禽波氏杆菌免疫PCR检测方法的建立及其亚单位疫苗的研制
|
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。
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 OMPs
In 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 avium
We 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 avium
Twenty-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 aviumstrains
After 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 avium
In 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 security
Prepared 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.
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。
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 OMPs
In 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 avium
We 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 avium
Twenty-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 aviumstrains
After 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 avium
In 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 security
Prepared 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.
引文
白丽，王晶，应用辛酸硫酸铵法提取小鼠腹水和血清中的IgG抗体[J]，大理医学院学报，2000，9(4):2-3
BW卡尔尼克，禽病学（第十版），北京：中国农业出版社，1999，347-365
曹娜娜，免疫PCR法检测肠出血性大肠埃希菌O157:H[D]，吉林大学，2010
曹军平，闵桂玲，两种简易高效的单克隆抗体提纯方法[J]，单克隆抗体通讯，1995，11(2)：52-54
陈佳，应用蛋白芯片和免疫PCR技术高效检测汉滩病毒的研究[D]，中国科学院，硕士学位论文，2005
陈敏，史久华，免疫增强剂的研究进展[J]，国外医学预防、诊断、治疗用生物制品分册，2001，24：65-67
高崧，刘秀梵，张如宽，一种快速提取禽源性大肠埃希氏菌外膜蛋白的方法[J]，微生物学通报，1996，23(2)：122-123
高崧，赵坤，徐大生，等，间接ELISA测定禽源性大肠杆菌膜蛋白抗体方法的建立[J]，中国兽医学报，1997，19(4)：326-328
黄昊洋，李逄慧，罗超，等，一例禽波氏杆菌病的治疗[J]，中国畜禽种业，2008，49-50
郭春祥，郭锡琼，介绍一种简单、快速、高效的辣根过氧化物酶标记抗体的过碘酸钠法[J]，上海免疫学杂志，2003，3(2)：55-58
华海涌，周永华，薛忠权，双抗体夹心免疫S678检测弓形虫循环抗原的实验研究[J]，中国现代医药杂志，2005，12(7)：1-2
胡晓娜，朱瑞良，刘红珍，等，禽波氏杆菌外膜蛋白的提取及其免疫原性的检测[J]，微生物学报，2007，47(4)：714-717
金文杰，崔治中，刘岳龙，等，传染性法氏囊病病料中MDV、CAV、REV的共感染检测[J]，中国兽医学报，2001，21(1)：6-9
蒋成淦，酶免疫测定法，北京：人民卫生出版社，1984，75~85；133~135
赖平安，杜春辉，鸡大肠杆菌致病力与某些生物特性的相关性探讨[J]，中国兽医科技，1992，22(8)：26-29
李福胜，赵风兰，胡军，鼠伤寒杆菌外膜蛋白免疫原性及免疫保护性研究[J]，中华微生物和免疫学杂志，1993，13(3)：156-158
李桂杰，刘思当，朱瑞良，等，禽波氏杆菌内毒素致病性的研究[J]，畜牧兽医学报，2000，31(4)：349-352
李亮，林跃智，那雷，等，猴免疫缺陷病毒p27蛋白单克隆抗体结合抗原表位的鉴定[J]，中国预防兽医学报，2011，33(9)：738-741
李康然，韦平，梁梅芳，用气管环培养中和试验对鸡传染性支气管炎病毒进行血清定型[J]，广西农学院学报，1991，10(3)：1-6
李新苍，朱瑞良，路建彪，等，AA肉鸡群中免疫抑制性病毒分子流行病学调查及对疫苗免疫效果影响分析[J]，中国兽医学报，2008，28(10)：1141-1144
廖立新，曹郁生，李国辉，等，一种快速粗提绿脓杆菌外膜蛋白的方法[J]，江西医学院学报，2003，42：60-61
廖立新，赵英，李国辉，等，革兰氏阴性杆菌的敏感性分析[J]，江西医学院学报，2001，41：117-118
梁米芳，宋干，杭长寿，等，用单克隆抗体分析流行出血热病毒的核蛋白抗原位点[J]，病毒学报，1989，5(1)：24-30
凌伟，沈茜，王靖，等，免疫一PcR法检测柯萨奇病毒B组抗原的实验研究[J]，第二军医大学学报，2001，22(3)：264-266
刘红芹，规模化种鸡场胚胎疾病病原学研究及禽波氏杆菌16SrRNA基因序列分析[D]，山东农业大学，2006
刘冠华，禽波氏杆菌OMP单克隆抗体的制备及其生物学特性研究[D]，山东农业大学，2009
刘晓波，蔡美英，王霞，一种简单实用纯化腹水McAb方法-辛酸/硫酸铵法[J]，华西医科大学学报，1999，30(4)：455-456
陆承平，兽医微生物学[M]，北京：中国农业出版社，第三版，2002：275-277
陆德源，医学微生物学[M]，第3版。北京，人民卫生出版社，1989：7-26
马荣德，禽波氏杆菌的生物学特性研究及血清分型的初步探索[D]，山东农业大学，2010
孟珊珊，崔治中，孙淑红，REV和ALV-J共感染鸡病毒血症及抗体反应的相互影响[J]，
中国兽医学报，2006，26：363-366
穆杨，周丽梅，侯宏虎，等，三种佐剂对鸡体液免疫影响的比较[J]，动物医学进展，2007，28(10)：37-39
乔生军，吴自荣，戚蓓静，等，中国科学，C辑，1998，28(1)：77-82
宋云扬，吴方晖，张靖，等，夹心免疫PCR方法检测金葡菌肠毒素B[J]，中国公共卫生，2004，20(7)：832-833
史俊岩，牟玲，罗恩杰，等，免疫-PCR检测肾综合征出血热患者血清中抗核衣壳蛋白抗体方法的建立[J]，中国人兽共患病杂志，2004，20(5)：397-400
王汉雷，用核酸探针和分子斑点杂交技术检测病毒的核酸[D]，山东农业大学，2007
王丽娜，免疫-PCR的研究进展[J]，国外医学临床生物化学与检验学分册，2003，24(6)：335-336
王晶钰，李健强，张国祥，等，鸡波德特氏菌病的研究[J]，西北农业大学学报，1997，25(4)：70-71
王世若，王兴龙，韩文瑜，等，动物现代免疫学[M]，吉林：科学技术出版社，2001：431-442
王雪敏，崔岩，禽波氏杆菌病研究进展[J]，动物医学，2004，21(1)：56-57
王玉燕，朱瑞良，禽波氏杆菌病原性研究及防治初探[J]，中国预防兽医学报，2001，23：117-120
翁立雪，鸡主要胚胎性疫病病原人工共感染的试验研究[D]，山东农业大学，2009
吴自荣，陈阜东，戚蓓静，通用型免疫-聚合酶链反应系统的建立及应用[J]，中华医学检验杂志，1997，20(3)：147
夏文浪，马宝骊，黄冬生，等，空肠弯曲菌67KD外膜蛋白的提取及其淋巴细胞的多克隆激活作用[J]，中国免疫学杂志，1992，8(1)：14-17
徐肇华，戊二醛前处理的聚苯乙烯塑料小孔在二点一步酶免疫法中的应用[J]，上海医学检验杂志，1989，2：104-106
严杰，方平楚，幽门螺杆菌脂多糖生物学活性的研究[J]，中华微生物和免疫学杂志，1994，14(3)：196
杨本升，刘玉斌，苟仕金，动物微生物学[M]，长春：吉林科学技术出社，1995，550
杨春晓，禽波氏杆菌OMP标记抗体检测方法的建立及相关毒素生物学特性的检测[D]，山东农业大学，2008
杨金耀，陆广富，王建业，等，32株禽波氏杆菌分离株特性的初步研究[J]，中国家禽，2008，30(6)：18-19
杨萍萍，禽波氏杆菌的定植规律、基因分型以及与REV共感染的协同致病作用研究[D]，山东农业大学，2012
庄国宏，朱国强，周继红，等，禽波氏杆菌病病原性的研究[J]，中国畜禽传染病，1998，(2)：71-75
朱瑞良，雏鸡禽波氏杆菌病的诊断[J]，畜牧与兽医，1993，(3)：124-125
朱瑞良，牛钟相，张绍学，病鸡眼中禽波氏杆菌的分离鉴定[J]，中国兽医科技，1994，24(4)：25-26
朱瑞良，万连英，李洁珍，广东省鸡波氏杆菌病血清学调查[J]，吉林畜牧兽医，1994，(5)：39-40
朱瑞良，张绍学，唐珂心，山鸡波氏杆菌病的诊断[J]，中国兽医杂志，1994，1(13)：13-14
朱瑞良，张绍学，唐珂心，鸡波氏杆菌病的研究初报[J]，山东农业大学学报，1991，22(1)：92-94
朱妍，李素，王文成，等，猪瘟病毒石门株与兔化弱毒疫苗株之间抗原差异的单克隆抗体分析[J]，中国兽医科学，2008，38(4)：293-296
朱中武，陈可毅，张晓梅，产蛋鸡大肠杆菌性生殖道病感染途径探讨[J]，中国兽医科技，1995，25(2)：20-21
张焕容，副猪嗜血杆菌抗原性研究进展[J]，动物医学进展，2010，31(7)：91-94
张青霞，免疫-PCR技术的策略，北华大学学报，2003，4(3)：204-208
张绍学，朱瑞良，唐珂心，鸡波氏杆菌病的流行病学调查[J]，中国畜禽传染病，1992，(2)：25-26
张晓华，Robertson P， Austin B，弧菌标准菌株外膜蛋白的比较研究[J]，微生物学报，1997，37：449-454
张永和，中国免疫学杂志，1986，2，76
张志，崔治中，姜世金，等，鸡肿瘤病料中马立克氏病病毒和禽网状内皮增生病病毒混合感染的研究[J]，中国预防兽医学报，2003，4：274-278
赵春燕，曹娜娜，王海河，等，免疫PCR技术检测肠出血型大肠埃希菌O157：H7[J]，临床检验杂志，2010，28(5)：332-333
赵香汝，杨汉春，细菌外膜蛋白的研究现状[J]，中国兽医杂志，1997，12(23)：41-42
赵萍，储岳峰，高鹏程，等，免疫佐剂的研究现状及发展动态[J]，中国兽禽种业，2008，(13)：66-68
郑姬，免疫PCR检测HIV-1p24的实验研究[D]，第三军医大学，2007
庄国庆，孙淑红，崔治中，等，鸡马立克氏病毒和网状内皮增生病病毒共感染商品代肉鸡时的相互作用[J]，中国病毒学，2006，21(2)：157-162
曾常茜，晏舒，王玉国，等，免疫-PCR方法在血清抗Sm抗体中的应用[J]，中国免疫学杂志，2001，5：263-265
AllenRC,RogeljS,CordovaSE,etal,An immuno-PCR method for detecting Bacillus Thuringiensis Cry1Aetoxin,J immunol Methods,2006,20(308):109-115
Arp LH,Cheville NF, Tracheal lesions in young turkeys infected with Bordetella avium [J],Am J Vet Res,1984,(45):2196-2200
Arp LH,Brooks EE, An in vivo model for the study of Bordetella avium Adherence to trachealmucosa in turkeys[J], Am J Vet Res1986,47(12):2614-2617
Arp LH,Mcdonald SM, Influence of temperature on the growth of Bordetella avium in turkeysand in vitro[J], Avian Dis1985,29:1066-1077
Arp LH,Cheville NF, Tracheal lesions in young turkeys infected with Bordetella avium [J],Am J Vet Res1984,(45):2196-2200
Bailey N C,Fidanza V,Mayer R,Mazza G,Fougereau M,Bona C, Activation of clonesproducing self-reacrive antibodies by foreign antigen and anti-idio type antibodycarrying the internal image of the antigen[J],Clin Invest,1989,84(3):744-756
Bhara S,Kurichh A,Miranda R,et al, Heterosubtypic immunity against human influenzaAviruses,including recently emerged avianH5andH9viruses,induced by FLU-ISCOMvaccine in mice requires both cytotoxic T-lymphocyte and macrophage function[J], Cellimmunol,2001,211(2):143-153
Bradford MM, A rapid and sensitive method of the quantitation of microgran quantities ofprotein utilizing the principle of protein-dye binding[J], Anal Biochem1976,72:248-254
Braun V,Gaisser S,Hermann C,Kampfenkel K,Killmann H,Traub I, Energy-coupled transportacross the outer membrane of Escherichia coli: ExbB binds ExbD and TonB in vitro,andleucine132in the periplasmic region and aspartate25in the transmembrane region areimportant for ExbD activity [J], J Bacteriol,1996,178(10):2836-2845
Braun V,Killmann H,Benz R, Energy coupled transport through the outer Membrane ofEscherichiacoli small deletions in the gating loop convert the Fhu Atransport protein intoa diffusion[J], FEBSL,1994,346(1):59-64
Byrd W,Hooke A, Irnmunization with temperature sensitive mutants of aetino bacillus PleuroPneumoniae induces Protective hemolysin neutralizing antibodies in mice [J],CurrentMicrobiology,1997a,34:149-154
Gentry-Weeks CR,Cookson BT,Goldman WE,Rimler RB,Porter SB,Curtiss R3rd,Dermonecrotic toxin and tracheal cytotoxin,putative virulence factors of Bordetellaavium[J], Infect Immun,1988,56:1698-1707
Cimiotti W,Glunder G,Hinz KH, Survival of the bacterial turkey coryza agent[J], Vet Rec,110:304-306
Blore PJ,Slavik MF,Neighbor NK, Detection of antibody to Bordetella avium using a particleconcentration fluorescence immunoassay (PCFIA)[J], Avian Dis,1991,35(4):756-60
Collier AM,Peterson LP,Baseman JB, Pathogenesis of infection with Bordetella pertussis inhamster tracheal organ culture[J], J Infect Dis,1977,36:196-203
Colwell WM,Lukert PO, Effects of avian infectious bronchitis virus(IBV)on tracheal organcultures[J], Avian Dis,1969,13:888-894
Connell TD,Dickenson A,Martone AJ,et,al, Iron starvation of Bordetella avium stimulatesexpression of five outer membrane proteins and regulates a gene involved in acquiringiron from serum[J], Infect Immun,1998,66(8):3597-3605
Cook JKA,Darbyshire JH,Peters RW, The use of chicken tracheal organ cultures for isolationand assay of avian infectious bronchitis virus[J], Archives of Virology,1976,50:109-118
Cookson BT,Goldman WE, Tracheal cytotoxin:a conserved virulence determinant of allBordetella species[J], J,Cell,Biochem,1987,11:124
Cui Z,Sun S,Wang J, Reduced serologic response to Newcastle disease virus in broilerchickens exposed to a Chinese field strain of subgroup J avian leukosis virus[J], AvianDiseases,2006,50:191-195
Cutter DL,Luginbuhl GH, Characterization of sulfonamide Resistance determinants andrelatedness of Bordetella avium Rplasmids[J], Plasmid,1991,26:136-140
Darbyshire JH,Cook JKA,Peters RW, Comparative growth kinetic studies on avian infectiousbronchitis virus in different systems[J], J Comp Pathol,1975,85:623-630
Darbyshire JH,Cook JK A,Peters RW, Organ culture studies on the efficiency of infection ofchicken tissuses with avian infectious bronchitis virus[J], Brit J Exp Pathol,1976,57:443-454
Edens FW,McCorkle FW,Simmons DG,et,al, Effects of Bordetella avium on lymphoid tissuemonoamine concentrations in turkey poults[J], Avian Dis,1987,31:746-751
Edens FW,Yersin AG,Simmons DG, TryptopHan methylester modulationof poultre sponses toBordetella avium[J], Poult Sci,1999,78(3):327-335
Eisen D,Russell ED,Tymms M,et al, Random amplified polymorphic DNA and plasmidanalyses used in investigation of an outbreak of multiresistant Klebsicalla pneumoniae[J],J Clin Microbiol,1995,33:713-717
Elaichorni A, Pseudomonas aeruginosasero type O12outbreak studied by arbitrary primerPCR[J], J Clin Microbiol,1994,March;32(3):666-671
El-Sukhon SN,Musa A,Al-Attar M, Studies on the bacterial etiology of airsacculitis ofbroilers in northern and middle Jordan with special reference to Escherichiacoli,Ornithobacterium rhinotracheale,and Bordetella avium[J], Avian Dis,2002,46(3):605-612
Endoh M,Amitan M,Nakase Y, Effect of purified heat-labile toxin of Bordetellabronchiseptica on the peripHeral blood vessels in guinea pigs or suckling mice[J],Microbiol Immunol,1986,30:1327-1330
Endoh M,Nagai M,Nakase Y, Effect of Bordetella heat-labile toxin on perfused lungpreparations of guinea pigs[J], Jpn J Med Sci Biol,1986,39:249
ER Hendriekson,TM Truby,RD Joerger,WR Majarian,and RC Ebersole, High sensitivitymultianalyte immuno assay using eovalent DNA labeled antlbodies and Polymeraseehain reaction[J],Nueleie Aeids Res,1995,23:522-529
Evans DG,Evans,DJ,Tjoa W, Hemagglutinationof human group A erythrocytes byenterotoxigenic Escherichia coli isolated from adults with diarrhea:correlation withcolonization factor[J], Infect Immun,1977,18:330-337
Fantinatti F,Silveira WD,Castro AFP, Characteristics associated with pathogenicity of avianaemic Es2cherichiacoli strains[J],Vet Microbiol,1994,41:75-86
Fedorka-Cray PJ,Stine DL,Greenwald JM,et al, The importance of secreted virulence factorsin Actinobacillus pleuropneumoniae bacterin preparation a comparison[J],Vet Microbiol,1993,37(1~2):85~100
Ficken MD,Edwards JF,Lay JC, Clearance of bacteria in turkeys with Bordetella aviuminduced trachetitis[J], Vet Rec,1986,30:352-357
Ficken MD, Antibiotic aerosolization for treatment of alcaligenesrhintracheit[J], Avian Dis,1983,27:545-548
Galan J,Timoney JF, Molecular analysis of the Mprotein of Streptococcus equi and cloningand expression of the M protein gene in Escherichia coli[J], Infect Immun,1987,55:3181-3187
Ganett JK,Davis RB,Ragland WL, An enzyme-linked immunosorbent assay for detection ofantibody to avian encepHalo myelitis virus in chickens[J], Avian Dis,1984,28(1):117-30
Gentry-Weeks CR,Hultsch AL,Kelly SM,Keith JM,Curtiss R, Cloning and sequencing of agene encoding a21-kilodalton outer membrane protein from Bordetella avium andexpression of the gene in Salmonella typhimurium[J], J Bacteriol,1992,174:7729-7742
Glunder G,van der Ven H,Foulman A, Links Studies on the efficacy of different adjuvants inlive stock specific bacterial vaccines for turkeys against Bordetella infection and onset ofantibody titers in respect to the age of the turkey poults[J], Pol J Vet Sci,2004;7(2):77-81
Goldman WE,Klapper DG,Baseman JB, Detection,isolation,and analysis of a releasedBordetellapertussis product toxic to cultured tracheal cells[J], Infect Immun,1982,36:782-794
González CR,Isibasi A,Ortiz-Navarrete V,Paniagua J,García JA,Blanco F,Kumate J,Lymphocytic proliferative response to outer-membrane proteins isolated fromSalmonella[J], Microbiol Immunol,1993,37(10):793-799
Gueirard P,Weber C,Coustumier AL,Guiso N, Human Bordetella bronchiseptica InfectionRelated to Contact with Infected Animals:Persistence of Bacteria in Host[J], J ClinMicrobiol,1995,33(8):2002-2006
Guo DQ,Tang C,Hai Q,Shao GQ,Yue H, Development of a universal plate-agglutination testfor detecting Haemophilus parasuis[J], J Vet Sci,2010,11:355-357
Guo XL,Zheng HQ,Li XL,Li Y,Gu ZL,Zheng CS,Wei ZH,Wang JS,Zhou RY,Li LH,Genetic variation of major histocompatibility complex BLB2gene exon2in Hebeidomestic chicken[J], Res Vet Sci,2012,92:76-79
Hai-Yuan Chao,A highly sensitive immuno-polymerase chain reaction assay for Clostridiumbotulinum neurotoxin type A[J],Toxicon,2004,43:27-34
Hancock REW, Role of porins in outer membrane perme ability[J], J Bacteriol1987,169(3):929-933
Harrington AT,Castellanos JA,Ziedalski TM,Clarridge III JE,Cookson BT, Isolation ofBordetella avium and novel Bordetella strain from patients with respiratory disease[J],Emerg Infect Dis,2009,15:72-74
Hatfield RM,Morris BA,Henry RR, Development of an Enzyme-linked Immunosorbent Assayfor the detection of humoral antibody to pasteurella anatiestifer[J], Avian pathology,1987,16:123-140
Hellwig DH,Arp LH,Fagerland JA,A cOMPArison of outer membrane proteins and surfacecharacteristics of adhesive and non-adhesive phenotypes of Bordetella avium[J], AvianDis,1988,32:787-792
He Y,Keen JE,Westerman RB, Littledike ET, Kwang J, Monoclonal antibodies fordetection of the H7antigen of Escherichiacoli[J], Appl Environ Microbiol,1996,62(9):3325-3332
Hinz KH,Glunder G,Romer KJ, A cOMPArative study of avian Bordetella-like strains,Bordetella bronchiseptica,Alcaligenes faecalis and other related nonfermentablebacteria[J], Avian Pathol,1983,12:263-276
Hioe CE,QiuH,Chend Pei-De,et al, Comparison of adjuvant formulation for cytotoxic T cellinduction suing synthetic peptides[J], Vaccine,1996,14(5):412-418
Hryncewicz-Gwó d A,Jagielski T,Dobrowolska A,Szepietowski JC,Baran E,2011,Identification and differentiation of Trichophyton rubrum clinical isolates usingPCR-RFLP and RAPD methods[J], Eur J Clin Microbiol Infect Dis,30:727-731
Hsueh PR,Teng LJ,Yang PC,Chen YC,Ho SW,Luh KT, Persistence of a multidrug-resistantPseu-domon as aeruginosa clone in an intensive care burn unit[J], J Clin Micro,1998,36:1347-1351
Huning L,Susan E,Cordova TL,et al, A highly sensitive immuno-PCR assay detecting groupastreptoeoeus[J], Nueleci Aeids Res,2003,279:101-110
Jackwood MW,Hilt DA,Dunn PA, Observations on colonial phenotypic variation inBordetella avium[J], Avian Dis,1991,31:782-786
Jackwood MW,Saif YM, Efficacy of acommercial turky coryza vaccine in turkey poults[J],Avian Dis,1985,29:1130-1139
Jackwood MW,Hilt DA,Dunn PA, Observations on colonial pheno typic variation inBordetella avium[J], Avian Dis,1991,31:782-786
Jackwood MW,Saif YM,Coplin DL, Isolation and characterization of Bordetella avium[J],Avian Dis,1987,31:782-786
Joerger RD,Truby TM,Hendrickson ER, Aanalyte detection with DNA-labeled antibodies andpolymerase chain reaction[J],Clin Chem,1995,41(9):1371-1377
Kann RKC,MT Kyaw-Tanner,JM Seddon,PR Lehrbach,RJG Zwijnenberg and JMeers,Molecular subtyping of feline immunodeficiency virus from domestic cats inAustralia[J], Aust Vet J,2006,84:112-116
KapurVetal, Outer memberane protein pattern smark clones of Escherichiacoli O2and O78strains that cause avian septicemia[J], Infect Immun1992,60(4):1687-1691
Kattar MM,Chavez JF,Limaye AP,Rassoulian-Barrett SL,Yarfitz SL,Carlson LC,HouzeY,Swanzy S,Wood BL,Cookson BT, Application of16SrRNA gene sequencing toidentify Bordetella hinzii as the causative agent of fatal septicemia[J], J Clin Microbiol,2000,38(2):789-794
Kersters K,Hinz KH,Hertle A,Segers P,Lievens A,Siegmann O,De Ley J, Bordetella aviumsp,nov isolated from the respiratory tracts of turkeys and other birds[J], Int J SystBacteriol,1984,34:56-70
Korn A,Rajabi Z,Wassum B,Ruiner W,Nixdorff K, Enhancement of uptake of lipopolysaccharide inmacrop Hages by the major outer membrane protein OMPA of Gramnegative bacteria[J], Infect Immun,1995,2697-2705
KaiWei,Zhenhong Sun,Zhengui Yan,et al, Effects of Taishan Pinus massoniana plooenpolysaccharide on immune response of rabbit haemorrhagic disease tissue inactivatedvaccine and on production performance of Rex rabbits[J],Vaccine,2011,29:2530-2536
Kirby AE,King ND,Connell TD, RhuR,an extracytoplasmic function sigma factor activator,isessential for heme-dependent expression of the outer membrane heme and hemoproteinreceptor of Bordetella avium[J], Infect Immun,2004,72:896-907
Kume K,Nakai T,Samejima Y,Sugimoto C, Properties of dermonecrotic toxin prepared fromsonic extracts of Bordetella bronchiseptica[J], Infect Immun,1986,52:370-377
Lathrop JT,Wei BY,Touchie GA, Kadner RJ, Sequences of the Escherichiacoli BtuB proteinessential for its insertion and function in outer membrane[J], J bacteriol,1995,77(23):6810-6815
Leclair D,Pagotto F,Farber JM,Cadieux B,Austin JW, Comparison of DNA fingerprintingmethods for use in investigation of type E Botulism outbreaks in the Canadian arctic[J], JClin Microbiol,2006,44:1635-1644
Leenaars M,Koedom M A,Hendriksen C F M,et al, Immune responses and side effects of fivedifferent oil-based adjuvants in mice [J], Veterinary Immunology and Immunopathology,1998,61:291-304
Liang H,Cordova SE,Kieft TL,et al, A highly sensitive immuno-PCR assay for detectinggroup asttococcus[J], J Immunol Methods,2003,279(1-2):101-110
Loker SB,Temple LM,Preston A,The Bordetella avium BAV1965-1962fimbrial locus isregulated by temperature and produces fimbriae involved in adherence to turkey trachealtissue[J], Infect Immun,2011,79:2423-2429
Luginbuhl GH,Cutter D,Campodonico G,Peace J,Simmons DG, Plasmid DNA of virulentAlcaligenes faecalis[J], Am J Vet Res,1986,47:619-621
Mahhubani MH,Schaefer FW,Jones DD, Detection giardia in environmental waters byimmuno-PCR application methods[J],Curr Microbiol,1998,36(2):107
Maia M,Takahashi H,Adle K, Development of a two-site immuno-PCR assay for hepatitis Bsurface antigen[J],J Virol Methods,1995,52(3):273
Manoil C, A genetic approach to defining the sites of interaction of a membrane protein withdifferent external agents[J], Mol Biol,1983,169(2):507-515
Mattoo S,Cherry JD, Molecular Pathogenesis,Epidemiology,and Clinical Manifesta tions ofRespiratory Infections Due to Bordetella pertussis and Other Bordetella Subspecies[J],Clinical Microbiology Reviews,2005,18(2):326-382
Maurice JHM,Wolfhagen ADC, Rapid detection of toxigenic clostridium difficile in fetalsamples by magnetic immuno-PCR assay[J],J Clin Microbio,1994,32(7):629
McKie A,Samuel D,Cohen B,et al,Development of a quantitative immuno-PCR assay and itsuse to detect mumps-specific IgG in serum[J], Immunol Methods,2002,261(1-2):55-59
Moll A,Manning PA,Timis KN, Plasmid determined rsistance to serum bactericidal activity:amajor outer membrane protein the traT product,is responsible for plasmi specifiedserum resistance in E,coli[J], Infect Immun,1980,28:359-367
Morandi S,Brasca M,Lodi R,Brusetti L,Andrighetto C,Lombardi A, Biochemicalprofiles,restriction fragment length polymorphism (RFLP),random amplifiedpolymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis(MLVA) for typing Staphylococcus aureus isolated from dairy products[J], Res Vet Sci,2010,88:427-435
Moser SA,Heiss LN,Unance ER, Inter leukinl is linked to the respiratory epithelialcytopathology of pertussis[J], Infect Immun,1993,61(8):3123-3128
Murphy ER,Sacco RE,Dickenson A,Metzger DJ,Hu Y,Orndorff PE,Connell TD, BhuR,avirulence-associated outer membrane protein of Bordetella avium,is required for theacquisition of iron from heme and hemoproteins[J], Infect Immun,2002,70(10):5390-5403
Najiah M,Lee KL,Noorasikin H,Nadirah M,Lee SW, Phenotypic and genotypic characteristicsof Mycobacterium isolates from fighting fish Betta spp, in Malaysia[J], Res Vet Sci,2011,91:342-345
Nikaido H,Rosenbery EY, Porin channels in Escherichiacoli:studies with liposomesreconstituted from purified proteins[J],J Bacteriol,1983,153:241-252
Nolan LK, Wooley RE,Giddings CW,et,al, Characteization of an avirulent mutant of avirulentavian Escherichcoli isolate[J], Avian Dis,1994,38(1):146-150
Obayashi Y,Fujita J,Ichiyama S,et al, Genomic fingerprinting of Staphylococcus aureus byrandom amplified polymorphic DNA[J], Diagn Microbiol Infect Dis,1997,28(2):53-56
Odugbo MO,Musa U,Ekundayo SO,Okewole PA,Esilonu J, Bordetella avium infection inchickens and quail in Nigeria: preliminary investigations[J], Vet Res Commun,2006,30:1-5
Ongor H,Kalin R,Acik MN, Detection of Mycoplasma ovipneumoniae from goats with nasaldischarge by culture and polymerase chain reaction[J], Pak Vet J,2011,31:244-248
Onovian O,Robison T, Detection of antibody against M,mycoides subspmycoides in cattle byan enzyme-linked immunosorbent assay[J], Veterinary Record,1979,105:165-167
Parasadarao NV,Wass CA,Weiser JN,Stins MF,Huang SH,Kim KS, Outer membrane proteinAof Escherichiacoli contributes toinvasion of brain microvas cular endothelial cells[J],Infect Immun,1996,64(1):146-153
Pettersson A,Ley PVD,Poolman JT,Tommassen J, Molecular characterization of the98,2kilodalton Iron regulated outer membrane protein of neisseria meningitides[J], InfectImmun,1993,61(1):4724-4733
Poorbaghi SL,Mohammadi A,Asasi K,Molecular detection of avian infectious bronchitis virusserotypes from clinically suspected broiler chicken flocks in Fars province of Iran[J], PakVet J,2012,32:93-96
Raffel TR,Register KB,Marks SA,Temple L, Prevalence of Bordetella avium infection inselected wild and domesticated birds in the eastern USA[J], J Wildlife Dis,2002,38:40-46,
Rapp VJ,Munson RS,Ross RF, Outer membrane protein profiles of Haemophilus pleurop-neum oniae[J],Infect Immun1986,52(5):414-420
Rapp VJ,Ross RF,,Antibody response of swine to outer membrane components of Haem-ophilus pleuropneumoniae during infection[J], Infect Immun,1986a,54(3):751-760
Riain UN,Cormican MG, PCR based fingerprinting of Enterobacter cloacae[J], J Hosp Infect,1994,27:237～240
RimLer RB,Kunkle RA, Bacterin-induced protection of turkeys against fowl cholerafollowing infection with Bordetella avium[J], Avian Dis,1998,42(4):752-756
RimLer RB,Simmons DG,Gray JG, Transmission of acute respiratory disease (rhinotrachetitis)of turkeys[J], Avian Dis,1979,23:132-138
Rosqvist R,Bolin I,Wolfwatz H, Inhibitions of pHagocytsis in Yersini apseu do Tuber culosis:a virulent plasmid encoded ability involving the Yopzb protein[J], Infect Immun,1988,56(8):2139-2143
Roy S,Das AB,Ghosh AN,Biswas T, Purification,poreforming ability and antigenicrelatedness of the major outer membrane protein of shigelladys enteriae type[J], InfectImmun,1994,62(10):4333-4338
Ruzicka V,Marz W,Rass A, Immuno-PCR with a commercially available avidinsystem[J],Science,1993(5108),260:698-699,
Sacco RE,Register KB,Nordholm GE,Restriction enzyme analysis and ribotyping distinguishBordetella avium and Bordetella hinzii isolates[J], Epidemiol Infect,2000,124:83-90
Saif YM,Moorhead PD,Dearth RN,Jackwood DJ, Observations on Alcaligenes faecalisinfection in turkeys[J], Avian Dis,1980,(24):665-684
Sam brook J,Russell D W, Molecular Cloning:A Laboratory Manual[M],3rded, Translated byHUANG Pei-tang,WANG Jia-xi,ZHU Hou-chu,et al, Beijing: Science Press,2002:1474-1480(in Chinese)
SanoT,CasandraL,SmithCL,et al, Immuno-PCR:very sensitive antigen deteetion by means ofspecific antibody DNA conjugate[J],Seience,1992,258(5079):120-122
Sano T,Cantor CR,A streptavidin protein a chimera that allows one-step production of avariety of specific antibody conjugates[J],Bio Technology,1991,9(12):1378-1381
SanoT,Smith CL,Cantor CR, Immuno-PCR:very sensitive antigen deteetion by means ofsPecific antibody DNA eonjugates[J],Science,1992,258(5079):120-122
S,B, Stockwell,H, Kuzmiak-Ngiam,N,M, Beach,et al, The autotransporter protein fromBordetella avium,Baa1,is involved in host cell attachment[J], MicrobiologicalResearch,2011,167:55-60
Sherman P,Cockerill F,Soni R,Brunton J, Outer membrane are competitive inhibitor ofEscherichiacoli O157BH7adherence to epithelial cell[J], Infect Immun,1991,59(3):890-899
Shelton CB,Temple LM,Orndorff PE, Use of bacterio-phage Ba1to identify propertiesassociated with Bordetella avium virulence[J], Infect Immun,2002,70(3):1219-1224
Shin EK,Seo YS,Han JH,Hahn TW,Diversity of swine Bordetella bronchiseptica isolatesevaluated by RAPD analysis and PFGE[J], J Vet Sci,2007,8:65-73,
Simmons DG,Gray JG, Transmission of acute respiratory disease (rhinotrarhetitis) ofturkeys[J], Avian Dis,1979,23:132-138
Simmons DJ,Dee JC,Rose LP, A heat-stable toxin isolated from the turkeycoryza agentBordetella avium[J], Avian Dis,1986,30:761-765
Skeeles,JK,Swafford WS,Wages DP,Hellwig HM,Slavik MF,Beasley JN,Houghten GE,BlorePJ,Crawford D, Studies on the use of a long-acting oxytetracycl;ine in turkeys: Efficacyagainst experimental infections with Alcaligenesfaecalis and Pasteurella multocida[J],Avian Dis,1983,27:1126-1130
Sonntang I,Schwarz H,Hirota Y,Henning U, Cell envelope and shape of Escherichiacoli:multiple mutants missing the outer membrane lipoprotein andother major outermembrane proteins [J], J Bacteriol,1978,136(1):280-285
Spears PA,Temple LM,Miyamoto DM,Maskell DJ,Orndorff PE, Unexpected similaritiesbetween Bordetella avium and other pathogenic bordetellae[J], Infect Immun,2003,71:2591-2597
Sperl J,Paliwal V,Ramabhadran,et al, Soluble Teellreee Ptors:detection and antitative assayin luidphase via ELISA or inununo-PCR[J], J immunol Methods,1995,186:181-194
Spilker T,Liwienski AA,Lipuma JJ, Identification of Bordetella spp, in respiratory specimensfrom individuals with cystic fibrosis[J], Clin Microbiol Infect,2008,14:504-506
Sun S,Sun A,Cui Z, Epidemiological and pathological studies of subgroup J avian leukosisvirus infections in Chinese local yellow chickens[J], Avian Pathol,2007,36(3):221-226
Suresh P,Arp LH, A monoclonal antibody-based latex bead agglutination test for the detectionof Bordetella avium[J], AvianDis,1993Jul-Sep;37(3):767-72
Tazumi A,Petry S,Hayashi K,Moore JE,Millar BC,Matsuda M, Molecular identification andcharacterization of the intervening sequences (IVSs) within23S ribosomal RNA (rRNA)genes of Taylorella asinigenitalis isolated in France[J], Res Vet Sci,2012,92,45-52
Temple LM,Miyamoto DM,Mehta M,Capitini CM,Stetina VS,Barnes HJ, ChristensenVL,Horton JH,Spears PA,Orndorff PE, Identification and characterization of twoBordetella avium gene products required for hemagglutination[J], Infect Immun,2010,78:2370-2376
Thanassi DG,Suh GSB,Nikaido H, Role of outer membrane barrier inefflux mediatedtetracycline resistance of Escherichiacoli[J], J Bacteriol,1995,177(4):998-1007,
Truscott WM,Hirsh DC, Demonstration of an outer membrane proteins with anti-pHagocyticfrom Pateurel lamultocida of avian origin[J], Infect Immun,1988,56(6):1538-1544
Van Couwenberghe CJ,Cohen SH,Tang YJ,Gumerlock PH,Silva J Jr, Genomicfingerprinting of epidemic and endemic trains of Stenotrophomonsa maltophilia(formerly Xanthomnsa mallophilia) by arbitrarily primed PCR[J], J ClinMicrobiol,1995,33:1289-1294
Vidotto MC,Muller EE,de Freitas JC,Alfieri AA,Guimar es IG,Santos DS, Virulence factorsof avian Escherichia coli[J], Avian Dis,1990,34:531-538
Vordermeier HM,Hoffmann P,Gombert FO,Jung G,Bessler WG, Synthetic peptide segmentfrom the Escherichiacoli porin OmpF constitute eukocyte activators[J], Infect Immun,1990,(8):2719-2724
Weiser JN,Gotschlich EC, Outer membrane proteinA(OMPA) contributes to serum resistanceand pathogenicity of Escherichiacolik21[J], Infect Immun,1991,59(7):2252-2258
Wikler MA, Methods for dilution antimicrobial susceptibility tests for bacteria that growaerobically: approved standard[J],Clinical and Laboratory Standards Institute,2006,26(2):431-442
Wulff G,Gram L,Ahrens P,Vogel BF, One group of genetically similar Listeria monocytogenesstrains frequently dominates and persists in several fish slaughter-and smokehouses[J],Appl Environ Microbiol,2006,72:4313-4322
XU Y J,VAN HUYSTEE R B,Identification of an antigenic determination anionic peanutperoxidase by monoclonal antibodies[J],J Exper Botany,1991,42(24):1-11
Yang X,Hou L,Ye J,He Q,Cao S, Detection of porcine circovirus type2(PCV2) in mosquitoesfrom pig farms by PCR[J], Pak Vet J,2012,32:134-135
Yersin,AG,Edens FW,Simmons DG, TryptopHan2,3-dioxyge-nase activity in turkey poultsinfected with Bordetella avium[J], Comp Biochem Physio,1990,197B:755-760
Yiang Z,Ertl HC, Manipulation of the immune response to a plasmidoncode viral antigen bycoinoculation with plasmids expressing cytokines [J],Immunity,1995,2:129-135
Yi Cao,Kathrin Kopplow,Guang Yaw Liu,et al.,In situ immuno-PCR to detect antigens [J],Lancet,2000,356(9234):1002-1003
Zhou H,Fisher RJ,Papas TS,Universal immuno-PCR for ultra-sensitive target proteindetection [J], Nucleic Acids Res,1993,21(25):6038-6041
BW卡尔尼克，禽病学（第十版），北京：中国农业出版社，1999，347-365
曹娜娜，免疫PCR法检测肠出血性大肠埃希菌O157:H[D]，吉林大学，2010
曹军平，闵桂玲，两种简易高效的单克隆抗体提纯方法[J]，单克隆抗体通讯，1995，11(2)：52-54
陈佳，应用蛋白芯片和免疫PCR技术高效检测汉滩病毒的研究[D]，中国科学院，硕士学位论文，2005
陈敏，史久华，免疫增强剂的研究进展[J]，国外医学预防、诊断、治疗用生物制品分册，2001，24：65-67
高崧，刘秀梵，张如宽，一种快速提取禽源性大肠埃希氏菌外膜蛋白的方法[J]，微生物学通报，1996，23(2)：122-123
高崧，赵坤，徐大生，等，间接ELISA测定禽源性大肠杆菌膜蛋白抗体方法的建立[J]，中国兽医学报，1997，19(4)：326-328
黄昊洋，李逄慧，罗超，等，一例禽波氏杆菌病的治疗[J]，中国畜禽种业，2008，49-50
郭春祥，郭锡琼，介绍一种简单、快速、高效的辣根过氧化物酶标记抗体的过碘酸钠法[J]，上海免疫学杂志，2003，3(2)：55-58
华海涌，周永华，薛忠权，双抗体夹心免疫S678检测弓形虫循环抗原的实验研究[J]，中国现代医药杂志，2005，12(7)：1-2
胡晓娜，朱瑞良，刘红珍，等，禽波氏杆菌外膜蛋白的提取及其免疫原性的检测[J]，微生物学报，2007，47(4)：714-717
金文杰，崔治中，刘岳龙，等，传染性法氏囊病病料中MDV、CAV、REV的共感染检测[J]，中国兽医学报，2001，21(1)：6-9
蒋成淦，酶免疫测定法，北京：人民卫生出版社，1984，75~85；133~135
赖平安，杜春辉，鸡大肠杆菌致病力与某些生物特性的相关性探讨[J]，中国兽医科技，1992，22(8)：26-29
李福胜，赵风兰，胡军，鼠伤寒杆菌外膜蛋白免疫原性及免疫保护性研究[J]，中华微生物和免疫学杂志，1993，13(3)：156-158
李桂杰，刘思当，朱瑞良，等，禽波氏杆菌内毒素致病性的研究[J]，畜牧兽医学报，2000，31(4)：349-352
李亮，林跃智，那雷，等，猴免疫缺陷病毒p27蛋白单克隆抗体结合抗原表位的鉴定[J]，中国预防兽医学报，2011，33(9)：738-741
李康然，韦平，梁梅芳，用气管环培养中和试验对鸡传染性支气管炎病毒进行血清定型[J]，广西农学院学报，1991，10(3)：1-6
李新苍，朱瑞良，路建彪，等，AA肉鸡群中免疫抑制性病毒分子流行病学调查及对疫苗免疫效果影响分析[J]，中国兽医学报，2008，28(10)：1141-1144
廖立新，曹郁生，李国辉，等，一种快速粗提绿脓杆菌外膜蛋白的方法[J]，江西医学院学报，2003，42：60-61
廖立新，赵英，李国辉，等，革兰氏阴性杆菌的敏感性分析[J]，江西医学院学报，2001，41：117-118
梁米芳，宋干，杭长寿，等，用单克隆抗体分析流行出血热病毒的核蛋白抗原位点[J]，病毒学报，1989，5(1)：24-30
凌伟，沈茜，王靖，等，免疫一PcR法检测柯萨奇病毒B组抗原的实验研究[J]，第二军医大学学报，2001，22(3)：264-266
刘红芹，规模化种鸡场胚胎疾病病原学研究及禽波氏杆菌16SrRNA基因序列分析[D]，山东农业大学，2006
刘冠华，禽波氏杆菌OMP单克隆抗体的制备及其生物学特性研究[D]，山东农业大学，2009
刘晓波，蔡美英，王霞，一种简单实用纯化腹水McAb方法-辛酸/硫酸铵法[J]，华西医科大学学报，1999，30(4)：455-456
陆承平，兽医微生物学[M]，北京：中国农业出版社，第三版，2002：275-277
陆德源，医学微生物学[M]，第3版。北京，人民卫生出版社，1989：7-26
马荣德，禽波氏杆菌的生物学特性研究及血清分型的初步探索[D]，山东农业大学，2010
孟珊珊，崔治中，孙淑红，REV和ALV-J共感染鸡病毒血症及抗体反应的相互影响[J]，
中国兽医学报，2006，26：363-366
穆杨，周丽梅，侯宏虎，等，三种佐剂对鸡体液免疫影响的比较[J]，动物医学进展，2007，28(10)：37-39
乔生军，吴自荣，戚蓓静，等，中国科学，C辑，1998，28(1)：77-82
宋云扬，吴方晖，张靖，等，夹心免疫PCR方法检测金葡菌肠毒素B[J]，中国公共卫生，2004，20(7)：832-833
史俊岩，牟玲，罗恩杰，等，免疫-PCR检测肾综合征出血热患者血清中抗核衣壳蛋白抗体方法的建立[J]，中国人兽共患病杂志，2004，20(5)：397-400
王汉雷，用核酸探针和分子斑点杂交技术检测病毒的核酸[D]，山东农业大学，2007
王丽娜，免疫-PCR的研究进展[J]，国外医学临床生物化学与检验学分册，2003，24(6)：335-336
王晶钰，李健强，张国祥，等，鸡波德特氏菌病的研究[J]，西北农业大学学报，1997，25(4)：70-71
王世若，王兴龙，韩文瑜，等，动物现代免疫学[M]，吉林：科学技术出版社，2001：431-442
王雪敏，崔岩，禽波氏杆菌病研究进展[J]，动物医学，2004，21(1)：56-57
王玉燕，朱瑞良，禽波氏杆菌病原性研究及防治初探[J]，中国预防兽医学报，2001，23：117-120
翁立雪，鸡主要胚胎性疫病病原人工共感染的试验研究[D]，山东农业大学，2009
吴自荣，陈阜东，戚蓓静，通用型免疫-聚合酶链反应系统的建立及应用[J]，中华医学检验杂志，1997，20(3)：147
夏文浪，马宝骊，黄冬生，等，空肠弯曲菌67KD外膜蛋白的提取及其淋巴细胞的多克隆激活作用[J]，中国免疫学杂志，1992，8(1)：14-17
徐肇华，戊二醛前处理的聚苯乙烯塑料小孔在二点一步酶免疫法中的应用[J]，上海医学检验杂志，1989，2：104-106
严杰，方平楚，幽门螺杆菌脂多糖生物学活性的研究[J]，中华微生物和免疫学杂志，1994，14(3)：196
杨本升，刘玉斌，苟仕金，动物微生物学[M]，长春：吉林科学技术出社，1995，550
杨春晓，禽波氏杆菌OMP标记抗体检测方法的建立及相关毒素生物学特性的检测[D]，山东农业大学，2008
杨金耀，陆广富，王建业，等，32株禽波氏杆菌分离株特性的初步研究[J]，中国家禽，2008，30(6)：18-19
杨萍萍，禽波氏杆菌的定植规律、基因分型以及与REV共感染的协同致病作用研究[D]，山东农业大学，2012
庄国宏，朱国强，周继红，等，禽波氏杆菌病病原性的研究[J]，中国畜禽传染病，1998，(2)：71-75
朱瑞良，雏鸡禽波氏杆菌病的诊断[J]，畜牧与兽医，1993，(3)：124-125
朱瑞良，牛钟相，张绍学，病鸡眼中禽波氏杆菌的分离鉴定[J]，中国兽医科技，1994，24(4)：25-26
朱瑞良，万连英，李洁珍，广东省鸡波氏杆菌病血清学调查[J]，吉林畜牧兽医，1994，(5)：39-40
朱瑞良，张绍学，唐珂心，山鸡波氏杆菌病的诊断[J]，中国兽医杂志，1994，1(13)：13-14
朱瑞良，张绍学，唐珂心，鸡波氏杆菌病的研究初报[J]，山东农业大学学报，1991，22(1)：92-94
朱妍，李素，王文成，等，猪瘟病毒石门株与兔化弱毒疫苗株之间抗原差异的单克隆抗体分析[J]，中国兽医科学，2008，38(4)：293-296
朱中武，陈可毅，张晓梅，产蛋鸡大肠杆菌性生殖道病感染途径探讨[J]，中国兽医科技，1995，25(2)：20-21
张焕容，副猪嗜血杆菌抗原性研究进展[J]，动物医学进展，2010，31(7)：91-94
张青霞，免疫-PCR技术的策略，北华大学学报，2003，4(3)：204-208
张绍学，朱瑞良，唐珂心，鸡波氏杆菌病的流行病学调查[J]，中国畜禽传染病，1992，(2)：25-26
张晓华，Robertson P， Austin B，弧菌标准菌株外膜蛋白的比较研究[J]，微生物学报，1997，37：449-454
张永和，中国免疫学杂志，1986，2，76
张志，崔治中，姜世金，等，鸡肿瘤病料中马立克氏病病毒和禽网状内皮增生病病毒混合感染的研究[J]，中国预防兽医学报，2003，4：274-278
赵春燕，曹娜娜，王海河，等，免疫PCR技术检测肠出血型大肠埃希菌O157：H7[J]，临床检验杂志，2010，28(5)：332-333
赵香汝，杨汉春，细菌外膜蛋白的研究现状[J]，中国兽医杂志，1997，12(23)：41-42
赵萍，储岳峰，高鹏程，等，免疫佐剂的研究现状及发展动态[J]，中国兽禽种业，2008，(13)：66-68
郑姬，免疫PCR检测HIV-1p24的实验研究[D]，第三军医大学，2007
庄国庆，孙淑红，崔治中，等，鸡马立克氏病毒和网状内皮增生病病毒共感染商品代肉鸡时的相互作用[J]，中国病毒学，2006，21(2)：157-162
曾常茜，晏舒，王玉国，等，免疫-PCR方法在血清抗Sm抗体中的应用[J]，中国免疫学杂志，2001，5：263-265
AllenRC,RogeljS,CordovaSE,etal,An immuno-PCR method for detecting Bacillus Thuringiensis Cry1Aetoxin,J immunol Methods,2006,20(308):109-115
Arp LH,Cheville NF, Tracheal lesions in young turkeys infected with Bordetella avium [J],Am J Vet Res,1984,(45):2196-2200
Arp LH,Brooks EE, An in vivo model for the study of Bordetella avium Adherence to trachealmucosa in turkeys[J], Am J Vet Res1986,47(12):2614-2617
Arp LH,Mcdonald SM, Influence of temperature on the growth of Bordetella avium in turkeysand in vitro[J], Avian Dis1985,29:1066-1077
Arp LH,Cheville NF, Tracheal lesions in young turkeys infected with Bordetella avium [J],Am J Vet Res1984,(45):2196-2200
Bailey N C,Fidanza V,Mayer R,Mazza G,Fougereau M,Bona C, Activation of clonesproducing self-reacrive antibodies by foreign antigen and anti-idio type antibodycarrying the internal image of the antigen[J],Clin Invest,1989,84(3):744-756
Bhara S,Kurichh A,Miranda R,et al, Heterosubtypic immunity against human influenzaAviruses,including recently emerged avianH5andH9viruses,induced by FLU-ISCOMvaccine in mice requires both cytotoxic T-lymphocyte and macrophage function[J], Cellimmunol,2001,211(2):143-153
Bradford MM, A rapid and sensitive method of the quantitation of microgran quantities ofprotein utilizing the principle of protein-dye binding[J], Anal Biochem1976,72:248-254
Braun V,Gaisser S,Hermann C,Kampfenkel K,Killmann H,Traub I, Energy-coupled transportacross the outer membrane of Escherichia coli: ExbB binds ExbD and TonB in vitro,andleucine132in the periplasmic region and aspartate25in the transmembrane region areimportant for ExbD activity [J], J Bacteriol,1996,178(10):2836-2845
Braun V,Killmann H,Benz R, Energy coupled transport through the outer Membrane ofEscherichiacoli small deletions in the gating loop convert the Fhu Atransport protein intoa diffusion[J], FEBSL,1994,346(1):59-64
Byrd W,Hooke A, Irnmunization with temperature sensitive mutants of aetino bacillus PleuroPneumoniae induces Protective hemolysin neutralizing antibodies in mice [J],CurrentMicrobiology,1997a,34:149-154
Gentry-Weeks CR,Cookson BT,Goldman WE,Rimler RB,Porter SB,Curtiss R3rd,Dermonecrotic toxin and tracheal cytotoxin,putative virulence factors of Bordetellaavium[J], Infect Immun,1988,56:1698-1707
Cimiotti W,Glunder G,Hinz KH, Survival of the bacterial turkey coryza agent[J], Vet Rec,110:304-306
Blore PJ,Slavik MF,Neighbor NK, Detection of antibody to Bordetella avium using a particleconcentration fluorescence immunoassay (PCFIA)[J], Avian Dis,1991,35(4):756-60
Collier AM,Peterson LP,Baseman JB, Pathogenesis of infection with Bordetella pertussis inhamster tracheal organ culture[J], J Infect Dis,1977,36:196-203
Colwell WM,Lukert PO, Effects of avian infectious bronchitis virus(IBV)on tracheal organcultures[J], Avian Dis,1969,13:888-894
Connell TD,Dickenson A,Martone AJ,et,al, Iron starvation of Bordetella avium stimulatesexpression of five outer membrane proteins and regulates a gene involved in acquiringiron from serum[J], Infect Immun,1998,66(8):3597-3605
Cook JKA,Darbyshire JH,Peters RW, The use of chicken tracheal organ cultures for isolationand assay of avian infectious bronchitis virus[J], Archives of Virology,1976,50:109-118
Cookson BT,Goldman WE, Tracheal cytotoxin:a conserved virulence determinant of allBordetella species[J], J,Cell,Biochem,1987,11:124
Cui Z,Sun S,Wang J, Reduced serologic response to Newcastle disease virus in broilerchickens exposed to a Chinese field strain of subgroup J avian leukosis virus[J], AvianDiseases,2006,50:191-195
Cutter DL,Luginbuhl GH, Characterization of sulfonamide Resistance determinants andrelatedness of Bordetella avium Rplasmids[J], Plasmid,1991,26:136-140
Darbyshire JH,Cook JKA,Peters RW, Comparative growth kinetic studies on avian infectiousbronchitis virus in different systems[J], J Comp Pathol,1975,85:623-630
Darbyshire JH,Cook JK A,Peters RW, Organ culture studies on the efficiency of infection ofchicken tissuses with avian infectious bronchitis virus[J], Brit J Exp Pathol,1976,57:443-454
Edens FW,McCorkle FW,Simmons DG,et,al, Effects of Bordetella avium on lymphoid tissuemonoamine concentrations in turkey poults[J], Avian Dis,1987,31:746-751
Edens FW,Yersin AG,Simmons DG, TryptopHan methylester modulationof poultre sponses toBordetella avium[J], Poult Sci,1999,78(3):327-335
Eisen D,Russell ED,Tymms M,et al, Random amplified polymorphic DNA and plasmidanalyses used in investigation of an outbreak of multiresistant Klebsicalla pneumoniae[J],J Clin Microbiol,1995,33:713-717
Elaichorni A, Pseudomonas aeruginosasero type O12outbreak studied by arbitrary primerPCR[J], J Clin Microbiol,1994,March;32(3):666-671
El-Sukhon SN,Musa A,Al-Attar M, Studies on the bacterial etiology of airsacculitis ofbroilers in northern and middle Jordan with special reference to Escherichiacoli,Ornithobacterium rhinotracheale,and Bordetella avium[J], Avian Dis,2002,46(3):605-612
Endoh M,Amitan M,Nakase Y, Effect of purified heat-labile toxin of Bordetellabronchiseptica on the peripHeral blood vessels in guinea pigs or suckling mice[J],Microbiol Immunol,1986,30:1327-1330
Endoh M,Nagai M,Nakase Y, Effect of Bordetella heat-labile toxin on perfused lungpreparations of guinea pigs[J], Jpn J Med Sci Biol,1986,39:249
ER Hendriekson,TM Truby,RD Joerger,WR Majarian,and RC Ebersole, High sensitivitymultianalyte immuno assay using eovalent DNA labeled antlbodies and Polymeraseehain reaction[J],Nueleie Aeids Res,1995,23:522-529
Evans DG,Evans,DJ,Tjoa W, Hemagglutinationof human group A erythrocytes byenterotoxigenic Escherichia coli isolated from adults with diarrhea:correlation withcolonization factor[J], Infect Immun,1977,18:330-337
Fantinatti F,Silveira WD,Castro AFP, Characteristics associated with pathogenicity of avianaemic Es2cherichiacoli strains[J],Vet Microbiol,1994,41:75-86
Fedorka-Cray PJ,Stine DL,Greenwald JM,et al, The importance of secreted virulence factorsin Actinobacillus pleuropneumoniae bacterin preparation a comparison[J],Vet Microbiol,1993,37(1~2):85~100
Ficken MD,Edwards JF,Lay JC, Clearance of bacteria in turkeys with Bordetella aviuminduced trachetitis[J], Vet Rec,1986,30:352-357
Ficken MD, Antibiotic aerosolization for treatment of alcaligenesrhintracheit[J], Avian Dis,1983,27:545-548
Galan J,Timoney JF, Molecular analysis of the Mprotein of Streptococcus equi and cloningand expression of the M protein gene in Escherichia coli[J], Infect Immun,1987,55:3181-3187
Ganett JK,Davis RB,Ragland WL, An enzyme-linked immunosorbent assay for detection ofantibody to avian encepHalo myelitis virus in chickens[J], Avian Dis,1984,28(1):117-30
Gentry-Weeks CR,Hultsch AL,Kelly SM,Keith JM,Curtiss R, Cloning and sequencing of agene encoding a21-kilodalton outer membrane protein from Bordetella avium andexpression of the gene in Salmonella typhimurium[J], J Bacteriol,1992,174:7729-7742
Glunder G,van der Ven H,Foulman A, Links Studies on the efficacy of different adjuvants inlive stock specific bacterial vaccines for turkeys against Bordetella infection and onset ofantibody titers in respect to the age of the turkey poults[J], Pol J Vet Sci,2004;7(2):77-81
Goldman WE,Klapper DG,Baseman JB, Detection,isolation,and analysis of a releasedBordetellapertussis product toxic to cultured tracheal cells[J], Infect Immun,1982,36:782-794
González CR,Isibasi A,Ortiz-Navarrete V,Paniagua J,García JA,Blanco F,Kumate J,Lymphocytic proliferative response to outer-membrane proteins isolated fromSalmonella[J], Microbiol Immunol,1993,37(10):793-799
Gueirard P,Weber C,Coustumier AL,Guiso N, Human Bordetella bronchiseptica InfectionRelated to Contact with Infected Animals:Persistence of Bacteria in Host[J], J ClinMicrobiol,1995,33(8):2002-2006
Guo DQ,Tang C,Hai Q,Shao GQ,Yue H, Development of a universal plate-agglutination testfor detecting Haemophilus parasuis[J], J Vet Sci,2010,11:355-357
Guo XL,Zheng HQ,Li XL,Li Y,Gu ZL,Zheng CS,Wei ZH,Wang JS,Zhou RY,Li LH,Genetic variation of major histocompatibility complex BLB2gene exon2in Hebeidomestic chicken[J], Res Vet Sci,2012,92:76-79
Hai-Yuan Chao,A highly sensitive immuno-polymerase chain reaction assay for Clostridiumbotulinum neurotoxin type A[J],Toxicon,2004,43:27-34
Hancock REW, Role of porins in outer membrane perme ability[J], J Bacteriol1987,169(3):929-933
Harrington AT,Castellanos JA,Ziedalski TM,Clarridge III JE,Cookson BT, Isolation ofBordetella avium and novel Bordetella strain from patients with respiratory disease[J],Emerg Infect Dis,2009,15:72-74
Hatfield RM,Morris BA,Henry RR, Development of an Enzyme-linked Immunosorbent Assayfor the detection of humoral antibody to pasteurella anatiestifer[J], Avian pathology,1987,16:123-140
Hellwig DH,Arp LH,Fagerland JA,A cOMPArison of outer membrane proteins and surfacecharacteristics of adhesive and non-adhesive phenotypes of Bordetella avium[J], AvianDis,1988,32:787-792
He Y,Keen JE,Westerman RB, Littledike ET, Kwang J, Monoclonal antibodies fordetection of the H7antigen of Escherichiacoli[J], Appl Environ Microbiol,1996,62(9):3325-3332
Hinz KH,Glunder G,Romer KJ, A cOMPArative study of avian Bordetella-like strains,Bordetella bronchiseptica,Alcaligenes faecalis and other related nonfermentablebacteria[J], Avian Pathol,1983,12:263-276
Hioe CE,QiuH,Chend Pei-De,et al, Comparison of adjuvant formulation for cytotoxic T cellinduction suing synthetic peptides[J], Vaccine,1996,14(5):412-418
Hryncewicz-Gwó d A,Jagielski T,Dobrowolska A,Szepietowski JC,Baran E,2011,Identification and differentiation of Trichophyton rubrum clinical isolates usingPCR-RFLP and RAPD methods[J], Eur J Clin Microbiol Infect Dis,30:727-731
Hsueh PR,Teng LJ,Yang PC,Chen YC,Ho SW,Luh KT, Persistence of a multidrug-resistantPseu-domon as aeruginosa clone in an intensive care burn unit[J], J Clin Micro,1998,36:1347-1351
Huning L,Susan E,Cordova TL,et al, A highly sensitive immuno-PCR assay detecting groupastreptoeoeus[J], Nueleci Aeids Res,2003,279:101-110
Jackwood MW,Hilt DA,Dunn PA, Observations on colonial phenotypic variation inBordetella avium[J], Avian Dis,1991,31:782-786
Jackwood MW,Saif YM, Efficacy of acommercial turky coryza vaccine in turkey poults[J],Avian Dis,1985,29:1130-1139
Jackwood MW,Hilt DA,Dunn PA, Observations on colonial pheno typic variation inBordetella avium[J], Avian Dis,1991,31:782-786
Jackwood MW,Saif YM,Coplin DL, Isolation and characterization of Bordetella avium[J],Avian Dis,1987,31:782-786
Joerger RD,Truby TM,Hendrickson ER, Aanalyte detection with DNA-labeled antibodies andpolymerase chain reaction[J],Clin Chem,1995,41(9):1371-1377
Kann RKC,MT Kyaw-Tanner,JM Seddon,PR Lehrbach,RJG Zwijnenberg and JMeers,Molecular subtyping of feline immunodeficiency virus from domestic cats inAustralia[J], Aust Vet J,2006,84:112-116
KapurVetal, Outer memberane protein pattern smark clones of Escherichiacoli O2and O78strains that cause avian septicemia[J], Infect Immun1992,60(4):1687-1691
Kattar MM,Chavez JF,Limaye AP,Rassoulian-Barrett SL,Yarfitz SL,Carlson LC,HouzeY,Swanzy S,Wood BL,Cookson BT, Application of16SrRNA gene sequencing toidentify Bordetella hinzii as the causative agent of fatal septicemia[J], J Clin Microbiol,2000,38(2):789-794
Kersters K,Hinz KH,Hertle A,Segers P,Lievens A,Siegmann O,De Ley J, Bordetella aviumsp,nov isolated from the respiratory tracts of turkeys and other birds[J], Int J SystBacteriol,1984,34:56-70
Korn A,Rajabi Z,Wassum B,Ruiner W,Nixdorff K, Enhancement of uptake of lipopolysaccharide inmacrop Hages by the major outer membrane protein OMPA of Gramnegative bacteria[J], Infect Immun,1995,2697-2705
KaiWei,Zhenhong Sun,Zhengui Yan,et al, Effects of Taishan Pinus massoniana plooenpolysaccharide on immune response of rabbit haemorrhagic disease tissue inactivatedvaccine and on production performance of Rex rabbits[J],Vaccine,2011,29:2530-2536
Kirby AE,King ND,Connell TD, RhuR,an extracytoplasmic function sigma factor activator,isessential for heme-dependent expression of the outer membrane heme and hemoproteinreceptor of Bordetella avium[J], Infect Immun,2004,72:896-907
Kume K,Nakai T,Samejima Y,Sugimoto C, Properties of dermonecrotic toxin prepared fromsonic extracts of Bordetella bronchiseptica[J], Infect Immun,1986,52:370-377
Lathrop JT,Wei BY,Touchie GA, Kadner RJ, Sequences of the Escherichiacoli BtuB proteinessential for its insertion and function in outer membrane[J], J bacteriol,1995,77(23):6810-6815
Leclair D,Pagotto F,Farber JM,Cadieux B,Austin JW, Comparison of DNA fingerprintingmethods for use in investigation of type E Botulism outbreaks in the Canadian arctic[J], JClin Microbiol,2006,44:1635-1644
Leenaars M,Koedom M A,Hendriksen C F M,et al, Immune responses and side effects of fivedifferent oil-based adjuvants in mice [J], Veterinary Immunology and Immunopathology,1998,61:291-304
Liang H,Cordova SE,Kieft TL,et al, A highly sensitive immuno-PCR assay for detectinggroup asttococcus[J], J Immunol Methods,2003,279(1-2):101-110
Loker SB,Temple LM,Preston A,The Bordetella avium BAV1965-1962fimbrial locus isregulated by temperature and produces fimbriae involved in adherence to turkey trachealtissue[J], Infect Immun,2011,79:2423-2429
Luginbuhl GH,Cutter D,Campodonico G,Peace J,Simmons DG, Plasmid DNA of virulentAlcaligenes faecalis[J], Am J Vet Res,1986,47:619-621
Mahhubani MH,Schaefer FW,Jones DD, Detection giardia in environmental waters byimmuno-PCR application methods[J],Curr Microbiol,1998,36(2):107
Maia M,Takahashi H,Adle K, Development of a two-site immuno-PCR assay for hepatitis Bsurface antigen[J],J Virol Methods,1995,52(3):273
Manoil C, A genetic approach to defining the sites of interaction of a membrane protein withdifferent external agents[J], Mol Biol,1983,169(2):507-515
Mattoo S,Cherry JD, Molecular Pathogenesis,Epidemiology,and Clinical Manifesta tions ofRespiratory Infections Due to Bordetella pertussis and Other Bordetella Subspecies[J],Clinical Microbiology Reviews,2005,18(2):326-382
Maurice JHM,Wolfhagen ADC, Rapid detection of toxigenic clostridium difficile in fetalsamples by magnetic immuno-PCR assay[J],J Clin Microbio,1994,32(7):629
McKie A,Samuel D,Cohen B,et al,Development of a quantitative immuno-PCR assay and itsuse to detect mumps-specific IgG in serum[J], Immunol Methods,2002,261(1-2):55-59
Moll A,Manning PA,Timis KN, Plasmid determined rsistance to serum bactericidal activity:amajor outer membrane protein the traT product,is responsible for plasmi specifiedserum resistance in E,coli[J], Infect Immun,1980,28:359-367
Morandi S,Brasca M,Lodi R,Brusetti L,Andrighetto C,Lombardi A, Biochemicalprofiles,restriction fragment length polymorphism (RFLP),random amplifiedpolymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis(MLVA) for typing Staphylococcus aureus isolated from dairy products[J], Res Vet Sci,2010,88:427-435
Moser SA,Heiss LN,Unance ER, Inter leukinl is linked to the respiratory epithelialcytopathology of pertussis[J], Infect Immun,1993,61(8):3123-3128
Murphy ER,Sacco RE,Dickenson A,Metzger DJ,Hu Y,Orndorff PE,Connell TD, BhuR,avirulence-associated outer membrane protein of Bordetella avium,is required for theacquisition of iron from heme and hemoproteins[J], Infect Immun,2002,70(10):5390-5403
Najiah M,Lee KL,Noorasikin H,Nadirah M,Lee SW, Phenotypic and genotypic characteristicsof Mycobacterium isolates from fighting fish Betta spp, in Malaysia[J], Res Vet Sci,2011,91:342-345
Nikaido H,Rosenbery EY, Porin channels in Escherichiacoli:studies with liposomesreconstituted from purified proteins[J],J Bacteriol,1983,153:241-252
Nolan LK, Wooley RE,Giddings CW,et,al, Characteization of an avirulent mutant of avirulentavian Escherichcoli isolate[J], Avian Dis,1994,38(1):146-150
Obayashi Y,Fujita J,Ichiyama S,et al, Genomic fingerprinting of Staphylococcus aureus byrandom amplified polymorphic DNA[J], Diagn Microbiol Infect Dis,1997,28(2):53-56
Odugbo MO,Musa U,Ekundayo SO,Okewole PA,Esilonu J, Bordetella avium infection inchickens and quail in Nigeria: preliminary investigations[J], Vet Res Commun,2006,30:1-5
Ongor H,Kalin R,Acik MN, Detection of Mycoplasma ovipneumoniae from goats with nasaldischarge by culture and polymerase chain reaction[J], Pak Vet J,2011,31:244-248
Onovian O,Robison T, Detection of antibody against M,mycoides subspmycoides in cattle byan enzyme-linked immunosorbent assay[J], Veterinary Record,1979,105:165-167
Parasadarao NV,Wass CA,Weiser JN,Stins MF,Huang SH,Kim KS, Outer membrane proteinAof Escherichiacoli contributes toinvasion of brain microvas cular endothelial cells[J],Infect Immun,1996,64(1):146-153
Pettersson A,Ley PVD,Poolman JT,Tommassen J, Molecular characterization of the98,2kilodalton Iron regulated outer membrane protein of neisseria meningitides[J], InfectImmun,1993,61(1):4724-4733
Poorbaghi SL,Mohammadi A,Asasi K,Molecular detection of avian infectious bronchitis virusserotypes from clinically suspected broiler chicken flocks in Fars province of Iran[J], PakVet J,2012,32:93-96
Raffel TR,Register KB,Marks SA,Temple L, Prevalence of Bordetella avium infection inselected wild and domesticated birds in the eastern USA[J], J Wildlife Dis,2002,38:40-46,
Rapp VJ,Munson RS,Ross RF, Outer membrane protein profiles of Haemophilus pleurop-neum oniae[J],Infect Immun1986,52(5):414-420
Rapp VJ,Ross RF,,Antibody response of swine to outer membrane components of Haem-ophilus pleuropneumoniae during infection[J], Infect Immun,1986a,54(3):751-760
Riain UN,Cormican MG, PCR based fingerprinting of Enterobacter cloacae[J], J Hosp Infect,1994,27:237～240
RimLer RB,Kunkle RA, Bacterin-induced protection of turkeys against fowl cholerafollowing infection with Bordetella avium[J], Avian Dis,1998,42(4):752-756
RimLer RB,Simmons DG,Gray JG, Transmission of acute respiratory disease (rhinotrachetitis)of turkeys[J], Avian Dis,1979,23:132-138
Rosqvist R,Bolin I,Wolfwatz H, Inhibitions of pHagocytsis in Yersini apseu do Tuber culosis:a virulent plasmid encoded ability involving the Yopzb protein[J], Infect Immun,1988,56(8):2139-2143
Roy S,Das AB,Ghosh AN,Biswas T, Purification,poreforming ability and antigenicrelatedness of the major outer membrane protein of shigelladys enteriae type[J], InfectImmun,1994,62(10):4333-4338
Ruzicka V,Marz W,Rass A, Immuno-PCR with a commercially available avidinsystem[J],Science,1993(5108),260:698-699,
Sacco RE,Register KB,Nordholm GE,Restriction enzyme analysis and ribotyping distinguishBordetella avium and Bordetella hinzii isolates[J], Epidemiol Infect,2000,124:83-90
Saif YM,Moorhead PD,Dearth RN,Jackwood DJ, Observations on Alcaligenes faecalisinfection in turkeys[J], Avian Dis,1980,(24):665-684
Sam brook J,Russell D W, Molecular Cloning:A Laboratory Manual[M],3rded, Translated byHUANG Pei-tang,WANG Jia-xi,ZHU Hou-chu,et al, Beijing: Science Press,2002:1474-1480(in Chinese)
SanoT,CasandraL,SmithCL,et al, Immuno-PCR:very sensitive antigen deteetion by means ofspecific antibody DNA conjugate[J],Seience,1992,258(5079):120-122
Sano T,Cantor CR,A streptavidin protein a chimera that allows one-step production of avariety of specific antibody conjugates[J],Bio Technology,1991,9(12):1378-1381
SanoT,Smith CL,Cantor CR, Immuno-PCR:very sensitive antigen deteetion by means ofsPecific antibody DNA eonjugates[J],Science,1992,258(5079):120-122
S,B, Stockwell,H, Kuzmiak-Ngiam,N,M, Beach,et al, The autotransporter protein fromBordetella avium,Baa1,is involved in host cell attachment[J], MicrobiologicalResearch,2011,167:55-60
Sherman P,Cockerill F,Soni R,Brunton J, Outer membrane are competitive inhibitor ofEscherichiacoli O157BH7adherence to epithelial cell[J], Infect Immun,1991,59(3):890-899
Shelton CB,Temple LM,Orndorff PE, Use of bacterio-phage Ba1to identify propertiesassociated with Bordetella avium virulence[J], Infect Immun,2002,70(3):1219-1224
Shin EK,Seo YS,Han JH,Hahn TW,Diversity of swine Bordetella bronchiseptica isolatesevaluated by RAPD analysis and PFGE[J], J Vet Sci,2007,8:65-73,
Simmons DG,Gray JG, Transmission of acute respiratory disease (rhinotrarhetitis) ofturkeys[J], Avian Dis,1979,23:132-138
Simmons DJ,Dee JC,Rose LP, A heat-stable toxin isolated from the turkeycoryza agentBordetella avium[J], Avian Dis,1986,30:761-765
Skeeles,JK,Swafford WS,Wages DP,Hellwig HM,Slavik MF,Beasley JN,Houghten GE,BlorePJ,Crawford D, Studies on the use of a long-acting oxytetracycl;ine in turkeys: Efficacyagainst experimental infections with Alcaligenesfaecalis and Pasteurella multocida[J],Avian Dis,1983,27:1126-1130
Sonntang I,Schwarz H,Hirota Y,Henning U, Cell envelope and shape of Escherichiacoli:multiple mutants missing the outer membrane lipoprotein andother major outermembrane proteins [J], J Bacteriol,1978,136(1):280-285
Spears PA,Temple LM,Miyamoto DM,Maskell DJ,Orndorff PE, Unexpected similaritiesbetween Bordetella avium and other pathogenic bordetellae[J], Infect Immun,2003,71:2591-2597
Sperl J,Paliwal V,Ramabhadran,et al, Soluble Teellreee Ptors:detection and antitative assayin luidphase via ELISA or inununo-PCR[J], J immunol Methods,1995,186:181-194
Spilker T,Liwienski AA,Lipuma JJ, Identification of Bordetella spp, in respiratory specimensfrom individuals with cystic fibrosis[J], Clin Microbiol Infect,2008,14:504-506
Sun S,Sun A,Cui Z, Epidemiological and pathological studies of subgroup J avian leukosisvirus infections in Chinese local yellow chickens[J], Avian Pathol,2007,36(3):221-226
Suresh P,Arp LH, A monoclonal antibody-based latex bead agglutination test for the detectionof Bordetella avium[J], AvianDis,1993Jul-Sep;37(3):767-72
Tazumi A,Petry S,Hayashi K,Moore JE,Millar BC,Matsuda M, Molecular identification andcharacterization of the intervening sequences (IVSs) within23S ribosomal RNA (rRNA)genes of Taylorella asinigenitalis isolated in France[J], Res Vet Sci,2012,92,45-52
Temple LM,Miyamoto DM,Mehta M,Capitini CM,Stetina VS,Barnes HJ, ChristensenVL,Horton JH,Spears PA,Orndorff PE, Identification and characterization of twoBordetella avium gene products required for hemagglutination[J], Infect Immun,2010,78:2370-2376
Thanassi DG,Suh GSB,Nikaido H, Role of outer membrane barrier inefflux mediatedtetracycline resistance of Escherichiacoli[J], J Bacteriol,1995,177(4):998-1007,
Truscott WM,Hirsh DC, Demonstration of an outer membrane proteins with anti-pHagocyticfrom Pateurel lamultocida of avian origin[J], Infect Immun,1988,56(6):1538-1544
Van Couwenberghe CJ,Cohen SH,Tang YJ,Gumerlock PH,Silva J Jr, Genomicfingerprinting of epidemic and endemic trains of Stenotrophomonsa maltophilia(formerly Xanthomnsa mallophilia) by arbitrarily primed PCR[J], J ClinMicrobiol,1995,33:1289-1294
Vidotto MC,Muller EE,de Freitas JC,Alfieri AA,Guimar es IG,Santos DS, Virulence factorsof avian Escherichia coli[J], Avian Dis,1990,34:531-538
Vordermeier HM,Hoffmann P,Gombert FO,Jung G,Bessler WG, Synthetic peptide segmentfrom the Escherichiacoli porin OmpF constitute eukocyte activators[J], Infect Immun,1990,(8):2719-2724
Weiser JN,Gotschlich EC, Outer membrane proteinA(OMPA) contributes to serum resistanceand pathogenicity of Escherichiacolik21[J], Infect Immun,1991,59(7):2252-2258
Wikler MA, Methods for dilution antimicrobial susceptibility tests for bacteria that growaerobically: approved standard[J],Clinical and Laboratory Standards Institute,2006,26(2):431-442
Wulff G,Gram L,Ahrens P,Vogel BF, One group of genetically similar Listeria monocytogenesstrains frequently dominates and persists in several fish slaughter-and smokehouses[J],Appl Environ Microbiol,2006,72:4313-4322
XU Y J,VAN HUYSTEE R B,Identification of an antigenic determination anionic peanutperoxidase by monoclonal antibodies[J],J Exper Botany,1991,42(24):1-11
Yang X,Hou L,Ye J,He Q,Cao S, Detection of porcine circovirus type2(PCV2) in mosquitoesfrom pig farms by PCR[J], Pak Vet J,2012,32:134-135
Yersin,AG,Edens FW,Simmons DG, TryptopHan2,3-dioxyge-nase activity in turkey poultsinfected with Bordetella avium[J], Comp Biochem Physio,1990,197B:755-760
Yiang Z,Ertl HC, Manipulation of the immune response to a plasmidoncode viral antigen bycoinoculation with plasmids expressing cytokines [J],Immunity,1995,2:129-135
Yi Cao,Kathrin Kopplow,Guang Yaw Liu,et al.,In situ immuno-PCR to detect antigens [J],Lancet,2000,356(9234):1002-1003
Zhou H,Fisher RJ,Papas TS,Universal immuno-PCR for ultra-sensitive target proteindetection [J], Nucleic Acids Res,1993,21(25):6038-6041
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |