各1条,建立了小反刍兽疫病毒鉴别实时荧光RT-PCR方法。结果显示,该方法对疫苗株和野毒株的检测灵敏度均可以检测至10拷贝/反应,在108至101拷贝/反应之间具有良好的线性关系,扩增效率均接近1。该方法具有良好的特异性,能够区分不同谱系的野毒株株和疫苗株。使用该方法对136份临床疑似样品检测,检出的阳性数量与普通RT-PCR相同,并且测序后的区分结果也相同。本方法的建立为该病的实验室鉴别检测和流行病学调查提供了技术支持。