禽流感高免球蛋白的制备及应用研究

应用H9N2亚型禽流感病毒(AIV)油乳剂灭活苗6个不同剂量免疫30周龄SPF蛋鸡,通过血凝抑制(HI)和琼脂免疫扩散(AGP)试验监测血清及卵黄抗体消长规律,确定2.0 mL为最适免疫剂量。首免后第18天血清与卵黄中抗体同时升至第一高峰,此时加强免疫。二免后1周,效价达到第二高峰,用常规抽提法从高抗体滴度卵黄中提取高免球蛋白(IgY)。中和试验(NT)表明:IgY具有中和病毒感染的作用,且中和抗体滴度随HI滴度的降低而降低;给鸡肌肉注射后6 h能检测出血清的HI抗体,24 h达到峰值,维持7 d,第13天血清中HI抗体消失。104个EID50的AIV(H9N2)攻毒,同时用HI价为214的IgY的10倍系列稀释物分5组进行保护试验,确定HI价为214的IgY保护范围在100倍稀释与10倍稀释之间。IgY系列保护试验表明HI价28为完全保护的最低滴度;在攻毒的前1天及至攻毒后第5天接种IgY均能100%保护。攻毒保存期试验证明,所制得的高免球蛋白4 ℃可以保存6个月,-20 ℃可保存1年。     

兔病毒性出血症病毒在兔体内分布情况的研究

兔病毒性出血症(RVHD)是由兔瘟病毒引起的一种急性败血传染病。该病自1984年2月在江苏无锡地区首次发现以来,人们作了大量的研究工作,但动态地研究该病病毒在体内的分布情况报道还较少。为此,本试验利用血凝(HA)、血凝抑制(HI)和SPA协同凝集试验动态系统地测出各组织器官的病毒滴度,探讨免病毒出血症的发病机理,进一步为本病的诊断提供依据。     

兔病毒性出血症灭活疫苗效力检验替代方法研究

为了采用血凝抑制(HI)试验方法替代兔病毒性出血症灭活疫苗效力检验的免疫攻毒方法,试验在制备了HI试验用兔出血症病毒血凝(HA)抗原、阳性血清、阴性血清的基础上,研究了免疫攻毒保护与免疫血清HI效价之间的平行关系,并进行了替代方法的应用。结果表明,当免疫血清HI效价不低于1∶32时,两种方法的检验结果符合率为100%,证明了该替代方法的可行性,也为下一步修订兔病毒性出血症灭活疫苗效力检验方法提供了数据支持。     

不同方法制备兔病毒性出血症高免血清效果比较

本试验采用两种方法制备兔病毒性出血症高免血清,并对其血清学效价进行了对比。结果,加福氏佐剂的灭活疫苗多次免疫注射获得的高免血清血凝抑制价(HI)为1:327 680,而注射甲醛灭活疫苗制备的高元血清 HI 仅为1:1280—1:2560。     

三峡库区兔病毒性出血症流行情况调查

为了调查三峡库区兔病毒性出血症(RHD)的流行情况,在三峡库区的开县、石柱县、渝北区等养兔示范县调查20个兔场,采集疑似RHD病料32份。采用血凝试验(HA)、血凝抑制试验(HI)对病料进行检测。结果表明HA试验检测到4株兔病毒性出血症病毒(RHDV),阳性率为12.5%,HA效价为8~12 lb;这4株RHDV血凝性都能被RHDV疫苗毒株(AV33)的抗血清抑制。在RHD疫苗普免的情况下,目前库区虽然没有RHD的大流行,但仍然存在RHD的散发,应引起兽医防疫部门和养兔场的高度重视。     

应用纸片沾血检测兔病毒性出血症病毒HI抗体

兔病毒性出血症(兔瘟)是近年发现的一种病毒性传染病,对本病的诊断,目前多采用临床综合判定,血凝(HA)和血凝抑制(HI)试验。为了摸索较简便的方法,我们采用纸片沾血来检测HI抗体。     

一株兔瘟病毒野毒株的分离与鉴定

为了对南部县某兔场疑似兔出血症病毒(RHDV)NB毒株进行鉴定,试验采用血凝(HA)及血凝抑制(HI)试验、家兔接种试验、家兔免疫攻毒试验、VP60基因的同源性比对。结果显示:NB毒株能凝集人"O"型红细胞,HA效价为12 log2,其血凝性能被RHDV疫苗毒株AV33株的抗血清抑制;NB毒株注射健康非免家兔,家兔在48h内死亡,具有典型的兔病毒性出血症(RHD)的临床症状和病理变化;RHDV(AV33)组织灭活疫苗免疫家兔后,家兔能抵抗NB毒株的攻击;NB毒株与AV33毒株的VP60基因同源性为96.12%,氨基酸序列同源性为97.59%。     

用血凝抑制法检测兔体内抗兔病毒性出血症抗体的消长动态

为了解兔病毒性出血症组织灭能苗的免疫效果,制定合理的免疫程序提供依据,我们于1986年3至10月间应用微量血凝抑制(HI)法,进行了组织灭能苗免疫兔体内抗体消长规律的试验。试验材料与方法 1.组织灭能苗:为本站生产的第13批疫苗,组织浓度为1:10;批号860113;经细菌、安全检查证明合格。 2.试验兔:为来自非疫区,未免疫的健康无病的西德杂交长毛兔,HI阴性。 3.强毒:系本区自然发病死亡的病兔     

兔出血症的免疫诊断及防治

兔病毒性出血症又称兔瘟。自1984年在我国江苏南部地区首次暴发后,短期内迅速蔓延至全国20几个省(市),严重地威胁着养兔生产。为了有效地控制此病,不少院所进行研究并取得了较好效果。本文就我国对此病免疫诊断及防治方面的研究概况综述如下:兔出血症的免疫诊断一、血凝(HA)及血凝抑制(HI)试验HA 和 HI 是目前诊断兔出血症最基本方法。刘胜江等(1984)使用高速离心的病兔肝悬液为待检物,以1%人○型红血球在96孔 U 型微量反应板上进行了 HA 和 HI 试验,对江苏南部地区暴发的病兔进行检测,证实本病在我国首次流行。张再清等(1989)用戍二醛处理人红细胞代替新鲜人红细胞     

抗兔出血症病毒单克隆抗体的应用

采用抗兔出血症病毒的单克隆抗体对来自全国不同地区的12株兔出血症病毒分别进行了血凝抑制(HI)试验,HI 效价达1:100×2~7～1:100×2~9.结果说明,该单抗对这12株病毒都能抑制其血凝现象.应用该单抗制备的荧光抗体检测了兔出血症病毒在感染兔体内主要脏器组织的分布动态。结果提示,该病毒在细胞核内增殖。经消化道感染兔时,病毒首先在入侵局部肠道粘膜内增殖。然后再侵入肝脏和脾脏。大量病毒生成后,又侵入其它的器官组织,病兔处于病毒血症状态。     

重组SEA增强H5亚型禽流感灭活疫苗对肉鸡的免疫效果研究

为评价重组葡萄球菌肠毒素A(rSEA)对H5亚型禽流感病毒(AIV)灭活疫苗免疫效果的影响,本研究以原核表达的rSEA为免疫增强剂,制备成含有rSEA高(167 μg/0.5 mL)、低(16.7 μg/0.5 mL)两种剂量的AIV油乳剂灭活苗,分别免疫7d龄肉鸡,通过检测免疫鸡AIV HI抗体滴度及外周血CD4+/CD8+值评价其免疫效果.HI 抗体检测结果显示,免疫后2周高剂量组HI抗体平均滴度为5.6 log2,低剂量组为4.2 log2,而无rSEA疫苗对照组为2.9 log2(p＜0.01);同时在免疫后前4周,rSEA免疫组的HI抗体平均滴度及峰值与对照组相比均差异显著(p＜0.05).T淋巴细胞亚群检测结果显示,肉鸡免疫高剂量rSEA的灭活苗后,其外周血CD4+百分含量及CD4+/CD8+值均显著高于对照组(p＜0.05).上述结果表明,rSEA作为AIV灭活苗免疫增强剂,能够快速诱导较高的HI抗体滴度,同时也可增强细胞免疫反应,有效缩短免疫空白期,使肉鸡短时间内产生较强的免疫应答反应.     

抗猪流行性腹泻病毒变异株卵黄抗体的制备、纯化及其活性影响因素的研究

为制备抗猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）变异株卵黄抗体并探索卵黄抗体的纯化方法，本试验采用灭活的PEDV变异株CH/JX01全病毒免疫蛋鸡，收集免疫后所产的鸡蛋，分别用水稀释法、PEG-6000法和水稀释-硫酸铵二次沉淀法对鸡蛋中IgY进行提纯，然后运用间接ELISA和Western blotting方法鉴定和比较3种方法的提纯效果，获得可用于规模化生产的提纯方法；同时研究不同温度、pH条件下抗体活性，寻找对提纯IgY活性具有保护作用的物质。结果显示，本试验成功制备了抗PEDV变异株的卵黄抗体。浓度检测结果表明，水稀释法提取的IgY浓度最高，为6.204 mg/mL；PEG-6000方法提纯的IgY浓度次之，为4.673 mg/mL；水稀释-硫酸铵二次沉淀法IgY浓度最低，为3.359 mg/mL。间接ELISA检测结果表明，水稀释-硫酸铵二次沉淀法提纯的IgY效价最高，为1：12 800；其次是PEG-6000，为1：6 400；而水稀释法最低，为1：1 600。综合3种方法所提纯的卵黄抗体浓度与效价，发现水稀释-硫酸铵二次沉淀法的提纯效果优于其他两种方法。活性影响因素试验结果表明，提纯的IgY在37~60℃活性稳定；pH为4.0~11.0时具有良好的活性；在酸性条件下，20%硫糖铝对IgY活性具有较好的保护作用。本试验结果将为卵黄抗体的大规模生产运用与储存提供了有效的参考依据。     

Influence of the mode of ventilation on ketamine/xylazine requirements in rabbits

OBJECTIVE: To evaluate the effect of the mode of mechanical ventilation (MV) on the dose of intravenous anesthetic during 3 hours of ketamine/xylazine anesthesia. STUDY DESIGN: Prospective laboratory study. ANIMALS: Sixty-one adult male New Zealand White rabbits. METHODS: Rabbits were anesthetized (ketamine/xylazine 35 + 5 mg kg(-1), IM), the trachea was intubated and randomized to four groups - (1) CMV-1 (n = 14), ventilated with traditional conventional volume-cycled MV [V(T) = 12 mL kg(-1), RR = 20, positive end-expiratory pressure (PEEP) = 0 cmH(2)O]; (2) CMV-2 (n = 13), ventilated with a modern lung-protective regimen of volume-cycled MV (V(T) = 6 mL kg(-1), RR = 40, PEEP = 5 cmH(2)O); (3) HFPV (n = 17) ventilated with high-frequency percussive ventilation [high-frequency oscillations (450 minute(-1)) superimposed on 40 minute(-1) low-frequency respiratory cycles, I:E ratio = 1:1], oscillatory continuous positive airway pressure (CPAP) of 7-10 cmH(2)O, and demand CPAP of 8-10 cmH(2)O. (4) A fourth group, spontaneously ventilating (SV, n = 17), was anesthetized, intubated, but not ventilated mechanically. FiO(2) in all groups was 0.5. Anesthesia was maintained at a surgical plane by IV administration of a ketamine/xylazine mixture (10 + 2 mg kg(-1), as necessary) for 3 hours after intubation. Total dose of xylazine/ketamine administered and the need for yohimbine to facilitate recovery were quantitated. RESULTS: The total dose of xylazine/ketamine was significantly higher in the HFPV and SV groups compared with CMV-1 (p < 0.01). Fewer animals required yohimbine to reverse anesthesia in the HFPV than CMV-1 group (p < 0.05). CONCLUSIONS: The HFPV mode of MV led to higher doses of ketamine/xylazine being used than the other modes of MV. CLINICAL RELEVANCE: In rabbits, anesthetic dose for the maintenance of anesthesia varied with the mode of MV used. Investigators should be aware of the possibility that changing the mode of ventilation may lead to an alteration in the amount of drug required to maintain anesthesia.     

Effect of first-pass hepatic metabolism on the disposition of levamisole after intravenous administration in rabbits

OBJECTIVE: To evaluate the contribution of first-pass hepatic metabolism of levamisole on levamisole disposition in rabbits. ANIMALS: 30 male New Zealand White rabbits. PROCEDURES: Rabbits were randomly placed into 2 groups. Rabbits in the first group received levamisole via the marginal ear vein at the following 3 doses: 12.5, 16, and 20 mg/kg (5 rabbits for each dose). Rabbits of the second group received levamisole via the jejunal vein at the same doses (5 rabbits each). During the following 240-minute period, plasma samples were obtained and quantified for levamisole concentrations by reversed-phase high-performance liquid chromatography. RESULTS: No significant differences were found between pharmacokinetic parameters calculated by compartmental or noncompartmental analysis. Mean hepatic extraction ratio ranged from -0.044 to 0.017 and from 0.020 to 0.081 when area under the plasma concentration-time curve values were obtained after compartmental or noncompartmental analysis, respectively. After compartmental analysis, plasma concentration decreased bi-exponentially. Mean pharmacokinetic parameter values were as follows for each dose (12.5, 16, and 20 mg/kg, respectively): after levamisole administration via the marginal ear vein, volume of distribution at steady state (Vss) = 4.26, 4.33, and 3.20 L/kg; total body clearance (CI) = 49.04, 43.77, and 39.26 mL/kg x min; and half-life associated with beta-phase (t1/2beta) = 77.93, 85.39, and 69.79 minutes. After levamisole administration via the jejunal vein, Vss = 4.38, 2.85, and 2.97 L/kg; CI = 48.14, 42.40, and 39.69 mL/kg x min; and t1/2b = 101.9, 76.71, and 76.13 minutes. CONCLUSIONS: Levamisole has a low degree of hepatic extraction in rabbits.     

“黄白散”对兔接种RHD疫苗后免疫功能的影响

在注射兔病毒性出血症 (RHD)疫苗后 ,30只兔随机分为 2组 ,试验组兔饲喂含 1 5 %黄白散的饲料。从接种到第 1 80天 ,每组取兔 8只 ,每隔 1 5d采血 1次 ,检测血凝抑制 (HI)抗体 ;在接种前 1天及接种后 5、 1 0、 1 7、 2 4、 31、 41、 5 1d ,每组取兔 1 0只 ,采血检测ANAE+淋巴细胞百分率 ;在 1 80、 2 4 0、 30 0d时 ,每组分别取兔 5只做攻毒保护试验。结果表明 :2组兔HI抗体峰值分别为 7 3log2和 9 8log2 ,差异极显著 (P <0 0 1 ) ;试验组疫苗保护期可延长 60d ;试验组ANAE+率从第 1 0天至 5 1天与对照组比较 ,差异极显著(P<0 0 1 )。所以黄白散可增强兔接种RHD疫苗后的特异性免疫功能 ,增强RHD疫苗的免疫效果。     

Pharmacokinetic-pharmacodynamic integration of moxifloxacin in rabbits after intravenous, intramuscular and oral administration

The pharmacokinetics of moxifloxacin was studied following intravenous (i.v.), intramuscular (i.m.) and oral dose of 5 mg/kg to healthy white New Zealand rabbits (n = 6). Moxifloxacin concentrations were determined by HPLC assay with fluorescence detection. The moxifloxacin plasma concentration vs. time data after i.v. administration could best be described by a two-compartment open model. The disposition of i.m. and orally administered moxifloxacin was best described by a one-compartment model. The plasma moxifloxacin clearance (Cl) for the i.v route was (mean +/- SD) 0.80 +/- 0.02 L/h.kg. The steady-state volume of distribution (Vss) was 1.95 +/- 0.18 L/kg. The terminal half-life (t(1/2lambdaz)) was (mean +/- SD) 1.84 +/- 0.12, 2.09 +/- 0.05 and 2.15 +/- 0.07 h after i.v., i.m. and oral, respectively. Minimal inhibitory concentration (MIC) assays of moxifloxacin against different strains of S. aureus were performed in order to compute pharmacodynamic surrogate markers. From these data, it is concluded that a 5 mg/kg dose moxifloxacin would be effective by i.m. and oral routes in rabbits against bacterial isolates with MIC < or = 0.06 microg/mL and possibly for MIC < or = 0.12 microg/mL, but in the latter case a higher dose would be required.     

Diatrizoate contrast agent study for luminal patency and visualization of the gastrointestinal tract in healthy rabbits (Oryctolagus cuniculus) and its influence on selected blood parameters and fecal quality

BackgroundThe aim of this study was to determine an effective dose of the contrast agent diatrizoate in healthy rabbits.MethodsThis study included 14 rabbits receiving three different dosages of a solution containing 300 mg iodine/mL and 600 mg diatrizoate/mL (Dose 1, 2 mL/kg; Dose 2, 7 mL/kg; Dose 3, 10 mL/kg). Full body radiographs were obtained at 0, 15, 30, 45 and 60 minutes, and 12 and 24 hours after oral administration. Blood samples 60 minutes and 24 hours after administration and fecal dry matter after 24 hours were compared to preliminary samples.ResultsRecommended doses and times for radiography for luminal patency and visualization of each gastrointestinal segment are given. The evaluated blood parameters were within their respective reference intervals throughout this study. However, significant changes (P < 0.05) could be observed. A decrease in hematocrit at all three doses administered, an increase in urea that persisted for more than 24 hours after administration of 2 mL/kg and 10 mL/kg, and a short-term increase in creatinine after administration of 7 mL/kg could be observed. Glucose and sodium were still increased after administration of 10 mL/kg after 24 hours, while potassium decreased after administration of all three doses. The chloride concentration was still increased after 24 hours after administration of 7 mL/kg. Administration of 7 mL/kg and 10 mL/kg resulted in a significant decrease in the dry matter of feces. Five rabbits developed self-limiting diarrhea after administration of 10 mL/kg.Conclusion and clinical relevanceDiatrizoate is a useful contrast agent in order to assess the passage through the intestine and achieve a good visualization of the gastrointestinal system.     

Egg yolk IgY against RHDV capsid protein VP60 promotes rabbit defense against RHDV infection

VP60 capsid protein is the major structural and immunogenicity protein of RHDV (Rabbit hemorrhagic disease virus, RHDV), and has been implicated as a main protein antigen in RHDV diagnosis and vaccine design. In this report, egg yolk antibody (IgY) against N-terminal of VP60 was evaluated and developed as a new strategy for RHDV therapy. Briefly, N-terminal of VP60 (～250aa) fragment was cloned and inserted into pET28a expression vector, and then the resultant plasmid, pET28a/VP60-N, was transformed into E. coli BL21(DE3) for recombinant VP60-N protein (rVP60-N) expression. Next, the rVP60-N was purified by Ni⁺-affinity purification chromatography and identified by Western blotting with RHDV antiserum. After immunizing the chickens with rVP60-N, the anti-rVP60-N IgY was isolated, and the activity and specificity of the IgY antibody were analyzed by ELISA and Western blotting. In our results, the rVP60-N could be expressed in E. coli as soluble fraction, and the isolated anti-rVP60-N IgY demonstrated a high specificity and titer (1:22,000) against rVP60-N antigen. For further evaluation of the IgY efficacy in vivo, rabbits were grouped randomly and challenged with RHDV, and the results showed that anti-rVP60-N IgY could significantly protect rabbits from virus infection and promote the host survival after a sustained treatment with anti-rVP60-N IgY for 5 days. Taken together, our study demonstrates evidence that production of IgY against VP60 could be as a novel strategy for the RHDV therapy.     

蛋鸡免疫重组禽流感病毒H_5亚型二价灭活疫苗抗体消长规律的研究

为了在临床上制定合理的禽流感疫苗免疫程序,用重组禽流感病毒H_5亚型二价灭活疫苗(H_5N_1,Re-5株+Re-4株)分别在14日龄、49日龄、105日龄免疫蛋鸡,免疫剂量分别为0.3ml/只、0.5ml/只、0.5ml/只。分别在第1、6、10、14、20、28、35、42、49、56、63、70、77、86、94、105、120、150、180日龄采血测定HI抗体效价。结果表明首免后HI抗体上升较慢较低,只有第二次免疫后,抗体才迅速升高。在70日龄(二免后3周)H5N1,Re-4HI抗体达到9.60log2,H_5N_1,Re-5HI抗体达到7.86log2。在120日龄(三免后2周)H_5N_1,Re-4HI抗体达到9.30log2,H_5N_1,Re-5HI抗体达到9.25log2。     

阿维菌素微囊在家兔体内的药动学研究

为了考察阿维菌素微囊在家免体内的药动学行为,将10只家兔随机分为2组,皮下注射阿维菌素微囊注射液（1．0mg／kg）和普通阿维菌素注射液（0．2mg／kg）,用高效液湘色谱法测定家兔血浆中阿维菌素的浓度,3P87药动学软件处理血浆药物浓度-时间数据。研究结果表明,阿维菌素微囊注射液和普通阿维菌素注射液药-时数据都符合一级吸收二室开放模型,阿维菌素微囊注射液的主要动力学参数分别是t1/2kα=21．72h;t1/2α=56．65h;t1／2β=239．43h;tmax=63．05h;Cmax=34．30ng／mL;AUC=11573．88ng／mL·h。普通阿维菌素注射液的主要动力学参数分别是t1/2kα=7．38h;t1/2α=14．59h;t1／2β=32．84h;tmax=19．78h;Cmax=20．88ng／mL;AUC=1482．04ng／mL·h。说明阿维菌素微囊化后。与普通阿维菌素注射泣相比,吸收慢,消除慢,作用时间长．生物利用度显著提高。