禽腺病毒QU分离株的致病性及细胞适应性研究

QU分离株是一株类似产蛋下降综合征病毒,属于鸭腺病毒1型病毒。通过人工感染和细胞增殖试验,结果显示QU分离株接种无特定病原雏鸡未出现临床病症及生长发育障碍,不致死鸡胚,对鸭胚的致死率明显比引起产蛋下降的HS株低。QU株在鸡胚肝细胞、鸭胚成纤维细胞及鸡胚成纤维细胞上生长良好,产生典型细胞病变,且在鸡胚肝细胞上的增殖滴度最高,但不适应鸡胚肾细胞。这些数据说明QU株系对鸡具有低毒力的腺病毒,有可能用作禽用基因疫苗或基因治疗的候选病毒载体。     

传染性支气管炎重组鸡痘病毒免疫鸡对LHLJ04X I株病毒攻击的免疫保护作用

将表达鸡IFN-γ基因和传染性支气管炎病毒S1基因的重组鸡痘病毒（rFPV-IFN-γ S1）接种SPF鸡后,用与S1基因亲本毒株具有相同致病型且遗传关系较近的现地分离株LHLJ04XI病毒进行攻击．评价重组疫苗对现地分离病毒的保护效果。结果显示,重组病毒在免疫后1周即可检出ELISA抗体,并且免疫组外周血CD4^＋、CD8^＋T淋巴细胞含量均有显著上升。当用LHLJ04XI株病毒及亲本毒LX4攻击后,免疫鸡的肝脏、肾脏、脾脏及肺脏等脏器的病理损伤程度明显轻于非免疫对照组,排毒时间缩短,攻毒后现地分离株攻击的免疫组的发病率为27.3％（3／11）,死亡率为9．1％（1／11）,亲本毒攻击的免疫组发病率为26．67％（4／15）,死亡率为6．67％（1／15）,两者没有明显差异,而非免疫对照组的发病率和死亡率分别为100％（11／11）和45．5％（5／11）,与免疫组相比差异显著,说明重组疫苗对免疫鸡具有较好的保护效果。体重测定结果表明,攻毒前重组疫苗免疫组和非免疫对照组的体重差异不显著（P〉0．05）,攻毒以后免疫组体重的增加幅度高于非免疫对照组。以上结果表明,共表达鸡IFN-^γ基因和传染性支气管炎病毒S1基因重组鸡痘病毒活载体疫苗能对异源现地分离株的攻击产生较好的免疫保护,同时也减轻了重组疫苗对体重增长的抑制作用。     

重组鸭肠炎病毒载体疫苗研究进展

重组病毒载体疫苗是一种以减毒活病毒为载体承载表达其他病原抗原性基因的新型疫苗,能感染宿主细胞、递呈外源基因所表达的抗原蛋白、诱导机体产生体液免疫应答和细胞免疫应答,从而有效地预防多种疾病。鸭肠炎病毒基因组长约160kb,包含多个非编码区和病毒复制非必需区,可携带多个或较大的外源基因,是构建重组活病毒疫苗的理想载体。随着分子生物学研究的不断深入和分子生物学实验技术的飞速发展,对鸭肠炎病毒基因组进行遗传修饰的技术手段已十分成熟,目前已通过这些技术成功构建了多株重组鸭肠炎病毒活载体疫苗。研究表明构建的重组鸭肠炎病毒活载体疫苗可成功抵抗鸭肠炎病毒和其他相应病原的攻击,具有作为重组病毒疫苗载体的潜力,并在未来的禽类新型疫苗研制中发挥重要作用。本文对以鸭肠炎病毒作为载体的重组病毒活载体疫苗的研究进展进行综述,以期为鸭肠炎病毒载体疫苗的研发提供参考。     

表达1型和3型鸭甲型肝炎病毒VP1基因重组新城疫病毒的构建及其免疫原性

为进一步研发安全有效的鸭甲型肝炎病毒(DHAV)多价疫苗,本研究以新城疫病毒(NDV)LaSota弱毒疫苗株为载体,构建出共表达DHAV-1和-3型VP1基因的重组病毒rLS-1VP1-2A-3VP1,并对其毒力、生物学特性进行鉴定。通过间接免疫荧光试验(IFA)验证DHAV-1和-3型VP1蛋白在细胞内能够有效表达。利用Western blot进一步验证重组病毒产生的中和抗体能和DHAV-1和DHAV-3VP1表达蛋白发生较强的反应。重组病毒rLS-1VP1-2A-3VP1保持了原重组疫苗株rLS-RFP对鸡胚的高滴度生长适应和低致病的特性。将重组病毒rLS-1VP1-2A-3VP1免疫种鸭1次,通过中和试验检测该重组病毒能够在鸭体内产生针对DHAV-1和DHAV-3的中和抗体,平均效价分别为1∶18.17和1∶16.60。本研究结果表明重组病毒rLS-1VP1-2A-3VP1具有作为鸭肝炎重组病毒活载体疫苗用于种鸭的潜力。     

共表达猪繁殖与呼吸综合征病毒GP5基因和猪瘟病毒E2基因的重组腺病毒的构建及其在小鼠模型上的免疫原性分析

本研究旨在以复制缺陷型人5型腺病毒为载体,构建一株同时表达猪繁殖与呼吸综合征病毒(PRRSV)变异株GP5蛋白和猪瘟病毒(CSFV)E2蛋白的重组腺病毒疫苗。首先利用重叠PCR将GP5和E2基因通过口蹄疫病毒(FMDV)2A序列连接,形成一个完整的ORF,并将其克隆到腺病毒穿梭载体中,通过细菌内同源重组构建共表达PRRSVGP5蛋白和CSFVE2蛋白的重组腺病毒(rAdV-GP52AE2)。间接免疫荧光试验和western blot检测证实2个外源基因均获得表达。在小鼠上进行的免疫效力评价结果显示,rAdV-GP52AE2免疫组针对CSFV的中和抗体滴度可达1∶128,针对PRRSV的中和抗体滴度为1∶16;在淋巴细胞增殖试验中,免疫组与阴性对照组在增殖指数上有显著差异,表明该重组腺病毒可以同时诱导抗PRRSV和CSFV的特异性体液免疫和细胞免疫。这些结果显示,利用具有自动剪切功能的FMDV2A多肽构建的重组腺病毒有望开发成一种同时预防PRRS和CSF的新型载体疫苗。     

雏鸭接种Ⅰ型鸭肝炎鸡胚化弱毒MY后血清生化指标的动态变化

用Ⅰ型鸭肝炎鸡胚化弱毒MY株人工接种4日龄雏鸭,于接种后第6、12、24、48、72、96、144、168、192h及14d采集接种鸭的血液,分离血清,进行生化指标测定,并与同期接种的弱毒疫苗HY、标准毒及对照组的血清生化指标进行比对。与对照组比较：MY组和HY组的白蛋白在接种后24h明显降低,48h差异极显著;血清谷丙转氨酶于24h、144h时段明显升高,且MY组持续至192h。标毒组则表现为接毒后,血清葡萄糖于24～96h降低,甘油三酯于72h、192h显著升高;血清总蛋白72h、96h、144h,白蛋白24—144h,球蛋白72～96h,144h明显降低;谷丙转氨酶于24～192h,血清谷草转氨酶在24～72h显著升高;尿酸72h呈一过性升高。结果表明,感染Ⅰ型鸭肝炎鸡胚化弱毒MY株与弱毒疫苗HY的血清生化变化规律相似。     

CAG和MLP启动子控制的重组禽腺病毒构建

根据鸡胚致死孤儿病毒(CELOV)的序列设计引物,通过PCR方法扩增了禽腺病毒FAVI-JS株的晚期启动子(MLP),序列与CELOV的MLP同源性为99%。分别构建了MLP和商品化启动子CAG控制的表达增强型绿色荧光蛋白(eGFP)基因的禽腺病毒转移载体。通过载体与禽腺病毒FAVI-JS株在原代鸡胚肾细胞内进行同源重组,分别获得了MLP和CAG控制的表达eGFP的重组禽腺病毒,为探讨不同启动子对禽腺病毒活载体疫苗免疫效果的影响提供了条件。     

表达鸡传染性支气管炎病毒S1基因重组鸡痘病毒对SPF鸡的免疫保护作用

将表达鸡传染性支气管炎病毒(IBV)S1基因的重组鸡痘病毒(rFPV-IBVS1)以5×106PFU剂量翅皮下注射接种4周龄的SPF鸡。血清抗体检测表明,该重组鸡痘病毒能刺激鸡体产生特异性抗体,外周血液中CD4 、TCRγδ T淋巴细胞比例显著高于对照组,而CD8 T淋巴细胞比例低于对照组。免疫后4周用1×103.0EID50的传染性支气管炎病毒LX4株进行攻毒,结果表明,重组鸡痘病毒免疫组与IB弱毒疫苗免疫保护率分别为12/16和13/16;攻毒后喉拭子IBV分离结果表明,IB弱毒疫苗和重组鸡痘病毒免疫组的IBV气管排毒时间比对照组缩短了2 d,病毒的分离率显著低于对照组;肾脏、气管、肺脏、腺胃、脾和肝脏的病理损伤也较对照组轻,而且病变持续时间缩短。     

表达犬瘟热病毒H基因重组新城疫病毒的构建及其免疫原性研究

为研制安全、有效的新型犬瘟热疫苗,本研究利用新城疫病毒(NDV) LaSota弱毒疫苗株反向遗传操作系统,构建出表达犬瘟热病毒(CDV)弱毒疫苗株Rockborn-20/8血凝素(H)蛋白的重组病毒rLa-CDV-H,并对其生物学特性进行鉴定,评估其作为犬瘟热活载体疫苗的安全性和有效性.通过免疫荧光和western blot试验证明了CDV H蛋白的正确表达;重组病毒株保持了LaSota亲本株的低致病性和高滴度鸡胚生长特性;重组病毒rLa-CDV-H接种12周龄比格犬后,可以诱导显著的CDV中和抗体反应.本研究表明重组病毒rLa-CDV-H具有作为犬瘟热重组病毒活载体疫苗的潜力.     

共表达传染性支气管炎病毒S1基因和鸡干扰素-γ基因的重组鸡痘病毒部分生物学特性测定

将共表达传染性支气管炎病毒S1基因和鸡干扰素-γ基因的重组鸡痘病毒(rFPV-IFNγS1)接种4周龄SPF鸡,免疫3周后用异源病毒LSC99I株攻击,考察重组疫苗对异源病毒攻击的保护作用。结果显示,疫苗接种后,免疫鸡血清中很快出现抗体;外周血中CD4 和CD8 T淋巴细胞的百分含量略高于非免疫对照组,但这种差异并不显著。LSC99I株IB病毒攻击后的保护率结果表明,免疫组的发病率为50%(5/10),死亡率为20%(2/10),非免疫对照组的发病率为100%(10/10),死亡率为50%(5/10);另外免疫鸡的病理损伤程度与非免疫对照组相比,二者差异不明显,在排毒时间和排毒量上也没有明显变化。体重分析结果显示,攻毒前免疫组与对照组相比,体重均没有显著变化,但攻毒后免疫组鸡体重增长的幅度要高于非免疫对照组。从以上结果可以说明,重组疫苗虽然能在动物体内很好地表达,但不能对实验动物产生很好的保护作用,而鸡干扰素-γ基因能在动物体内表达并发挥其免疫调节作用,减轻了重组疫苗对体重增长的影响。     

Comparative pathogenesis in specific-pathogen-free chickens of two strains of avian hepatitis E virus recovered from a chicken with Hepatitis–Splenomegaly syndrome and from a clinically healthy chicken

Avian hepatitis E virus (avian HEV) is the primary causative agent of Hepatitis–Splenomegaly (HS) syndrome in chickens. Recently, a genetically unique strain of avian HEV, designated avian HEV-VA, was recovered from healthy chickens in Virginia. The objective of this study was to experimentally compare the pathogenicity of the prototype strain recovered from a chicken with HS syndrome and the avian HEV-VA strain in specific-pathogen-free chickens. An infectious stock of the avian HEV-VA strain was first generated and its infectivity titer determined in chickens. For the comparative pathogenesis study, 54 chickens of 6-week-old were assigned to 3 groups of 18 chickens each. The group 1 chickens were each intravenously inoculated with 5 × 10^2.5 50% chicken infectious dose of the prototype strain. The group 2 received the same dose of the avian HEV-VA strain, and the group 3 served as negative controls. Six chickens from each group were necropsied at 2, 3 and 4 weeks post-inoculation (wpi). Most chickens in both inoculated groups seroconverted by 3 wpi, and the mean anti-avian HEV antibody titers were higher for the prototype strain group than the avian HEV-VA strain group. There was no significant difference in the patterns of viremia and fecal virus shedding. Blood analyte profiles did not differ between treatment groups except for serum creatine phosphokinase levels which were higher for prototype avian HEV group than avian HEV-VA group. The hepatic lesion score was higher for the prototype strain group than the other two groups. The results indicateded that the avian HEV-VA strain is only slightly attenuated compared to the prototype strain, suggesting that the full spectrum of HS syndrome is likely associated with other co-factors.     

Gallid-1 herpesvirus infection in the chicken. 3. Reinvestigation of the pathogenesis of infectious laryngotracheitis in acute and early post-acute respiratory disease

Specific-pathogen-free chickens were infected via the trachea when 4 weeks old with 2000 plaque-forming units (PFU) of the virulent Australian infectious laryngotracheitis (ILT) virus strain CSW-1. Titers of ILT virus in the trachea were greatest (10(7.0) PFU/ml in washings, 10(6.0) PFU/g of tissue) 2-4 days postinfection (PI). Infectivity then declined rapidly, to become undetectable by 7 days PI, although highly localized areas of ILT antigen in the tracheal epithelium were occasionally observed by fluorescent antibody staining at 7 and 8 days PI. Tracheal organ cultures established 7 and 8 days PI provided no evidence of latent ILT virus infection at this immediate post-acute stage of pathogenesis. ILT virus was not isolated from peripheral blood leukocytes or lymphoid organs (spleen, bursa, thymus). ILT virus was found in the trigeminal ganglia and/or brain in 14 of 36 chickens (40%) examined between 4 and 7 days after intratracheal inoculation, but it was not in these tissues in five chickens examined at 8 days PI. Virus was also detected at 6 days PI in the trigeminal ganglia in one of five chickens infected by the conjunctival route. These data indicate that the early pathogenesis of ILT (CSW-1) infection frequently involves the tissues of the nervous system. In acute ILT in 4-week-old chickens, interferon-alpha/beta activity was not detectable in serum or tracheal exudates within 14 days PI, but tracheal washings contained significant virus-neutralizing activity by 7 and 8 days PI. In 3-day-old chickens infected via the trachea with 200 PFU of ILT CSW-1, the clearance of ILT virus from the trachea was similar to that observed in 4-week-old chickens, but ILT virus spread systemically to the livers of 20% by 5-7 days PI.     

鸡病毒性关节炎弱毒苗的研制 Ⅱ.弱毒株的免疫原性

应用具有良好安全性的鸡病毒性关节炎病毒（AVAV）弱毒JN-1株免疫1日龄肉鸡40只,分别用琼脂扩散试验、间接ELISA和中和试验（NT）检测免疫鸡血清中的AVAV抗体。中和抗体于免疫后7d开始出现,14d后全部阳转;ELISA抗体也于免疫后7d开始显示阳性,21d后全部阳转;琼脂扩散抗体于免疫后14d开始阳转,28d以后全部显示阳性。分别在免疫后7、14、21和 28d于足掌皮下用强毒攻击免疫鸡,临床保护率分别为2/10、9/10、10/ 10、10/10,病理保护率分别为0/10、7/10、9/10、10/10。体外抗病毒谱试验显示,弱毒株的免疫血清能中和AVAV J-1株、S_(1133)株、FDO株和H_(9307)株。上述实验结果表明,AVAV弱毒株具有良好的免疫原性。     

Betaine Alleviates Heat Stress-Induced Hepatic and Mitochondrial Oxidative Damage in Broilers

The aim of this study was to evaluate the effects of dietary betaine (BET) on growth performance, redox state, and related gene expression in broilers under heat stress (HS). A total of 144 21-day-old male broiler chickens with similar body weights were assigned randomly to three treatments with six replicates (eight chickens per replicate cage). Broilers in the control (CON) group were kept at thermoneutral (TN, 22±1°C) conditions and fed a basal diet until they were 42 days of age. Broilers in the other two groups (defined as HS and HS + BET) were exposed to HS (34±1°C, 8 h/day) and fed the basal diet without or with 1000 mg/kg BET, respectively. Rectal and cockscomb temperature of broilers was increased (P<0.05) in HS and HS + BET groups compared with the CON group, whereas there was no difference between HS and HS + BET groups. Dietary BET supplementation restored (P<0.05) average daily gain (ADG) and average daily feed intake (ADFI) of broilers and reversed (P<0.05) the increase in serum alanine transaminase (ALT) activity and malondialdehyde (MDA) content in the liver tissue of broilers under HS. The HS + BET group had higher (P<0.05) activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in the liver tissue and mitochondria than the HS group, and the same pattern was observed for glutathione (GSH) and GSH/glutathione disulphide (GSSG) in the liver tissue. The decreased mRNA levels of GPX1 and uncoupling protein (UCP) in the liver induced by HS were restored by BET supplementation. In conclusion, dietary BET supplementation can alleviate HS-induced hepatic and mitochondrial oxidative damage of broilers by regulating mRNA expressions of GPX1 and UCP.     

Cloning and expression in Escherichia coli of Mycoplasma gallisepticum antigens recognized by sera from infected chickens

A clone bank of Mycoplasma gallisepticum (MG) strain A5969 DNA was prepared in the expression vector phage lambda gt11. Approximately 75% of the resulting phages were recombinants, based upon the insertional inactivation of the lacZ gene of the vector. Clones were screened immunologically with serum prepared from specific-pathogen-free white leghorn chickens that had been infected with aerosolized MG. Approximately 250 clones, or less than 1% of the recombinant phage, reacted positively to various degrees with the test serum and failed to react with serum from uninfected specific-pathogen-free control chickens. A single clone was chosen at random for comparison with a vector control by western immunoblot, revealing a polypeptide of 140,000 molecular weight in the clone profile but not the control profile that reacted with immune serum. Clones expressing MG antigens recognized during infection may provide an improved means for antigen preparation for serologic diagnosis of mycoplasmosis.     

传染性法氏囊病免疫保护试验中3种评定指标的比较

100只SPF鸡均分为5组，A、B、c3组免疫后攻毒，接种3批次自制的IBD基因工程重组亚单位油乳剂疫苗；D组不免疫不攻毒，E组不免疫而攻毒。免疫后第22天，感染IBDV强毒株BC-6／85。攻毒后第4天，将所有存活的鸡只以颈脱臼致死，收集法氏囊，以3种方法和指标（法氏囊眼观病变；法氏囊显微病变；法氏囊中IBDV抗原检测）进行分析，以评定免疫保护率。结果显示，以这3种方法和指标评定，A组免疫保护率为90％，B、C组免疫保护率均为95％；试验鸡A3、A14、B7、C16、E1～E20，法氏囊眼观病变明显，法氏囊显微病理损伤评分为3～5分，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阳性；其他试验鸡，法氏囊眼观无明显异常，法氏囊显微病理损伤评分在3分以下，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阴性。结果表明，这3种方法和指标在IBD疫苗免疫保护试验评定中具有很好的一致性。     

Effect of broiler chicken age on susceptibility to experimentally induced Cryptosporidium baileyi infection

Clinical signs of respiratory tract disease were observed in chickens that were inoculated intratracheally with 1 x 10(6) oocysts of Cryptosporidium baileyi at 2 or 14 days of age (10 chickens/group), but not in chickens inoculated at 28 or 42 days of age (10 chickens/group). Orally inoculated chickens in all age groups (10 chickens/group) did not develop clinical signs of disease. Orally and intratracheally inoculated chickens in all age groups were infected, as determined by the finding of cryptosporidia in tissue sections of the trachea, bursa of Fabricius, and cloaca, and by the recovery of oocysts from their feces. Chickens inoculated at 2 and 14 days of age excreted oocysts for a longer period and had greater numbers of cryptosporidia in their tissues, compared with chickens inoculated at 28 and 42 days of age.     

口服重组鸡α-干扰素对鸡新城疫病毒感染和疫苗免疫的影响

通过点眼、滴鼻的方式使SPF鸡感染新城疫La Sota株,感染8 h后在饮水中添加1000 U/mL重组鸡α-干扰素,每只鸡每天饮用干扰素剂量为20000 U,分别饮用不同时间。于感染后第7天采集气管组织,制备病理切片。结果显示,连续3 d以上饮用重组鸡α-干扰素的试验鸡,其气管的病理损伤可得到明显改善;检测饮用重组鸡α-干扰素后试验鸡的新城疫抗体效价,结果表明口服高剂量的干扰素可明显下调新城疫抗体的滴度。