二乙烯亚胺对猪细小病毒的灭活作用

使用新型灭活剂二乙烯亚胺(binary ethylenimine,BEI)对猪细小病毒(Porcine parvovirus,PPV)进行了灭活试验,通过ST传代细胞接种法观察病毒灭活后是否出现细胞病变,并结合血凝试验检测灭活效果,确定最佳灭活方法。用3～5日龄乳鼠检测BEI灭活后的病毒培养物和相应制备疫苗的安全性,并用豚鼠检测该灭活工艺制备疫苗的效果,与传统甲醛灭活进行了比较。结果显示,终浓度为1‰的BEI在32℃情况下经20 h即可彻底灭活PPV病毒;BEI灭活的病毒制备的疫苗免疫豚鼠较甲醛灭活病毒产生较高的血凝抑制抗体。     

二乙烯亚胺对猪伪狂犬病病毒灭活效果研究

为研究二乙烯亚胺(BEI)对猪伪狂犬病病毒的灭活效果,使用终浓度为0.03%、0.04%和0.05%的BEI在30℃条件下对猪伪狂犬病病毒(DQ株)进行了灭活试验,通过ST传代细胞接种法观察病毒的灭活效果,用2~2.5 kg大耳白兔和断奶仔猪检测BEI灭活后的病毒液所制备疫苗的安全性,用小鼠检测该灭活工艺制备疫苗的免疫效果,并与传统甲醛灭活进行了比较。结果表明,终浓度为0.05%的BEI在30℃情况下经24 h即可彻底灭活PRV病毒;BEI灭活的病毒制备的疫苗接种大耳白兔和猪的安全性良好,免疫小鼠较甲醛灭活病毒能提供更高的保护率。本研究可为猪伪狂犬病灭活疫苗灭活工艺研究提供依据。     

两种灭活方法制备的猪肺炎支原体灭活疫苗的免疫效果比较

为对比使用甲醛、二乙烯亚胺（BEI）两种灭活方法制备的猪肺炎支原体灭活疫苗对猪的呼吸道发病率、肺部病变记分、饲料转化率、日增重、以及猪血清抗体水平等指标的影响,将120头健康仔猪随机分成三个试验组和一个对照组,于7、21日龄对试验组的仔猪进行疫苗接种,所有猪于20、50、80及140日龄采血分离血清,检测血清中猪肺炎支原体抗体水平。结果显示,用BEI灭活的疫苗在呼吸道发病率、肺部病变记分、饲料转化率、日增重、以及猪血清抗体水平等各项指标均好于甲醛灭活的疫苗（P<0.05）,与进口疫苗无显著差异。试验表明,使用BEI灭活方法制备的猪肺炎支原体灭活疫苗的免疫效果好。     

猫杯状病毒不同分离株致病性的比较

为确定猫杯状病毒(Feline calicivirus,FCV)SH、JL-1、JL-2分离株毒力强弱,将其分别人工接种6~11周龄健康非免疫家猫,分成接种组和对照组。接种前后分别测定各组猫体温、体质量,观察发病情况及临床症状,按照欧洲药典对症状进行计分。于感染后14d麻醉处死,制备病料组织切片,观察组织器官的病理变化。结果显示,家猫感染FCV后总发病率为100%,死亡率为66.7%;SH、JL-1、JL-2组临床症状平均得分分别为8、4、9,对照组得分为1。病理解剖观察发现,猫感染FCV后,以鼻、眼分泌物增加,口腔溃疡为特征,消化道病变较轻微,肺部出现不同程度的变性、出血,呈明显病理性肉样变;病理组织学观察发现,肺脏有数量不等的肺泡上皮细胞和巨噬细胞脱落,伴随少量纤维蛋白渗出和弥漫性肺泡损伤。结果表明,FCV SH、JL-1、JL-2分离株均有一定的致病性,其中以JL-2株的毒力最强。     

卵黄抗体中外源病毒的不同灭活方法比较

为防止提取的卵黄抗体混入外源病毒,确保提取卵黄抗体的安全性,比较了甲醛和二乙烯亚胺对卵黄抗体的灭活效果。在卵黄抗体中,人工混入鸡新城疫病毒、禽流感病毒（H9型）、禽传染性法氏囊病病毒、减蛋综合征病毒和禽脑脊髓炎病毒,分别加入终浓度为0.02％二乙烯亚胺;0.03％、0.05％和0.08％甲醛, 25℃放置24小时。结果显示,0.02％二乙烯亚胺和0.03％甲醛只能灭活部分病毒,而0.05％和0.08％甲醛能完全灭活上述病毒并且不降低抗体活性。因此,可使用0.05％甲醛灭活卵黄抗体中潜在病毒。     

猫杯状病毒自然弱毒株的分离鉴定及对猫的免疫试验

在进行猫狂犬病流行病学调查时,从猫的唾液中发现了杯状病毒.通过对病毒进行分离和形态学、理化学、动物感染试验和分子生物学等鉴定,证明该病毒为猫杯状病毒(FCV)自然弱毒株.免疫试验证明,该毒株可以诱导猫产生较高水平的中和抗体,且能持续50周以上,并能抵抗FCV强毒株攻击而不发病.     

四种不同灭活剂对新城疫病毒的灭活效果研究

为探索甲醛、β-丙内酯(BPL)、二乙烯亚胺(BEI)和盐酸聚六亚甲基胍(PHMG)对新城疫病毒(NDV)的最佳灭活条件,试验采用不同浓度梯度的甲醛、BPL、BEI和PHMG对NDV进行不同时间的灭活处理,通过血凝(HA)试验、无菌检验和灭活检验确定灭活效果。结果显示,37℃下,NDV的最佳灭活条件为0.1%～0.2%的甲醛灭活22～24h、0.02%～0.025%的BPL灭活9～10h、2%～3%的PHMG灭活16～20h,该研究为筛选规模化生产ND疫苗所需的理想灭活剂提供了依据。     

猫杯状病毒、猫细小病毒、猫流感病毒和猫疱疹病毒多重PCR检测方法的建立和应用

为建立快速检测猫科动物重要传染病的多重PCR检测方法,根据GenBank中登录的猫杯状病毒(feline calicivirus, FCV)、猫细小病毒(feline parvovirus, FPV)、猫流感病毒(feline influenza virus, FIV)和猫疱疹病毒1型(feline herpesvirus type 1,FHV-1)的相对高保守性基因ORF2、NSP1、M和UL27,分别设计4对特异性引物。通过对反应体系的摸索及优化,建立了可同时扩增FCV(257 bp)、FPV(380 bp)、FIV(519 bp)和FHV-1(761 bp)的四重PCR反应条件及体系。结果显示：能够在同一体系中用同一反应扩增出以上4种病毒基因,其重复性及特异性均良好,敏感性可达到10 copies/μL。用该方法检测69份口鼻拭子,结果发现FCV和FPV混合感染的阳性率为10.14%,FCV和FHV-1混合感染的阳性率为1.45%,FCV、FPV和FIV混合感染的阳性率为10.14%。结果表明：研究建立的四重PCR检测方法可对常见猫科动物病毒性传染病进行快速准确的鉴别诊断,而且为...     

一例猫杯状病毒和支原体混合感染的诊断与治疗

1只5岁雄性英国短毛猫,就诊前一星期连续出现打喷嚏、流浓稠鼻涕、呼吸不畅等症状,并拒绝饮食,就诊当天表现为口腔黏膜苍白、颜面轻微溃烂。经血常规检查、生化检查、猫呼吸道五联荧光定量PCR检测,患猫确诊为猫杯状病毒与猫支原体混合感染。根据诊断结果对症治疗,通过分泌物清理、点眼滴鼻、全身抗菌消炎以及抗病毒治疗,同时加强营养供给,连续半个月后,患猫面部溃烂逐渐结痂恢复,血液各项相关指标逐渐回归到正常水平,贫血状况明显好转,呼吸道病原学检测全部转为阴性。     

Onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus

The induction of a quick onset of immunity against feline parvovirus (FPV), feline herpesvirus (FHV) and feline calicivirus (FCV) is critical both in young kittens after the decline of maternal antibodies and in cats at high risk of exposure. The onset of immunity for the core components was evaluated in 8–9 week old specific pathogen free kittens by challenge 1 week after vaccination with a combined modified live (FPV, FHV) and inactivated (FCV) vaccine. The protection obtained 1 week after vaccination was compared to that obtained when the challenge was performed 3–4 weeks after vaccination. The protocol consisted of a single injection for vaccination against FPV and two injections 4 weeks apart for FHV and FCV.At 1 week after vaccination, the kittens showed no FPV-induced clinical signs or leukopenia following challenge, and after FCV and FHV challenges the clinical score was significantly lower in vaccinated animals than in controls. Interestingly, the relative efficacy of the vaccination was comparable whether the animals were challenged 1 week or 3–4 weeks after vaccination, indicating that the onset of protection occurred within 7 days of vaccination. Following the 1-week challenge, excretion of FPV, FHV and FCV was significantly reduced in vaccinated cats compared to control kittens, confirming the onset of immunity within 7 days of vaccination.     

Carrier-state infection of feline T-lymphoblastoid cells with feline calicivirus

The susceptibility of feline T lymphocytes to feline calicivirus (FCV) in vitro was investigated using feline T-lymphoblastoid cell lines, namely MYA-1 and FL74 cells. The virus titers of supernatants in FCV-infected MYA-1 and FL74 cell cultures increased rapidly, and FCV antigens were also detected in the FCV-infected cells. There were slight differences in the molecular weights of capsid proteins expressed in FCV-infected MYA-1, FL74 and Crandell feline kidney cells. MYA-1 and FL74 cells were productively and persistently infected with FCV, and FCV antigens were observed in the FCV-infected cells for more than one month. At 3 months post infection, FCV-infected FL74 cells that stopped producing infectious FCV could be reinfected with FCV. However, no cytopathic effects were observed.     

豹源猫杯状病毒ORF2基因的扩增、克隆及原核表达

为制备猫杯状病毒血清学检测抗原,本研究根据实验室保存的1株豹源猫杯状病毒基因序列设计引物,扩增了开放阅读框2（ORF2）中1734 bp的核苷酸序列,将其克隆至表达载体pET-28a上,构建了pET-28a-FCV1734重组质粒。将该质粒转化大肠杆菌,诱导表达产物经SDS-PAGE和Western blot检测,证明所表达的蛋白具有良好的反应原性。     

嵌合猫杯状病毒F9株感染性克隆的构建及病毒拯救

为构建猫杯状病毒(FCV)弱毒株F9的感染性克隆,本研究利经PCR分段扩增了FCV各基因片段,并拼接出FCVF9全基因组序列。经测序显示,其1823位多出一个碱基造成该序列存在移码突变,经替换为pOK-2280(已构建出的FCV强毒株的感染性克隆)相应序列并对差异氨基酸逐个突变,最终获得嵌合FCVF9全基因组的重组质粒pOK-F9H。将线性化的pOK-F9H体外转录,获得的加帽RNA转染F81细胞,得到了拯救病毒rFCVF9。经过对拯救病毒与亲本病毒的蚀斑形成能力、CPE形成能力和多步生长曲线分析,结果显示rFCVF9与亲本株在复制能力和体外生长能力方面基本一致,表明构建了嵌合FCVF9株的感染性克隆并拯救出重组病毒。本研究构建的嵌合FCVF9株感染性克隆可以为FCV的基因功能研究以及新型FCV疫苗的研发奠定基础。     

Prevalence of feline calicivirus and the distribution of serum neutralizing antibody against isolate strains in cats of Hangzhou,China

BackgroundFeline calicivirus (FCV) is a common pathogen of felids, and FCV vaccination is regularly practiced. The genetic variability and antigenic diversity of FCV hinder the effective control and prevention of infection by vaccination. Improved knowledge of the epidemiological characteristics of FCV should assist in the development of more effective vaccines.ObjectivesThis study aims to determine the prevalence of FCV in a population of cats with FCV-suspected clinical signs in Hangzhou and to demonstrate the antigenic and genetic relationships between vaccine status and representative isolated FCV strains.MethodsCats (n = 516) from Hangzhou were investigated between 2018 and 2020. The association between risk factors and FCV infection was assessed. Phylogenetic analyses based on a capsid coding sequence were performed to identify the genetic relationships between strains. In vitro virus neutralization tests were used to assess antibody levels against isolated FCV strains in client-owned cats.ResultsThe FCV-positive rate of the examined cats was 43.0%. Risk factors significantly associated with FCV infection were vaccination status and oral symptoms. Phylogenetic analysis revealed a radial phylogeny with no evidence of temporal or countrywide clusters. There was a significant difference in the distribution of serum antibody titers between vaccinated and unvaccinated cats.ConclusionsThis study revealed a high prevalence and genetic diversity of FCV in Hangzhou. The results indicate that the efficacy of FCV vaccination is unsatisfactory. More comprehensive and refined vaccination protocols are an urgent and unmet need.     

6例猫杯状病毒病例的诊断及治疗

上海市某宠物店15只猫中有6只患猫出现呼吸道症状.经临床检查发现,6只患猫体温、呼吸、心率都正常;1#、2#、3#、5#和6#患猫血常规检测正常,4#患猫血常规显示有轻度脱水.对6只患猫的样品进行猫杯状病毒核酸检测和基因测序,结果均为猫杯状病毒阳性.对6只患猫进行抗菌、点眼、清洁口腔等治疗,患猫均在10～14 d内恢复...     

蜂胶对NDV的灭活作用效果观察

本文通过鸡胚接种的方法就蜂胶乙醇浸出物对NDV的灭活作用进行了试验观察,结果表明常温下3分钟,含蜂胶乙醇浸出物0.1mg/ml的95%乙醇稀释液、含蜂胶乙醇浸出物10mg/ml的47.5%乙醇稀释液、含蜂胶乙醇浸出物10mg/ml的生理盐水稀释液;37℃3分钟,含蜂胶乙醇浸出物0.1mg/ml的95%乙醇稀释液、含蜂胶乙醇浸出物0.1mg/ml的47.5%乙醇稀释液、含蜂胶乙醇浸出物10mg/ml的生理盐水稀释液均对ND克隆-30具有完全灭活作用;常温下3分钟,1mg/ml的47.5%乙醇稀释液对ND克隆-30具有灭活作用。