免疫与未免疫鸡感染MDV后血液部分指标及血浆MDA含量与SOD活性的动态变化

为探讨丙二醛(MDA)和超氧化物歧化酶(SOD)在鸡马立克氏病发病过程中的作用机制,研究了经火鸡疱疹病毒疫苗(HVT疫苗)免疫和未免疫的1日龄健康罗曼雏鸡在感染马立充氏疱疹病毒(MDV)后血液部分指标及血浆MDA含量与SOD活性的动态变化.结果表明:攻毒后外周血液红细胞数(RBC)、白细胞数(WBC)、红细胞压积(PCV)、网织红细胞比例(RCC)和血红蛋白含量(Hb)均呈上升趋势,非免疫攻毒组(Ⅱ组)和免疫攻毒组(Ⅲ组)的WBC、RCC和PCV明显高于非免疫对照组(C组)和免疫对照组(Ⅰ组),Ⅱ和Ⅲ组的RBC和Hb明显低于C和Ⅰ组,Ⅰ和Ⅲ组的RBC和WBC低于C和Ⅱ组,Ⅱ组的PCV除了在第7天显著高于C组外,其余均无显著差异;免疫组(Ⅰ和Ⅲ组)血浆MDA含量在攻毒后第7天低于非免疫组(C和Ⅱ组),第14天后便高于或显著高于非免疫组;而免疫组血浆SOD活性在攻毒后第7天低于或显著低于非免疫组,第14天后免疫组和非免疫组之间无显著差异.表明雏鸡感染MDV后血细胞的发育成熟受阻,而免疫对红细胞的发育成熟和白细胞的生成具有促进作用;同时发现雏鸡血浆中MDA含量的降低与SOD活性的提高对雏鸡抗MDV感染有增强作用.     

感染“白点病”番鸭抗氧化功能的变化

用“白点病”病毒人工感染雏番鸭，于感染后第1、3、5、7、10和14d测定了雏番鸭血浆中丙二醛(MDA)的含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性。结果，雏番鸭感染“白点病”病毒后血浆中MDA的含量、SOD和GSH-Px活性发生明显变化，其中攻毒组雏番鸭在攻毒后第3d MDA含量开始升高，第5和第10d显著高于对照组；攻毒组雏番鸭SOD活性在攻毒后第3d开始显著低于对照组，随后在低于或显著低于对照组的范围内波动；GSH-Px的活性在攻毒后第7d开始下降，攻毒后第10d攻毒组显著低于对照组。提示，自由基可能在雏番鸭“白点病”的发病过程中起关键作用。     

IBV感染雏鸡血清中SOD,GSH-Px活性与MDA含量的研究

将80只15日龄雏鸡随机分为对照组和试验组。试验组雏鸡用传染性支气管炎病毒尿囊液滴鼻染毒,攻毒后1,3,6,9,12d分别测定各组血清中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性与丙二醛(MDA)含量,研究血清中氧自由基在鸡传染性支气管炎发病过程中的动态变化。结果表明:试验组血清SOD,GSH-Px活性自攻毒后明显降低(P<0.05或P<0.01);MDA含量在攻毒后开始上升,且在感染后第6、第9天差异极显著(P<0.01)。提示氧化损伤可能参与调节了传染性支气管炎的发生发展过程。     

硒缺乏对雏鸡空肠黏膜中SOD活性和MDA含量的影响

将40只1日龄雏鸡随机分为两组(n=20),对照组,饲料硒含量为0.40 mg/kg,试验组,饲料硒含量为0.01 mg/kg。分别与试验的第7天、21天、35天处死雏鸡并取空肠黏膜(各时间点均为6只),测定其中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果表明,与对照组相比,第7天试验组空肠黏膜中SOD活性显著低于对照组(P<0.05),MDA含量无明显变化。第21天试验组空肠黏膜中SOD活性极显著高于对照组(P<0.01),MDA含量显著高于对照组(P<0.05)。第35天试验组空肠黏膜中SOD活性极显著低于对照组(P<0.01),MDA含量极显著高于对照组(P<0.01)。这表明,硒缺乏会降低雏鸡空肠黏膜中SOD的活性和提高MDA的含量。     

抗性选育协同疫苗免疫对鸡羽囊马立克病毒载量的影响

为了研究马立克病(MD)火鸡疱疹病毒(HVT)疫苗接种和抗性选育对鸡马立克病毒(MDV-1)攻毒后羽囊中病毒载量的影响,选择普通霞烟鸡及其MD抗性品系,利用MDV-1攻毒后第7、14、21、28和35天(DPI)五次采集并分离的全部试验鸡的羽囊为材料,采用Real-time FQ-PCR技术对这些材料中meq基因进行绝对定量分析以确定其病毒载量,用来分析比较MDV-1在MD抗性/普通鸡及HVT疫苗免疫/HVT疫苗未免疫鸡体内的增殖情况。结果显示:1)从MD抗性对病毒载量的影响看,一方面,普通非免疫攻毒组(F组)的MDV-1含量于14、21DPI时显著(P0.05)高于MD抗性非免疫攻毒组(T组);另一方面,MD抗性免疫攻毒组(R组)的MDV-1含量总体上却和普通免疫攻毒组(G组)相当。2)从HVT疫苗免疫对病毒载量的影响看,一方面,MD抗性非免疫攻毒组(T组)的MDV-1含量于28DPI时显著(P0.05)高于MD抗性免疫攻毒组(R组);另一方面,普通非免疫攻毒组(F组)的MDV-1含量于14、21DPI时显著(P0.05)高于普通免疫攻毒组(G组)。3)从MD抗性协同HVT疫苗免疫对病毒载量的影响看,普通非免疫攻毒组(F组)的MDV-1含量于14(P0.05)、28(P0.01)、35(P0.05)DPI时显著高于MD抗性免疫攻毒组(R组)。结果表明,鸡MD抗性选育协同HVT疫苗接种可显著降低鸡羽囊的病毒载量,这将会降低MDV-1传播风险,并提高鸡的存活机会。     

小花棘豆总生物碱对小鼠氧自由基的影响

为了探讨小花棘豆主要有毒成分———总生物碱对动物机体内氧自由基的影响,采用紫外分光光度法分别测定了小白鼠血浆、肝和脑中超氧化物歧化酶(Superoxidedismutase,SOD)和谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-PX)的活性以及脂质氧化代谢终末产物丙二醛(Malonalde-hyde,MDA)的含量。结果表明,攻毒组肝、脑中SOD、GSH-PX活性显著低于对照组(P<0.01),血浆中GSH-PX活性显著低于对照组(P<0.05),SOD活性与对照组则无显著性差异(P>0.05);攻毒组脑、血浆中MDA含量显著高于对照组(P<0.01),但肝脏中的MDA含量却无显著性差异。表明小花棘豆主要有毒成分能显著降低抗氧化酶的活性和提高MDA的含量,并能诱发机体内氧自由基的产生。     

抗马立克氏病新品系选育第四代雏鸡MDV攻毒试验结果

F4雏鸡MDV攻毒试验结果显示,亲本父母代鸡均携带No614血型基因的选育试验组,60日龄死亡率显著低于不经血型基因选育的对照组(P＜0.05);其内脏器官组织肿瘤病变率也低于该对照组,但差异不显著(P＞0.05);选育试验组鸡的肝脏的肿瘤病变率仍显著低于血型基因选育对照组和非选育对照组鸡(P＜0.05).提示No614血型基因具有一定程度的马立克氏病(MD)抗性.     

鸡马立克氏病(MD)与淋巴细胞糖皮质激素受体(GR)相关性研究Ⅰ.MDV对鸡外周血淋巴细胞GR及T细胞转化率的影响

本实验对马立克氏病病毒(MDV)引起鸡外周血淋巴细胞(PBL)糖皮质激素受体(GR)含量及T细胞对丝裂原(PHA)刺激反应的变化规律作了观察,同时比较了注射MD二价疫苗对以上指标的影响.结果表明:(1)攻毒鸡PBL GR含量及T细胞对PHA刺激转化率均明显降低,而且两者变化之间呈现正相关关系(γ=0.8580,P<0.01);(2)攻毒后出现特征性MD肿瘤病变鸡的PBL GR含量较未出现病变鸡的更低,但攻毒鸡不论是否出现特征性病变,其T细胞对PHA刺激转化率均明显降低;(3)注射MD二价疫苗鸡的PBL GR含量及T细胞对PHA刺激转化率都显著高于攻毒鸡;(4)攻毒鸡血浆皮质醇含量不升高,提示攻毒鸡PBL GR含量下降与激素负反馈调节无关.     

抗马立克氏病新品系选育Ⅲ.第四代雏鸡MDV攻毒试验

F4 雏鸡MDV攻毒试验结果显示 ,亲代父母鸡均携带№ 6 14血型基因的选育试验组 ,6 0日龄死亡率显著低于不经血型基因选育的对照组 (P <0 .0 5 ) ;其内脏器官组织肿瘤病变率差异不显著 (P >0 .0 5 ) ;而肝脏的肿瘤病变率 ,选育试验组仍显著低于血型基因选育对照组和非选育对照组 (P <0 .0 5 )。提示№ 6 14血型基因具有一定程度马立克氏病(MD)抗性。     

超强马立克病病毒株的致病性研究

拟对一株超强马立克病病毒(MDV)SD2012-1株的致病性进行研究。将60只SPF鸡平均分成未免组、HVT免疫组和CVI988疫苗免疫组3组。于1日龄时对其进行马立克病(MD)疫苗免疫,于10日龄时进行SD2012-1攻毒;每天观察攻毒鸡临床症状,对病死鸡进行病理剖检和组织学观察;用PCR反应对感染鸡进行MDV跟踪监测。病理学研究结果显示:攻毒后第2周,试验鸡有轻微组织病变;攻毒后第6周,试验鸡有眼观病变,镜检有散在的肿瘤细胞团块;攻毒后第9周,试验鸡有明显的眼观肿瘤病变,镜检有大量肿瘤细胞聚集;SD2012-1可以突破CVI988疫苗免疫,引起高达30%的鸡发病。PCR跟踪监测结果显示:攻毒后第5天,未免组和HVT免疫组可检出MDV;攻毒后第10天,未免组和HVT免疫组阳性检出率均为100%,CVI988免疫组阳性检出率为30%;攻毒后第20天,3组试验鸡阳性检出率均为100%;用PCR检测病死鸡的肝、肾、肌胃、肠系膜、十二指肠、心和法氏囊样品的结果均为MDV阳性;攻毒300d后,健康存活鸡的羽髓PCR检测结果均为MDV阳性。结果表明,HVT疫苗对于SD2012-1株几乎无保护作用,超强马立克病病毒SD2012-1株能够长期在免疫鸡体内存在,突破免疫保护,引起发病,这对国内防控鸡马立克病提出了严峻挑战。     

Early posthatch protection against Marek's disease in chickens vaccinated in ovo with a CVI988 serotype 1 vaccine

CVI988, a serotype 1 Marek's disease virus (MDV), was used as an in ovo vaccine in specific-pathogen-free chickens to determine if this virus induces early posthatch protection against Marek's disease as has been shown previously for turkey herpesvirus. MDV CVI988 was injected at embryonation day (ED) 17 (group 1) or at hatch (group 2). A third group (group 3) was left unvaccinated. At 1, 2, 3, 4, 5, and 7 days of age, chickens from each group were sampled and examined as follows: a) single-cell suspensions of spleen were inoculated onto chicken embryo fibroblast monolayers to isolate the virus; b) sections of bursal tissues were stained by indirect immunofluorescence assays with anti-pp38 monoclonal antibody to identify viral antigen expression; and c) chickens were exposed intra-abdominally to MDV RB1B, a virulent serotype 1 MDV. Results revealed that in chickens given MDV CVI988 at ED 17, virus and virus-encoded protein were not detected until chickens were 3 and 2 days old after hatching, respectively. Results also indicated that during the first 4 days after hatch, the chickens given MDV CVI988 at ED 17 were better protected against virulent MDV than those given MDV CVI988 at hatch (P < or = 0.001). These results suggested that MDV CVI988 proteins were adequately expressed in the embryo to initiate prehatch immunologic response. Additional efforts with more sensitive techniques than used in this study are needed to identify the nature of viral expression in embryos.     

牛病毒性腹泻病毒对新西兰白兔致病性分析及E2蛋白免疫效果的评价

【目的】 研究牛病毒性腹泻病毒（BVDV）感染对新西兰白兔的致病性以及BVDV E2重组蛋白的免疫效果。【方法】 将实验室培养保存的BVDV病毒纯化并按照Reed-Muench法测定其病毒滴度。在致病性试验中,将10只新西兰白兔随机分为感染组和对照组,每组5只。感染组用1 mL纯化的BVDV病毒攻毒（滴鼻500 μL、耳缘静脉注射500 μL）,对照组用等体积的生理盐水处理,连续3 d,每天1次,每天观察各组兔的临床症状并测量体温;分别于接种病毒后第6、9、12、15、17天通过耳缘静脉采集血液检测血常规;感染病毒第17天采集鼻拭子进行RT-PCR鉴定,采集后剖杀并采集气管、肺脏、脾脏和小肠组织,制备病理切片观察病理变化。在免疫效果评价试验中,将10只新西兰白兔随机分为免疫组和对照组,每组5只,免疫组用E2重组蛋白（1 mg/只）与佐剂混合后经肌内多点注射免疫新西兰白兔,对照组接种等体积生理盐水;共免疫2次,2次免疫间隔为14 d。在一免后0、7、14、21、28 d采集血清,通过间接ELISA方法检测血清中抗重组蛋白特异性抗体水平;在一免后第28天按致病性试验中方法攻毒,在攻毒第17天采集鼻拭子进行RT-PCR鉴定,采集气管、肺脏、脾脏和小肠组织制备病理切片观察病理变化及免疫组织化学检测。【结果】 纯化后BVDV的病毒滴度为4.16×10⁶ TCID₅₀/mL。与对照组相比,感染组部分新西兰白兔6 d内活动减少,采食略微减少,6 d后逐渐恢复正常,在感染第13天出现腹泻症状,从第5天开始体温略微升高,但均在正常范围内波动。与对照组相比,在攻毒第6和9天,感染组白细胞和血小板分别显著和极显著降低（P<0.05;P<0.01）;在攻毒第12、15和17天,感染组白细胞、血小板和淋巴细胞均极显著降低（P<0.01）。鼻拭子RT-PCR检测为阳性,气管、肺脏、脾脏及小肠组织表现出轻度至重度的组织病理学变化。间接ELISA检测结果表明,在一免后7 d时,血清抗体滴度为1：16~1：32;在一免后28 d时,血清抗体滴度为1：256~1：512;免疫攻毒组新西兰白兔鼻拭子经RT-PCR检测为阴性;组织病理学观察显示,免疫攻毒组气管及肺脏表现出轻微的组织病理学变化。免疫组化检测结果显示,免疫组结果均呈阴性,对照组结果均为阳性。【结论】 通过滴鼻及耳缘静脉注射BVDV的方式可以构建新西兰白兔致病模型,BVDV E2亚单位疫苗能够刺激机体产生特异性抗体,起到免疫防御的作用。     

日粮添加黄芪多糖对蛋鸡机体抗氧化能力和鸡蛋品质的影响

选用540只27周龄海兰褐蛋鸡,随机分为4个处理,每个处理5个重复,每个重复27只鸡。采用单因素完全随机分组设计,对照组饲喂基础日粮,试验组饲喂在基础日粮中分别添加100、200、300mg／kg黄芪多糖的日粮,试验期为70d,探讨日粮中添加不同水平的黄芪多糖对蛋鸡机体抗氧化能力和鸡蛋品质的影响。结果显示：黄芪多糖能不同程度地提高（P〈0．05）血清和蛋黄SOD、血清GSH—Px和T-AOC活性,显著降低（P〈0．05）血清和蛋黄MDA含量、蛋黄胆固醇质量浓度。随着受试时间的延长,各组内血清sOD活性显著升高（P〈0．05）,血清MDA含量和T-AOC活性显著降低（P〈0．05）;对照组的蛋黄MDA含量显著升高（P〈0．05）,而血清GsH—Px活性显著降低（P〈0．05）。结果表明,黄芪多糖能改善蛋鸡机体及其产品抗氧化能力,对蛋黄胆固醇质量浓度有降低作用。     

鸡传染性贫血病毒与马立克氏病病毒共感染SPF鸡群免疫抑制协同作用的研究

将鸡传染性贫血病毒（Chicken infectious anemia virus,CIAV or CAV）和马立克氏病病毒（Marek s dis-ease virus,MDV）人工单一和共同感染1日龄的SPF鸡,感染后分别于14、21、28、35日龄检测鸡体红细胞压积的变化,并检测鸡群疫苗免疫3周后的抗体反应,以探讨CAV与MDV共感染对鸡体的免疫抑制是否有协同作用。结果表明,在血液分析方面,CAV与MDV共感染组较病毒单一感染组与对照组差异极显著,共感染不仅加重了鸡群贫血现象,而且延长了贫血的病理症状;而在禽流感病毒（Avian influenza virus,AIV）H5/H9疫苗、新城疫病毒（Newcastle disease virus,NDV）疫苗和传染性法氏囊病毒（Infectious bursal disease virus,IBDV）疫苗免疫后3周的抗体检测中,CAV与MDV共感染组较其它各实验组差异极显著,抗体滴度大大低于其它实验组;此外,CAV与MDV共感染组,鸡体生长状况明显差于实验各组,有6只鸡只死亡（6/25）,比病毒单一感染时的死亡率大大增加。综上研究证明,CAV与MDV共感染在免疫抑制作用上有协同作用。     

牛初乳粉预防大鼠高血糖的实验研究

目的:探讨牛初乳粉对大鼠糖尿病的预防作用。 方法:给大鼠灌胃3个不同剂量的牛初乳粉(BCP)溶液15d后腹腔注射链脲霉素(STZ)55mg/kg。注射后第7、14天测定各项指标(平均饮水量在注射后第6、13天测量)。血糖用Roche公司血糖仪测定,血脂用Beckman公司全自动生化分析仪测定,肝肾组织SOD、GSH-Px、NOS及MDA均用南京建成生物工程研究所相应试剂盒测定。结果:注射STZ后第7、14天中剂量BCP组的血糖均显著低于STZ组(P<0.05,P<0.01)。第13天中剂量组的平均饮水量的数值小于STZ组。第14天BCP各组的体重、胸腺指数与STZ组相比差异均无显著性,5个组之间的血脂、肝MDA、肝肾SOD、GSH-Px、NOS差异均无显著性。 结论:预防性给予BCP可预防STZ所致的高血糖,并使STZ大鼠平均饮水量减少(中剂量);但不能使STZ大鼠降低了的体重及胸腺指数恢复。     

Effects of reticuloendotheliosis virus and Marek's disease virus infection and co-infection on IFN-gamma production in SPF chickens

Two experiments were used to examine the potential role of IFN-gamma in chickens infected with reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). First, chickens were infected with REV and/or MDV at 5 days of age and examined from 3 to 50 days post-infection (dpi). In REV+MDV co-infection chickens, IFN-gamma ELISA demonstrated a 3-fold increase at 7 dpi compared to the controls, while REV alone caused a 5-fold increase, the IFN-gamma levels peaked, and then gradually decreased. IFN-gamma levels significantly decreased in MDV infection at 3 dpi and 15 dpi. Second, experiments were designed to determine the effects of different viruses and ConA on IFN-gamma production. For REV- or MDV-infected chickens, the IFN-gamma levels decreased slightly after adding ConA. This is the first report of IFN-gamma production in SPF chickens infected with REV and MDV measured by directly quantitative method.     

The early pathogenesis in chicken inoculated with non-pathogenic serotype 2 Marek's disease virus.

A newly cloned serotype 2 Marek's disease virus (MDV), strain ML-6, was inoculated via the nasal cavity in specific-pathogen-free chicks to examine early virus replication and the expression of Marek's disease (MD)-related antigens. Following inoculation, viral intracellular antigens (VIAs) were detected in lymphoid organs (bursas and spleens) between 5 and 14 days post inoculation (PI), in feather follicles between 14 and 30 days PI, and in lungs at 3 days PI by the immunohistopathological staining of avidin-biotin-peroxidase complex method. But, very few VIAs were expressed in the thymuses between 5 and 14 days PI. However, MD tumor-associated surface antigens were not detected in any organs. Viruses were isolated from separated spleen cells at 14 and 30 days PI. Fluorescent antibodies of convalescent sera were also detected after 10 days PI. As most of the VIAs were detectable in B-cells in bursas and spleens. B-cells were considered to be the main first target cells for the serotype 2 MDV infection.     

Interaction of Marek's disease virus and Cryptosporidium baileyi in experimentally infected chickens

Histocompatible B13/B13 white specific-pathogen-free leghorn chickens were used to investigate the effect of coinfection with Cryptosporidium baileyi and the HPRS 16 strain of Marek's disease virus (MDV) in chickens and to assess the pathogenicity of C. baileyi when MDV is given before or after the parasite. Groups of chickens concurrently infected with C. baileyi orally inoculated at day (D)4 and MDV inoculated at hatching (C4M0 group) or at D8 (C4M8 group) were compared with relevant control groups inoculated with only C. baileyi at D4 (C4 group), only MDV at hatching (M0 group) or at D8 (M8 group), and an uninoculated control group (UC group). The chickens were kept in isolator units until the end of the experiment at D62. Our results showed a considerable synergistic effect in concurrently infected chickens and more severe consequences when chickens received MDV before C. baileyi infection. In fact, except for a slight transitory weakness, the chickens in C4 group remained free of overt clinical signs and there was no mortality. However, coinfection with both pathogens induced more lasting or permanent oocyst shedding. Severe clinical cryptosporidiosis with weakness, anorexia, depression, growth retardation, and chronic and severe respiratory disease causing death occurred in all chickens in the C4M0 group between D12 and D43 and in 67% of the chickens in the C4M8 group between D17 and D57. Eighty-two percent and 33%, respectively, died before the development of specific Marek's disease lesions. Mortality rates were 27% and 33% in the M0 and M8 groups, respectively. The presence of MDV enhanced the establishment of more lasting cryptosporidial infection in the respiratory tract, esophagus, crop, proventriculus, and kidneys (only in C4M0 group) as well as in bursa of Fabricius, ceca, and cloaca. Serologic analysis showed that chickens with chronic cryptosporidiosis in the C4M8 group had an increased level of C. baileyi-specific immunoglobulin A. Our results may explain some cases of mortality in chickens naturally infected with MDV and Cryptosporidium.     

马立克氏病病毒感染鸡羽髓上皮细胞差异蛋白质组学研究

为鉴定鸡羽髓上皮细胞感染马立克氏病病毒(MDV)前后差异表达的蛋白,本研究以MDV强毒GA株人工感染SPF鸡,并通过双向电泳技术进行分析.结果显示:在病毒感染后4 d、7 d、14 d和21 d显著差异表达的蛋白点分别有2个、8个、25个和9个;而通过质谱技术鉴定出29种蛋白质,其中包括能量代谢相关蛋白、增殖和凋亡相关蛋白、细胞骨架蛋白、信号传导蛋白、转录相关蛋白、免疫相关蛋白和其他功能蛋白质.本实验首次对鸡羽髓上皮细胞感染MDV后各时期蛋白表达水平的变化进行研究,鉴定了多种差异表达蛋白质,为进一步揭示MDV与宿主的相互关系、感染性病毒粒子的成熟和致病机制提供了依据.