绵羊痘病毒、山羊痘病毒及羊口疮病毒多重PCR检测方法的建立和应用

为了建立鉴别绵羊痘病毒(SPPV)、山羊痘病毒(GTPV)和羊口疮病毒(ORFV)的多重PCR检测方法,针对GenBank中3种病毒的基因组序列,合成了3对引物,通过优化多重PCR反应条件,建立了鉴别检测3种病毒的多重PCR方法。特异性试验表明,应用该方法可分别扩增出3种病毒对应的目的片段,对大肠埃希菌、沙门菌、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、Vero细胞、正常羊组织的DNA和灭菌双蒸水均无扩增;敏感性试验表明,该方法最低检测量分别为30.46pg/μL的绵羊痘病毒、28.9pg/μL的山羊痘病毒和26.94pg/μL的羊口疮病毒基因组DNA;应用本方法对85份临床病料进行检测,结果与其他已建立的单项PCR检测方法结果一致,说明该方法可以用于临床上SPPV、GTPV和ORFV的鉴别诊断。     

禽流感、新城疫、传染性支气管炎三联没乳剂灭活疫苗的研制

以禽流感病毒（AIV）H5N4株，新城疫病毒（NDV）LaSota株与传染性支气管炎病毒（IBV）M41株为抗原研制了AI－ND－IB三联油乳剂灭活疫苗，并对疫苗的物理性状，安全性，免疫效力，保存期及抗体消长规律进行了检测，结果表明，疫苗在免疫后3周后6个月内，对AIV－H5N4，NDV－北京株的攻击均获全部保护，在免疫后52周内，免疫鸡箅清中AIV－N5N4，MDV，IBV的HI抗体也保持较高的水平，疫苗在4℃保存15个月，其免疫力没有下降。     

Virus, bacteriophages and water purification

Water can be a vector of viral disease, but direct virological analysis of water has logistic and practical limitations. Viruses of major importance for water hygiene (e.g. hepatitis and gastro-enteritis viruses) cannot yet be grown in tissue culture. Therefore, as in bacteriological quality procedures, model organisms are required for the evaluation of virological quality of water and the effectiveness of virus removal by water treatment processes. On the basis of published information, the F specific RNA (FRNA) phages have been chosen for this purpose. For the enumeration of the phages a particular Salmonella typhimurium strain with an artificially introduced F plasmid was developed as a host strain and was found to give accurate and reliable results. FRNA phages were found in very high numbers (10(2)-10(5) pfu/ml) in all types of waste water investigated. FRNA phages are seldom found in non-faecally contaminated waste water. Surprisingly low numbers are found in faeces. FRNA phages in waste water effluent were found to be highly resistant to chloramines and relatively resistant to UV inactivation. The FRNA phages can thus effectively be used as indicator organisms for human pathogenic viruses in the evaluation of disinfection processes for water treatment plants.     

Virus,Bacteriophages and Water purification

Summary

Water can be a vector of viral disease, but direct virological analysis of water has logistic and practical limitations. Viruses of major importance for water hygiene (e.g. hepatitis and gastro‐enteritis viruses) cannot yet be grown in tissue culture. Therefore, as in bacteriological quality procedures, model organisms are required for the evaluation of virologicalquality of water and the effectiveness of virus removal by water treatment processes. On the basis of published information, the F specific RNA (FRNA) phages have been chosen for this purpose. For the enumeration of the phages a particular Salmonella typhimurium strain with an artificially introduced F plasmid was developed as a host strain and was found to giveaccurate and reliable results. FRNA phages were found in very high numbers (102–105 pfu/ml) in all types of waste water investigated. FRNA phages are seldom found in non‐faecally contaminated waste water. Surprisingly low numbers are found in faeces.

FRNA phages in waste water effluent were found to be highly resistant to chloramines and relatively resistant to UV inactivation. The FRNA phages can thus effectively be used as indicator organisms for human pathogenic viruses in the evaluation of disinfection processes for water treatment plants.     

应用多重聚合酶链反应同时检测鉴别鸡新需疫病毒,鸡传染性支气管炎病毒,鸡

本文建立了一种同时检测ＤＮＶ、ＩＢＶ、和ＭＧ４种病原体的多重ＰＣＲ技术。根据ＮＤＶ、ＩＢＶ、ＩＬＴＶ和ＭＧ的基因文库,设计了４对分别与ＮＤＶ、ＩＢＶ、ＩＬＴＶ和ＭＧ某段基因序列互补的引物,用这４对引物对同一样品中的ＮＤＶ、ＩＢＶ、ＩＬＴＶ、ＭＧＲＶＡ和ＤＮＡ模板进行多重ＰＣＲ扩增,结果均同时得到了４条特异性大小与实验设计相符的３１０ｂｐ（ＮＤＶ）、１７２０ｂｐ（ＩＢＶ）、６４７ｂｐ（ＩＬＴＶ）和７３２ｂｐ（ＭＧ）多重ＰＣＲ扩增带,而对其他６种禽病病原的ＰＣＲ扩增结果均为阴性;敏感性测定结果表明,该多重ＰＣＲ技术难检出１０ｐｇ的ＩＢＶ、１ｂｇ的ＮＤＶ ＲＮＡ模板和ＩＬＴＶ、１ｐｇ的ＭＧ ＤＮＡ模板。     

猫细小病毒、犬细小病毒、貂细小病毒的特征比较

猫细小病毒、犬细小病毒和貂细小病毒是3种极为相似的细小病毒。最初人们主要根据患病水貂、猫、犬临床症状相似的特点,注意到它们之间可能有密切关系。时至今日,对这3种病毒的许多方面都已进行了深入的研究。作者从猫细小病毒、犬细小病毒、貂细小病毒病共同特性、生物学差异和进化机制等方面对这3种病毒的特征进行了比较和综述。     

多重RT-PCR检测猪口蹄疫病毒、猪水泡病病毒和猪水疱性口炎病毒的研究

建立了一种同时检测猪口蹄疫病毒(FMDV)、猪水泡病病毒(SVDV)和猪水疱性口炎病毒(VSV)三种病原体的多重RT-PCR方法。参照文献报道的基因序列,设计合成了三对特异性引物;PCR扩增条件进行优化后,用这三对引物对同一样品中的FMDV、SVDV、VSVRNA模板进行扩增,结果同时得到了三条特异性条带,大小与试验设计相符:FMDV(208bp)、SVDV(862bp)、VSV(638bp),且对猪瘟病毒(CSFV)、猪繁殖与呼吸综合症病毒(PRRSV)和猪传染性胃肠炎病毒(TGEV)核酸扩增结果为阴性;三种病毒RNA模板检出的最小量均为10fg。试验证明,此方法经济、快速、敏感、特异,可用于FMDV、SVDV和VSV这三种猪水泡性疾病的鉴别诊断及流行病学调查。     

绵羊感染伪狂犬病的病理学观察与病毒分离鉴定

为了确诊1例疑似绵羊伪狂犬病病例,本试验以上海郊区某羊场发病绵羊的病料为研究对象进行了病理学观察、病毒分离和鉴定。病理组织学变化显示,发病羊大脑组织神经元发生广泛性变性、坏死并伴有嗜神经现象,神经元周围出现胶质细胞增生。病料接种BHK-21细胞,细胞出现病变,间接免疫荧光试验和荧光定量PCR检测结果证实所分离的病毒为伪狂犬病病毒。流行病学调查结果表明,绵羊伪狂犬病可能是由猪伪狂犬病病毒感染引起的。通过免疫接种猪伪狂犬病弱毒疫苗,绵羊伪狂犬病疫情得到及时控制。     

肉种禽网状内皮增生症、鸡传染性贫血和禽白血病血清学调查

对不同种鸡场不同周龄肉种鸡进行REV、CAV和ALV（A、B亚群）抗体检测。在572份血清样品中，除了一个8．1周龄和15周龄鸡群CAV抗体阳性率分别为13．3％和75％外，其它鸡群无论是否进行CAV疫苗免疫，抗体阳性率均为100％。在212份血清样品中，REV抗体阳性率在16．7％～62．5％之间；ALV（A、B亚群）抗体阳性率在0％～75％之间。本研究结果表明，所检测种鸡群中存在3种免疫抑制性病毒的感染或混合感染。     

Investigation and Analysis of Porcine Epidemic Diarrhea Virus,Transmissible Gastroenteritis Virus and Pseudorabies Virus of Swine Diarrhea Cases in Shandong Province

To understand porcine viral diarrhea prevalence in the large-scale pig farms of Shandong province, a total of 3 035 clinical samples were detected by PCR from January, 2014 to December, 2016.Those samples were detected for porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and pseudorabies virus (PRV). The results showed that the detection rate of PEDV, PRV and TGEV were 67.49%, 9.33% and 3.29%, respectively.During the past three years, the lowest detection rate of PEDV was 48.15% in the fourth quarter of 2014,and the highest was 88.57% in the fourth quarter of 2015.In 2016,the detection rate represented fluctuate declining compared with 2015.The highest positive rate of TGEV was 18.52% in the fourth quarter of 2014,and in the third quarter of 2015 was 15.38%.The lowest positive rate of TGEV was 6.67% in the first quarter of 2016 and TGEV was not detected in the other quarters. The highest detection rate of RPV was 15.68% in the second quarter of 2016,and the lowest was 2.56% in the second quarter of 2014,except the first quarter of 2014 that the PRV was 0. By detecting three kinds of viruses in 69 clinical samples collected passively, the results showed that the detection rate of PEDV,TGEV and PRV were 86.96%,5.80% and 37.68%,respectively. The total single infection rate was 69.57%,the single infection rates of PEDV,TGEV and PRV were 57.97%,1.45% and 10.14%, respectively;The total mixed infection rate was 30.43%,the mixed infection rates of PEDV/PRV,PEDV/TGEV and TGEV/PRV were 26.09%,2.90% and 1.45%, respectively;Obviously, the total single infection rate was higher than the total mixed infection rate. The results showed that the PEDV, PRV and TGEV were prevailing in Shandong province. There were PEDV/TGEV, TGEV/PRV, PEDV/PRV mixed infection, and the number of PEDV/PRV mixed infection was in the majority. However, there was no PEDV/PRV/TGEV mixed type infection. At present, PEDV was the major pathogen of porcine viral diarrhea and the test results could provide the reference to the diagnosis of porcine viral diarrhea.     

山东省猪腹泻病例中PEDV、TGEV和PRV的感染调查与分析

为了解山东省规模化猪场由猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）和猪伪狂犬病毒（PRV）引起猪病毒性腹泻的流行情况,自2014年1月至2016年12月,对来自山东省各地规模化猪场的猪腹泻病料（共3 035份）进行PCR检测。结果显示,PEDV、PRV和TGEV阳性率分别为67.49%、9.33%和3.29%;3年间,PEDV阳性率在2014年第四季度最低,为48.15%,2015年第四季度阳性率最高,为88.57%,2016年各季度阳性率相对2015年呈波动下降趋势;TGEV阳性率在2014年第四季度最高,为18.52%,2015年第三季度阳性率为15.38%,2016年第一季度阳性率为6.67%,其他季度未检测出阳性病料;PRV阳性率在2016年第二季度最高,为15.68%,除2014年第一季度未检出阳性病料外,2014年第二季度阳性率最低,为2.56%。通过对69份被动送检的病料进行PEDV、TGEV和PRV混合感染检测发现,这部分病料中PEDV、TGEV、PRV阳性率分别为86.96%、5.80%和37.68%;总单独感染率为69.57%,PEDV、TGEV和PRV单独感染率分别为57.97%、1.45%和10.14%;总混合感染率为30.43%,PEDV/PRV、PEDV/TGEV和TGEV/PRV混合感染率分别为26.09%、2.90%和1.45%;总单独感染率比总混合感染率高。结果表明,山东省存在PEDV、PRV和TGEV 3种病毒流行,存在PEDV/TGEV、TGEV/PRV和PEDV/PRV的混合感染,混合感染中主要为PEDV/PRV混合感染,不存在PEDV/PRV/TGEV的混合感染。目前PEDV是引起山东省猪病毒性腹泻的主要病因,本试验结果可为山东省猪病毒性腹泻的诊断和控制提供参考。     

猪瘟、猪伪狂犬病及猪链球菌病混合感染的诊治

今年1－4月，在鄂、豫两省三个规模化养猪场暴发了以母猪繁殖为主要特征的疫情后，采集病料进行了病理部检及实验室检验，结合临床症状及发病情况，确诊为猪瘟、猪伪狂犬及猪链球菌病混合感染，并通过采取有效的综合防治措施，使疫情及时地得到控制。     

施马伦贝格病毒的病原学、流行病学及临床症状与检测方法

本文对2011年引起德国奶牛疾病的一种新病毒——施马伦贝格病毒的病原学、流行病学、临床症状及检测方法进行了阐述。。该病毒可引起牛、山羊、绵羊等家畜发病,造成动物发热、腹泻、乏力等临床症状,可导致动物早产或难产,给畜牧业带来了巨大的危害．     

Reciprocal Antibody and Complement Responses of Two Chicken Breeds to Vaccine Strains of Newcastle Disease Virus, Infectious Bursal Disease Virus and Infectious Bronchitis Virus

Serum antibody responses and haemolytic complement activity were evaluated in White Leghorn (WLH) and Rhode Island Red (RIR) chickens that were vaccinated with live-attenuated vaccines of Newcastle disease virus, or infectious bronchitis virus, or infectious bursal disease virus by means of ocular challenge at 10 times the normal vaccination dose. Complement titres in non-vaccinated birds were significantly higher in WLH birds compared to RIR birds. The lentogenic viral infection resulted in an immediate stimulation of complement activity, followed by a decrease to initial complement levels within 2 weeks post vaccination, when the antibody response took over immune defence. As compared to WLH chickens, RIR birds mounted a faster and significantly higher antibody response to the vaccine viruses used. In WLH hens, significantly higher haemolytic complement activity post vaccination was found as compared to RIR hens. Possible consequences of the observed differences in immune responsiveness of the two breeds to viral vaccines are discussed.     

猪繁殖与呼吸综合征、猪圆环病毒2型、猪伪 狂犬病病毒混合感染的检测分析

本研究采用RT-PCR方法对2010年12月至2011年11月采集于豫西地区的170份疑似PRRSV感染猪病料进行病原检测,得到102份高致病性PRRSV阳性样本,在此基础上应用PCR方法检测PCV2和PRV的感染情况,并计算混合感染率。试验结果显示,豫西地区PRRS发病猪主要疫病的总混合感染率为58.52%,二重混合感染率为39.21%,三重混合感染率为19.61%,其中PRRSV/PCV2型二重混合感染最严重,混合感染率达33.33%;春夏和秋冬总混合感染率分别为48.57%、81.25%,而且不管是二重还是三重混合感染,秋冬均比春夏更严重,尤其是PRRSV/PCV2/PRV型三重混合感染,秋冬与春夏季节的混合感染率相差较大,分别为37.50%、11.43%;PRRS发病猪从哺乳期到育肥期都有混合感染,混合感染率分别为42.10%、50.00%、100.00%,混合感染程度逐渐加重,主要集中在育肥期;不同发病时期的最高混合感染型也有所不同,其中哺乳期最高混合感染型为PRRSV/PCV2,混合感染率为42.10%,保育期最高混合感染型为PRRSV/PCV2、PRRSV/PCV2/PRV,混合感染率均为22.73%,育肥期最高混合感染型为PRRSV/PCV2/PRV,混合感染率为50.00%。本研究反映了豫西地区PRRS发病猪群与猪圆环2型及猪伪狂犬病的混合感染情况和规律,为该地区PRRS及其混合感染的临床诊治和区域性防控工作的进行提供了理论依据和指导。     

IBV广西分离株GX-YL5在鸡体内动态分布和排毒规律及组织嗜性研究

鸡传染性支气管炎病毒(IBV)广西分离株GX-YL5通过滴鼻点眼人工感染14日龄健康非免疫雏鸡,对接毒后1、3、5、7、10、14、21、28、35和42 d试验鸡的气管、肺脏、肾脏、胸腺、腺胃、盲肠扁桃体和肝脏等组织及泄殖腔棉拭子进行IBV的反转录巢式PCR检测,同时对接毒后不同时间的气管、肺脏、肾脏、胸腺、腺胃、法氏囊、脾脏和肝脏进行组织病理学观察。结果表明接毒后的3～21 d,气管、肺脏、肾脏、胸腺、腺胃、盲肠扁桃体和肝脏的病毒检测均为阳性,接毒后42 d肾脏和气管检测结果仍为阳性;接毒后3～42 d泄殖腔棉拭子检测结果为阳性。组织病理学观察见到气管黏液,气管和细支气管纤毛脱落,炎症细胞浸润,肺充血,淋巴细胞浸润,肾脏间质性肾炎以及法氏囊水肿。研究结果表明,GX-YL5株对雏鸡具有广泛的组织嗜性,主要脏器带毒时间21 d或更长,泄殖腔排毒持续至少39 d。     

猪伪狂犬病毒DQ株的分离鉴定和生物学特性的研究

从黑龙江大庆某猪场采集流产死亡的仔猪大脑、扁桃体等病料组织接种BHK-21细胞,分离到1株病毒,经PCR检测、直接荧光法检测,血清中和试验,证实分离到的病毒为伪狂犬病毒,命名为PRV DQ株,该病毒经克隆纯化后测得其毒价为108.36TCID50/ml,对热、胰蛋白酶、氯仿、乙醚敏感,15日龄哺乳仔猪接种该分离株后发病死亡。     

牛病毒性腹泻病(BVD)的危害及防制

牛病毒性腹泻病（BVD）．是由牛病毒性腹泻病毒（BVDV）引起的一种极为复杂、有多种临床表现的疾病，又称粘膜病。Olafson。MacCallum和Fox等人于1946年在纽约首次发现该病．并将其描述为严重的腹泻性胃肠炎。Ramsev和Chivers在1953年描述了美国爱荷华州及周边州区不同年龄阶段肉牛和奶牛的粘膜病。与Olafson所描述的腹泻和粘膜溃疡临床症状相似。起初认为此病与病毒性腹泻病不是同一种病．但通过奶牛的免疫学和细胞培养法的研究．结果表明病毒性腹泻病和粘膜病的病原都是BVDV．粘膜病可能是病毒性腹泻病的激发病症。     

新城疫病毒变异、进化与跨种感染

新城疫病毒是一种变异、进化速度很快的病原,近年来新城疫病毒感染的宿主范围有不断扩大的趋势,不仅可跨种感染多种水禽,并有跨种感染猪的报道。本文通过新城疫病毒F基因和HN基因变异规律分析,探讨了基因变异对新城疫病毒毒力及跨种感染能力的影响。