白鹭源新城疫病毒的分离与鉴定

从1只患病的白鹭的咽喉、泄殖腔棉拭子中分离到1株病毒,经血凝、血凝抑制试验和RT-PCR法鉴定为新城疫病毒。根据该毒株对鸡胚平均致死时间、鸡胚半数致死量、鸡胚半数感染量的测定和新城疫强弱毒鉴别的RT-PCR检测,表明该分离株为新城疫强毒株。     

鸡毒支原体(MG)疫苗株和野毒株的PCR鉴定

针对由于鸡毒支原体(Mycoplasma gallisepticum, MG)不同毒株(针对TS-11、6/85、F株疫苗株和S6、R株野毒株)的特定序列,设计出特异性检测引物,对临床免疫MG疫苗株TS-11的鸡群以及父母代(A～H场)鸡群和祖代(I场)鸡群采集毛蛋气囊拭子,A场父母代种鸡群和I场祖代鸡群同时采集咽喉拭子进行MG的PCR鉴别诊断,对临床送检免疫6/85的客户鸡群(J场)采集病料,F株疫苗株进行MG的鉴别诊断,同时采集SPF鸡群咽喉拭子作为阴性对照群,验证该鉴别引物的特异性。结果显示:鉴别引物具有非常好的特异性,SPF鸡群MG的检测均为阴性,不存在假阳性的弊端;临床免疫MG疫苗株6/85的鸡群(J场)存在MG野毒株S6的感染,在免疫后1个月左右采集病料中仍能检出MG 6/85疫苗株。对免疫TS-11的父母代种鸡群(A～H场)和祖代种鸡群(I场)死亡的毛蛋气囊拭子,用PCR检测MG阳性率,结果显示:祖代鸡群(I场)后代死亡的毛蛋气囊评估结果为优,毛蛋气囊拭子MG的PCR鉴别引物检测均为阴性;而父母代鸡群后代死亡的毛蛋气囊从50周龄开始出现浑浊,MG的PCR鉴别引物可同时检出TS-11疫苗毒和野毒株,部分鸡群同时存在R株和S6株两种野毒感染;采集A场父母代种鸡群的咽喉拭子和后代啄壳死亡毛蛋气囊拭子,PCR检测结果显示种鸡群和后代可同时检出野毒株S6和疫苗株TS-11。这表明种鸡群MG的感染,可通过垂直传播感染其后代。本研究设计的MG鉴别引物,可将不同MG疫苗株(TS-11、F株、6/85)与野毒株(S6株、R株)区分,为临床MG的鉴别诊断和种鸡群的MG感染状态评估提供积极的指导意义。     

2株野禽源新城疫病毒的分离鉴定及F基因序列分析

新城疫是一种由新城疫病毒引起的鸡和多种禽类发病的急性、高度接触性传染病,广泛分布于世界各地,给养禽业造成巨大经济损失。本研究检测113份禽类泄殖腔拭子,包括10份蓑羽鹤拭子,6份鸳鸯拭子,27份鸭拭子,33份黑天鹅拭子,37份雁拭子。其中2份黑天鹅拭子(命名为W53和W76)鉴定为新城疫病毒阳性。对这2株毒的F基因进行测序和遗传进化分析,结果表明,W53和W76与我国NDV经典强毒株F48E9同属Class Ⅱ中的基因IX毒株,其F蛋白裂解位点氨基酸序列均为112-RRQRRF-117,符合NDV强毒株的典型分子特征。     

禽流感重组灭活疫苗(H5N1,Re-6)在鸡体内外的分布及分析

本研究以重组禽流感病毒灭活疫苗(H5N1亚型,Re-6株)对雏鸡进行免疫,于免疫后不同时间点采用实时荧光RT-PCR方法对试验鸡的泄殖腔拭子、血清及内脏组织进行病毒RNA检测,同时于免疫前后对动物房及周围环境进行检测,旨在动态研究疫苗在鸡体内外的分布规律,据此对疫苗的免疫效果及安全性进行初步的分析研究。结果可见,该疫苗在24 h分布于全身,并能在血液中持续分布,表明疫苗免疫效果较好;免疫后试验鸡临床表现正常,表明疫苗安全性较好,但可在泄殖腔拭子及环境中检测到病毒核酸,可能存在灭活疫苗通过机体排出现象,致体外环境污染。     

荧光RT-PCR检测活禽和禽产品中新城疫病毒中强毒株的研究

本研究中，采用TaqMan方法，根据新城疫病毒F基因核苷酸序列，设计合成多对引物，在上游引物和下游引物之间设计多条探针，通过对引物、探针的筛选，反应条件的选择和优化，建立了检测活禽和禽产品中中强毒力新城疫病毒的荧光RT-PCR方法。经对10株倍比稀释的中强毒力新城疫病毒的尿囊液进行检测后，表明所建立的荧光RT-PCR方法的检测极限在10^-5～10^-7”之间，略低于鸡胚病毒分离方法（10^-8～10^-9）；对收集到的所有新城疫病毒株和常见禽类病毒（包括禽流感病毒H5、H9亚型、IBDV、IBV、KIBV、CAAV、鸭瘟病毒、鸭肝炎病毒）进行检测，结果表明建立的方法不能检出其他的常见禽类病毒，特异性良好。进一步用建立的荧光RT-PCR检测人工感染SPF肉鸡的组织脏器、咽喉及泄殖腔拭子及临床样品中的中强毒力的新城疫病毒，并同鸡胚分离结果比较，结果表明荧光RT-PCR的敏感性同鸡胚分离试验基本一致。由于荧光RT-PCR方法快速，从处理样品开始到出结果只需不到4小时，而且检测样品量大，这就充分显示了其快速、敏感、特异的优势。在强调口岸快速通关的今天，该方法的建立无疑为活禽或禽产品的快速检验检疫提供了有效的手段。     

应用荧光RT-PCR方法检测H7N9亚型禽流感病毒

为了验证荧光RT-PCR方法检测H7N9亚型禽流感病毒的可行性,应用建立的荧光RT-PCR方法检测H7N9亚型禽流感抗原、H9亚型禽流感抗原、H5亚型禽流感抗原、新城疫抗原评估检测方法的特异性,检测不同稀释度的H7N9亚型禽流感抗原,并用建立的方法检测采自三鸟批发市场100份泄殖腔、喉拭子。结果是建立的荧光RT-PCR方法,H7体系能检测出1:10-10抗原稀释度,N9体系能检测1:10-9抗原稀释度,对H9亚型禽流感抗原、H5亚型禽流感抗原、新城疫抗原无交叉反应;检出100份泄殖腔、喉拭子结果均为阴性。结果表明,荧光RT-PCR方法H7N9亚型禽流感病毒具有快速、灵敏、特异的特点,适合对禽类的大规模监测。     

鉴别犬瘟热病毒强弱毒株SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

为建立犬瘟热强弱毒株SYBR Green Ⅰ荧光定量RT-PCR检测方法,根据Gen Bank公布的犬瘟热毒株的全基因序列并参考相关文献,选择M基因保守区域合成了1对通用引物M1/M4及鉴别犬瘟热强弱毒株的特异引物M2和M3。试验证明,该方法重复性良好;检测强弱毒株的最低检出限度在10拷贝;对新城疫病毒、水貂肠炎病毒、阿留申病毒、犬腺病毒、犬细小病毒5种病毒的特异性检测均为阴性;对175份犬瘟热疑似病料进行荧光定量RT-PCR,检出127份阳性样品,其中强毒株98份,弱毒疫苗株29份,常规RT-PCR检出112份阳性样品。结果表明,该方法的敏感性高于常规RT-PCR检测,并能有效的鉴别强弱毒株。     

基于Taqman-MGB探针的猪瘟病毒野毒株荧光RT-PCR检测方法的研究

应用MGB荧光RT-PCR技术对猪瘟病毒野毒株进行实时定量检测,以实现对猪瘟病毒野毒株与疫苗株的鉴别诊断。以猪瘟病毒5’端非编码区为扩增靶区,通过鉴别诊断位点的筛选、引物、探针和反应条件的优化,建立了猪瘟病毒野毒株荧光RT-PCR检测方法。试验结果表明,该方法的检测灵敏度为10拷贝/反应或3.0 TCID50/反应;对猪瘟病毒野毒株的检测结果均为阳性,而对猪瘟病毒疫苗株和其他猪源病毒的检测结果均为阴性,表明该方法具有较好的特异性;对515份临床样品的检测结果表明,该方法与RT-nPCR测序法的检测符合率为98.3%;猪瘟病毒人工感染猪实验结果表明,猪瘟野毒感染猪发病前2-4天即可被检测出阳性,疫苗免疫株检测结果始终为阴性。     

肠炎沙门菌疫苗株Sm24/Rif12/Ssq在蛋鸡体内的定殖规律

为明确肠炎沙门菌疫苗株Sm24/Rif12/Ssq在蛋鸡体内的定殖规律,选取50只海兰褐蛋鸡,于0日龄(出雏当日)滴口免疫1羽份(1×10⁸ CFU/只)Sm24/Rif12/Ssq疫苗(首免),分别于免疫后1、3、5、7d采集咽拭子、泄殖腔拭子以及盲肠和肝脏样品测定疫苗株定殖水平,并于免疫后30 d采集血液测定抗体水平;随后选取30只经过首免的6周龄蛋鸡和30只经过二免的16周龄蛋鸡,分别滴口免疫1羽份(1×10⁸ CFU/只)Sm24/Rif12/Ssq疫苗,即为二免和三免,于免疫后4 d采集泄殖腔拭子,测定疫苗株定殖水平,并于免疫后30、60、100 d采集血液测定抗体水平。结果显示,首免后咽拭子、泄殖腔拭子和盲肠样品中疫苗株载量呈先上升后下降趋势,疫苗株核酸检出率60%～100%,肝脏样品中疫苗株载量呈逐步下降趋势,疫苗株核酸检出率50%-90%,但未从血液样品中检出抗体;二免和三免后泄殖腔拭子中疫苗株核酸检出率为17%～20%,血清抗体阳性率呈逐步上升趋势。结果表明,Sm24/Rif12/Ssq疫苗株易定殖于0日龄蛋鸡肠道,而较难定...     

番鸭呼肠孤病毒弱毒株在免疫番鸭体内的分布及排毒规律

番鸭呼肠孤病毒(Muscovy duck reovirus,DRV)B37弱毒株经肌内免疫1日龄雏鸭,应用RT-PCR检测病毒核酸,以阐明病毒在体内分布及排毒规律。结果表明,B37株免疫雏鸭后4h,即可在血液、心、肝、脾组织中检出DRV RNA;接种后8h,血液、心、肝、脾、肺、肾、胰腺均可检测到DRV RNA;接种后3d,喉头和泄殖腔棉拭子可检出DRV RNA;接种后14d,喉头和泄殖腔棉拭子已不能检出DRV RNA;接种后28d,肺、肾和胰腺均已不能检出DRV RNA;接种后42d,血和心脏已不能检出DRV RNA;接种后49d,所有组织均已不能检出DRV RNA。因此,DRV弱毒在血液、心脏中分布时间为接种后4h～35d,在肝、脾组织中分布时间为4h～42d;肺、肾和胰腺的分布时间为8h～25d;向外界排毒时间为3～14d。     

Characterization of a battery of monoclonal antibodies for differentiation of Newcastle disease virus and pigeon paramyxovirus-1 strains

Twenty monoclonal antibodies (MCAs) prepared against the velogenic GB-Texas strain of Newcastle disease virus (NDV) and the type 1 pigeon paramyxovirus (PPMV-1) were characterized and examined as potential immunodiagnostic reagents. All MCAs generated were found to bind specifically, but with varying reactivity, to various NDV strains in direct binding assays. In addition, MCA 15C4 neutralized and inhibited hemagglutination (HA) of all lentogenic, mesogenic, and velogenic NDV strains tested but not the PPMV-1 strain. Antibody 10D11 also inhibited HA activity, but inhibition was more selective and limited to the mesogenic and domestic or indigenous velogenic strains of NDV. MCA 79 reacted in all serologic assays with an antigenic site common to all serotype 1 avian paramyxoviruses. Passive immunization studies involving three different neutralizing MCAs (35, 79, and 15C4) showed that enhanced, but not complete, protection against virulent NDV challenge was provided when the three MCAs were administered in combination.     

荧光定量RT-PCR检测中、强毒力新城疫病毒方法的建立及验证

根据新城疫病毒F基因的裂解位点序列,设计合成一对引物和TaqMan探针,以本室构建并保存的鹅源新城疫病毒ZJ1株F基因阳性重组质粒作为中、强毒力新城疫病毒RNA定量检测的标准品,建立检测方法。结果表明本试验建立的标准曲线循环阈值（Ct值）与模板浓度具有良好的线性关系,相关系数为0.999,灵敏度约为3拷贝/μL,对禽流感病毒和其他禽病病毒无交叉反应,特异性好、重复性佳,为中、强毒力新城疫病毒检测提供了一种快速高效的定量检测方法。对28株标准分离株强弱毒力的检测与经典病毒分离方法符合率达100%,对187份临床样品的检测,二者结果符合率接近90.0%。在新城疫病毒临床样品快速检测、流行病学监测等方面显示良好的应用前景。     

Unexpected newcastle disease virus in day old commercial chicks and breeder hen

Newcastle disease virus (NDV) specific antigen in the gut contents and NDV specific antibody in blood circulation were seen in day old chicks belonging to nine different commercial hatcheries of Tamil Nadu, India. Antigen disappeared by 4th week and antibody by 6th week of age. Fourteen NDV isolates obtained from the gut contents of day old chicks of different commercial hatcheries, one NDV isolate from dead in shell eggs and one NDV isolate from breeder hen were characterized and grouped under velogenic, mesogenic and lentogenic pathotypes. Four isolates were grouped under F and another four isolates were grouped under E based on reaction with monoclonal antibodies (Mabs) but found to be velogenic based on pathogenicity tests. In one particular flock velogenie NDV was isolated from breeder hen, dead in shell embryos and day old chicks and they all belong to Mabs group E. Vertical transmission of velogenic, mesogenic and lentogenic NDVs and role of NDVs in the gut contents have been discussed.     

基于锁核酸探针的双重荧光实时RT-PCR检测方法鉴别新城疫中强毒株与弱毒株

为鉴别新城疫病毒(NDV)强毒株和弱毒株,本研究建立了基于新型锁核酸(LNA)探针的实时荧光RT-PCR检测方法(Duplex LNA rRT-PCR)。该方法针对NDVF基因裂解位点设计了两条新型LNA探针,通过对11株NDV株进行大将军脂duplex LNA rRT-PCR检测方法检测,验证该方法的特异性;通过对副粘病毒I型(APMV-1)和NDV中强毒株(vNDV)不同浓度病毒液进行检测,确定该方法的灵敏度,并与TaqMan实时荧光RT-PCR检测方法进行比较。结果显示本研究所建立的方法对11株NDV检测的特异性为100%(11/11),优于TaqMan实时荧光RT-PCR检测方法(10/11);所建立的duplex LNA rRT-PCR方法检测中强毒株F48E9和弱毒株LaSota的灵敏度分别为10个EID50和0.1个EID50,比美国农业部推荐的TaqMan实时荧光RT-PCR检测方法低10倍。本研究利用新型LNA探针技术,建立了鉴别NDV中强毒株与弱毒株的duplex LNA rRT-PCR检测方法,可以特异性检测NDV并有效区分中强毒株与弱毒株,适合用于鸡场和进出境动物产品中NDV的快速检测。     

鹅副黏病毒与新城疫病毒对鸡胚和鹅胚成纤维细胞的感染性分析

用鸡新城疫病毒F48E9,Komarov, LaSota,V4/66株和鹅副黏病毒SF02株分 别感染鸡胚和鹅胚成纤维细胞,用MTT显 色方法检测存活细胞数目。结果表明,不同 毒力新城疫病毒致细胞病变的作用不一样, 鸡新城疫强毒F48E9株和鹅副黏病毒SF02 株均引起两种细胞几乎全部死亡,感染动物 时,SF02与NDV强毒株的致病性有显著的 区别,但感染细胞时,二者并无明显差别; NDV与GPMV在感染水禽时的致病性差异 并不是因为诸如细胞膜受体等细胞水平的因 素不同造成的,可能与干扰素途径有关。     

Velogenic viscerotropic Newcastle disease virus strains in Morocco

Isolates of Newcastle disease virus (NDV) in Morocco were characterized as velogenic. Two isolates were from tracheal swabs taken at a Moroccan live poultry market, and four isolates were from field cases. Infection of 8-week-old chickens showed that these isolates and previously characterized Moroccan isolates were of the viscerotropic pathotype. Based on hemagglutinin thermostability and the capacity to agglutinate equine erythrocytes, the Moroccan velogenic viscerotropic NDV isolates were classified as belonging to at least three distinct strains.     

Comparison of Various Diagnostic Methods in Characterizing Newcastle Disease Virus Isolates from Desi Chickens

Eleven Newcastle disease viruses (NDV), isolated from apparently healthy and ailing Desi chickens were subjected to both conventional and modern characterization techniques. The virulence and strain differentiating experiments placed 10 isolates in the velogenic group and one in the mesogenic group. In MDBK cells, 9 isolates produced characteristic cytopathogenic effects up to 5 and 2 up to 3 passages. Molecular characterization with a 21-mer oligonucleotide probe placed all the isolates in the velogenic/mesogenic group. The results of this study clearly indicated that the isolates obtained are either velogenic or mesogenic but not lentogenic.     

THE RESPONSE OF AUSTRALIAN CHICKENS NATURALLY INFECTED WITH AVIRULENT NEWCASTLE DISEASE VIRUS TO CHALLENGE WITH VELOGENIC NEWCASTLE DISEASE VIRUS

SUMMARY Sixty-eight breeder chickens, 4 to 12 months of age, were taken from Australian flocks that had been naturally infected with avirulent Newcastle disease virus (NDV) and transported by air to Malaysia. Nearly all the breeders had haemagglutination inhibition antibodies to NDV, at titres of from 2 to 128. Thirty-two were inoculated intranasally with an Asian, velogenic, viscerotropic strain of NDV and all survived this challenge. Thirty-six were exposed to contact infection with the same velogenic NDV and 2 died of Newcastle disease within 14 days. The levels of haemagglutination inhibition antibodies against NDV increased in the surviving breeders after challenge, reaching 2048 or greater in a few birds. Velogenic NDV was isolated from a cloacal swab from one clinically normal breeder 10 days after challenge by contact. Cloacal swabs taken 7 to 10 days after challenge from another 23 breeders yielded no NDV. Twenty-four broilers, 7 weeks of age, were also transported from Australia to Malaysia. All lacked detectable haemagglutination inhibition antibody to NDV and they were from a flock with no detectable antibody to NDV. Twelve were challenged with velogenic NDV intranasally and 12 were subjected to contact challenge. All broilers died of Newcastle disease within 13 days.     

Antigenetically Unusual Newcastle Disease Virus from Racing Pigeons in India

Newcastle disease virus isolated from an outbreak in racing pigeons in India was found to be velogenic, based on the mean time to death in 10-day-old embryonated hen's eggs, the intravenous pathogenicity index in 6-week-old chickens and the pathogenesis in chickens and pigeons. The virus induced disease in chickens without prior adaptation in chickens. The virus was antigenically unusual since it could not be grouped with the available panel of monoclonal antibodies at the World Reference Laboratory for Newcastle disease, UK. However, commercially available lentogenic and mesogenic vaccines provided 100% protection to chickens against this antigenically unusual NDV.