【作者】 刘冠华；

【导师】 朱瑞良；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2013， 博士

【摘要】 禽波氏杆菌（Bordetella avium, B. avium）最早于1967年分离自火鸡，该菌引起的禽波氏杆菌病是一种高度接触传染性疫病，具有高度上呼吸道接触传染性，1周龄内的雏禽最易感，主要引起禽类的鼻炎、眼炎等呼吸道症状，造成种鸡场孵化率降低、死胎率高和弱雏增加。在中国，朱瑞良教授在1991年首次报道了鸡波氏杆菌病，随后从病鸡眼中分离得到了禽波氏杆菌（朱瑞良，1994）。禽波氏杆菌可垂直传播，又可水平传播。禽波氏杆菌感染的同时，常常继发或并发感染免疫抑制病毒（ALV、REV、CIAV等）、大肠杆菌、肺炎克雷伯氏菌和荧光假单胞菌，使禽波氏杆菌病的危害不断放大。近几年，在全国不同地区经流行病学调查发现禽波氏杆菌的感染率在10%到50%之间，给我国的养禽业已经造成了巨大的经济损失，对该病做出及时快速的诊断及防控十分必要，因此，禽波氏杆菌快速灵敏的诊断方法的建立和疫苗的制备在预防禽波氏杆菌感染和及时诊断治疗方面显得尤为重要。本研究由以下五个部分组成：1.禽波氏杆菌外膜蛋白单克隆抗体的抗原识别位点分析对本试验室冻存的3株杂交瘤细胞株（3G10、4A3和4E8）进行复苏，经过3次有限稀释法克隆后，制备腹水。通过间接ELISA试验证明，冻存后复苏的3株杂交瘤细胞株均能稳定的分泌抗体，经检测得出产生的腹水效价较高，杂交瘤细胞培养上清的单抗效价为1：320～1：640，腹水中单抗效价为1：6400～1：12800。测定单克隆抗体的饱和工作浓度，绘制单克隆抗体的饱和曲线图，根据曲线得出单克隆抗体3G10、4A3、4E8的饱和工作浓度分别为1∶80、1∶80和1∶160。然后用ELISA相加试验分析单克隆抗体的抗原结合位点。通过计算相加指数AI来分析抗原位点的结合情况，本试验复苏的3株杂交瘤细胞产生的单克隆抗体，进行相加ELISA试验，3G10、4A3和4E8的相加指数均小于50%，说明3株单克隆抗体识别的抗原位点可能相同或者比较接近。经western-blot试验进行鉴定，3株单克隆抗体均特异性识别禽波氏杆菌外膜蛋白58KD上的抗原表位。本试验通过对单克隆抗体识别抗原位点的分析，为特异性诊断试剂的研制奠定了基础。2.免疫PCR技术检测禽波氏杆菌方法的建立选择抗体效价最高的单克隆抗体4E8株作为包被抗体，包被在酶标板上，使用禽波氏杆菌菌体抗原免疫家兔制备多克隆抗体，以多克隆抗体作为检测抗体。以pUC19质粒为模板，用生物素标记的引物，进行PCR扩增，得到生物素标记的DNA报告分子。使用链霉亲和素作为连接分子，将生物素标记的报告DNA连接到生物素标记的羊抗兔IgG分子上，利用PCR扩增报告DNA分子，经过琼脂糖凝胶电泳后观察出现特异性的扩增条带的情况，如果出现特异性的扩增条带，那表明待检样品中含有禽波氏杆菌。试验中对生物素标记的报告DNA和链霉亲和素的工作浓度进行了测定，得出生物素标记的报告DNA最适工作浓度为10ng/mL，链霉亲和素最适工作浓度为10ng/mL。对传统的间接ELISA方法与建立的免疫PCR的灵敏度进行比较，间接ELISA方法检测出禽波氏杆菌菌液的最低浓度为103CFU/mL，免疫PCR法的最低检测浓度为1CFU/mL。因此，本试验建立的免疫PCR方法的灵敏度比间接ELISA方法提高了近103倍。然后利用禽波氏杆菌、大肠杆菌、鸡白痢沙门氏菌、鸡奇异变形杆菌、支气管败血波氏杆菌和绿脓杆菌对建立的免疫PCR方法进行特异性检测，结果说明该方法具有高度特异性。3.免疫PCR检测禽波氏杆菌的应用研究对试验室保存的1991年至今分离到的22株禽波氏杆菌进行免疫PCR的检测，所有菌株均扩增出了特异性条带，说明该方法可以用于对禽波氏杆菌的检测。在对患病鸡场中采集病料的检测中，常规的细菌分离培养和鉴定，确切的鉴定某种细菌的感染最少需要3-4d的时间，费时费力。免疫PCR方法能够特异性的检测出禽波氏杆菌的感染，而且仅需要20h左右，节省了时间。对患病鸡场采集的130份血清的检测中，使用了平板凝集试验、间接ELISA试验和免疫PCR试验三种方法，并对三种试验进行比较。平板凝集试验结果有82份血清样本为阳性，25份可疑，阳性率为63.1%；间接ELISA试验结果有96份血清样本为阳性，阳性率为73.8%；而免疫PCR方法检测结果显示，经其它两种方法检测的阳性血清全部显示阳性结果，从判断为可疑或是阴性的检查样品中也检测出了阳性样品，阳性率为80.7%。三种检验方法相比，免疫PCR比平板凝集试验、间接ELISA试验更加敏感、检出率更高。在对禽波氏杆菌感染雏鸡后在鸡体内定植规律的检测中，感染后1d，能够在气管和肺脏中经PCR扩增出特异性条带，并且一直持续存在至第6d采样结束；感染4d后，在心脏、肝脏和脾脏中扩增出特异性条带；感染后6d，在肾脏中扩增出特异性条带。检测结果优于细菌涂板培养，检出时间提前。4.禽波氏杆菌分离株抗原性与免疫原性差异分析首先将复壮的22株禽波氏杆菌免疫动物制备高免血清，利用凝集试验测定各菌株血清效价的高低，结果显示了各菌株抗原性的大小。根据凝集试验的结果，按照血清型和基因型的不同，选择出8株抗原性较高的禽波氏杆菌来进行交叉凝集试验和免疫琼脂扩散试验，分析菌株之间是否存在共同的抗原成分。然后进行免疫保护试验，分析各菌株间的交叉免疫保护情况。凝集试验结果显示，22株分离株的血清抗体效价在1:320～1280之间，均能刺激机体产生高效价的抗体，抗原性较好。交叉凝集试验和免疫琼脂扩散试验结果显示，选择的8株代表株之间存在共同的抗原成分，但是交叉凝集抗体效价均比较低。免疫保护试验结果说明免疫菌苗对相应菌株的攻击具有较高的免疫保护率，对于同实验组内其它菌株的攻击，存在交叉免疫保护，但是保护率较低。其中Ba15株制备的疫苗对于同型菌株或者异型菌株的攻击，均表现出较高的免疫保护率，Ba11株和Ba8株次之。5.禽波氏杆菌亚单位疫苗的研制及免疫效果的研究本试验利用提取的禽波氏杆菌外膜蛋白作为免疫原，选择松花粉多糖、蜂胶和白油作为免疫佐剂来进行比较。将松花粉多糖设计成10mg/mL、20mg/mL和40mg/mL等3个剂量，把等量的禽波氏杆菌外膜蛋白与不同剂量的松花粉多糖、蜂胶、白油分别组合，制备不同免疫佐剂的禽波氏杆菌亚单位疫苗。将制备的疫苗进行无菌检验和安全性检验，结果证明，本试验制备的不同免疫佐剂的禽波氏杆菌亚单位疫苗无菌性和安全性良好。将制备的疫苗免疫雏鸡，检测免疫后不同时间雏鸡的体液免疫和细胞免疫的各项指标。结果表明，免疫后，各组疫苗均能刺激机体产生不同程度的抗体水平，并且能够持续存在。而对于各项细胞免疫指标的测定说明，制备的亚单位疫苗能够提高免疫动物的细胞免疫水平。在对各种佐剂的比较中，试验结果证明III组（40mg/ml松花粉多糖）各项指标显著高于其他各组（P<0.05），IV组（蜂胶佐剂）指标高于I、II、V组，但差异不显著。免疫保护试验结果显示III组（含40mg/ml松花粉多糖）保护率最高，IV组（蜂胶佐剂）保护率较好，优于I、II、V组。不同佐剂组的免疫指标与免疫保护试验的保护率呈平行关系，说明40mg/ml松花粉多糖可以作为禽波氏杆菌亚单位疫苗的免疫佐剂，与禽波氏杆菌外膜蛋白混合，用于预防禽波氏杆菌的感染。总之，本研究结果表明试验室保存的三株禽波氏杆菌外膜蛋白的单克隆抗体均特异性识别禽波氏杆菌外膜蛋白58KD处的抗原位点，位置相同或者相邻。用4E8株和禽波氏杆菌多克隆抗体建立了双抗体夹心免疫PCR检测方法，实验证明该方法具有较高的灵敏度和特异性，在临床应用上，该方法在检测时间和检出率方面均优于传统的检验方法，能够检测出禽波氏杆菌的早期感染。通过对禽波氏杆菌分离株抗原性与免疫原性差异的分析，选择两株分离株进行亚单位疫苗的研制，根据各项免疫指标证明40mg/ml松花粉多糖作为免疫佐剂，与禽波氏杆菌外膜蛋白组合研制成亚单位疫苗，可以更好的预防禽波氏杆菌的感染。更多还原

【Abstract】 Bordetella avium (B. avium) was first isolated from young turkeys in1967. The disease(Bordetellosis) caused by this bacillus was a kind of height contacted infective epidemicdisease. It mainly causes respiratory diseases, including ophthalmia and rhinitis, in poultries.It has high upper respiratory tracts to keep in touch, the chicks in age of one week can easilyinfected. Bordetellosis in poultry is clinically characterized by death of chicken embryos, lowhatchability, rapid chick death, and ophthalmia in adult chickens. In China, Professor Ruiliang Zhu reported bordetellosis for the first time in1991, then he isolated B.avium fromdiseased embryos and chicks. Bordetellosis undergoes both vertical and horizontaltransmission, so the danger of this disease can be enlarged constantly. In recent years,investigate poultry’s infection rate in the national different areas is between10%and50%byepidemiology. This has already caused the enormous economic losses to raising the poultryindustry of our country. So fast sensitive of diagnostic method and preparation of vaccineseems particularly important to treat in time.This research is made up of five parts:1. Analysis of the antigenic epitope recognized by three monoclonalantibodies against Bordetella avium OMPsIn this experiment, hybridomas (3G10,4A3and4E8) preserved in our laboratory weresubcloned for three rounds using the limiting dilution method. Then prepared ascites. IndirectELISA test proved that three hybridoma cell lines after recovery can secrete antibodiessteadily and the antibodies of ascites have high titres. Antibody titers in the hybridoma cellculture fluid were1:3201:640, and the titers in ascitic fluid were1:64001:12800,respectively. Determine the saturated working concentration of monoclonal antibody, anddraw the saturation curves of monoclonal antibody. According to the saturation curves, thesaturated concentration of3G10,4A3,4E8were1:80,1:801:160, respectively. Then analyzeantigen binding sites of monoclonal antibody through the ELISA additivity test analysis.Calculated additive index AI,3G10,4A3and4E8were all smaller than50%. It was provedthat three strains of monoclonal antibodies recognize the antigen sites may be the same orrelatively close. In western blot analysis, all mAbs recognized a58kDa protein band. In thisexperiment, the analysis of the antigenic sites recognized by monoclonal antibodies, laid the foundation for the development of specific diagnostic reagents.2. Establishment of immuno-PCR methods for detecting Bordetella aviumWe choosed the strain of4E8which has the highest antibody titre as the coated antibody.An ELISA microplate was coated with4E8in carbonate buffer. Rabbit polyclonal antibodiesagainst B. avium were used as detection antibodies. pUC19plasmid as a template, withbiotin-labeled primers for PCR amplification,we prepared the biotin-reporter DNA. Thebiotin-reporter DNA was connected to the biotin-labeled goat anti-rabbit IgG by streptavidinas the linker. Using PCR amplification reports DNA molecule, then, specific amplified bandswere observed after agarose gel electrophoresis. If there is a specific amplified band, it isproved that the detected sample contained B. avium.In this experiment，we determined optimization concentration of the reporter DNA andthe streptavidin. The optimization concentration of the reporter DNA is10ng/mL. Theoptimization concentration of the streptavidin is10ng/mL. Compared the traditional indirectELISA method to the immune PCR with sensitivity, the results was the sensitivity of theindirect ELISA was103CFU/mL, but the immune PCR is1CFU/mL. So, the sensitivity of theimmune PCR method in this test has raised nearly104times. B. avium, chicken P. mirabilis, E.coli, Bordetella bronchiseptica, Pseudomonas aeruginosa and Salmonella were used in the immunePCR to determine the specificity. The results shows that the immune PCR has highlyspecificity.3. Application of immuno-PCR methods for detecting Bordetella aviumTwenty-two strains of Bordetella avium which were preserved in our laboratory weredetected by immuno-PCR. All strains were amplified specific bands. The result showed thatthis method can be used for the detection of Bordetella avian.Samples collected from diseased chicken were detected in this experiment. Theconventional bacterial isolates culture and identification need3or4days to identify thebacterial. It is time-consuming and laborious. Immuno-PCR method can specifically detectavian Bordetella infection, and only need about20hours.Detection of130serum samples collected from diseased chicken were used the method ofplate agglutination test, indirect ELISA and immune PCR. Then we compared this threemethods according to their results. Plate agglutination test resulted of82serum samples werepositive and25equivocal. The positive rate was63.1%; Indirect ELISA test resulted of96serum samples were positive and the positive rate was73.8%; The results of Immuno-PCRshowed that all positive sera detected by the other two methods showed positive and also detected positive sera from suspicious or negative samples. The positive rate was80.7%. Sothe immuno-PCR is more sensitive and has a higher detection rate than the other twomethods.Colonization time of Bordetella avium after infected chicken were detected byimmuno-PCR. The result showed that the trachea and lungs were amplified specific bands byPCR one day after infection; the heart, liver and spleen were amplified specific bands4daysafter infection; the kidney were amplified specific bands6days after infection.4. Comparision of antigenicity and immunogenicity of Bordetella aviumstrainsAfter rejuvenation of22strains, we prepared Bordetella avium hyperimmune serum byimmunizing animals.The antibody titre of each strain was detected by agglutination test. Theresult showed the antigenicity of each strain. According to the different serotypes andgenotypes, we choosed8strains of Bordetella avium which had higher antibody titre. Weanalyzed the existence of a common antigen component between8strains bycross-agglutination test and immune agar diffusion test. Then we analyzed cross-protectiveimmunity among the8strains by protective immunity test.Agglutination test results showed that the22strains of serum antibody titer was between1:320and1280. It proved that all strains can stimulate the body to produce high titerantibodies. The result of cross-agglutination test and immune agar diffusion test showed thatthere was common antigen component between8strains. But cross agglutination antibodytiter is lower than the agglutination test.Immune protection test results showed that the vaccine had a higher immune attack rate ofprotection on the corresponding strain. There is cross-protective immunity when attacked byother strains within the same experimental group, but the rate of protection was lower. TheBa15strain vaccine prepared for the same type of strain or atypical strains attack, showed ahigher immune protection, Ba11and Ba8strains followed.5. Study on subuit vaccine of Bordetella aviumIn this experiment, the omps of Bordetella avium were used as immunogen, and pinepollen polysaccharide, propolis and shortening were used as immune adjuvant. Pine pollenpolysaccharide designed to three doses of10mg/mL,20mg/mL and40mg/mL. Differentimmune adjuvant subunit vaccine of Bordetella avium were prepared by different doses ofpine pollen polysaccharide, propolis and white with the same amount of Bordetella aviumomps. The preparaed vaccines were detected by sterility and safety test. The result showed that they were aseptic and securityPrepared subunit vaccine were used to vaccinate chickens, then detected the immunehumoral and cellular immune indicators of chicks at different times after immunized. Thelevel of antibody in the serum can reflect the state of the humoral immune. The ratio of bloodlymphocytes, lymphocyte subsets in blood IL-2, IFN-content and spleen lymphocytetransformation rate that reflects the level of cell-mediated immunity. The results showed thateach group vaccine can stimulate the body to produce different degrees of antibody levels andpersistent after immunization. Description for the determination of the cellular immuneindicators, the preparation of subunit vaccines were possible to improve the level of immunecells of the immunized animal. In the comparison of various adjuvants, the test results showedthat the indicators of the Group III (40mg/ml pine pollen polysaccharide) was significantlyhigher than the other groups (P <0.05). Group IV (Propolis adjuvant) were higher I (10mg/mlpine pollen polysaccharide), II (20mg/ml pine pollen polysaccharide), V (mineral oil adjuvant)group, but the difference was not significant. Protective immunity test results showed thatGroup III (40mg/ml pine pollen polysaccharide) had the highest rates of protection, and groupIV (propolis adjuvant) had better protection, higher than I, II, and V group. Different adjuvantgroup of immune indicator and immune protection test protection rates were parallel. Itproved that40mg/ml pine pollen polysaccharide can be used as adjuvant of Bordetella aviumsubunit vaccine for the prevention of Bordetella avium infection.In a word, the results of this study showed that three Bordetella avium omps monoclonalantibodies which were preserved in our laboratory can specifically recognize58kD antigenicsites of Bordetella avium omps. They recognized at the same location or adjacent. Weestablished double antibody sandwich immuno-PCR method by4E8strain and polyclonalantibody of Bordetella avium. Experiments showed that this method had high sensitivity andspecificity. In the clinical application, the method was better than traditional testing methodsin the detection time and detection rate. And the immuno-PCR can detect theearly infection ofBordetella avium. According to the antigenic and immunogenic differences analysis of Bordetella avium,we choosed two strain to prepare Bordetella avium subunit vaccine. The results proved that40mg/ml pine pollen polysaccharide can be used as adjuvant of Bordetella avium subunitvaccine for the prevention of Bordetella avium infection.更多还原