兔支气管败血波氏杆菌的分离鉴定及其外膜蛋白免疫效果的研究
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摘要
兔支气管败血波氏杆菌病是由支气管败血波氏杆菌引起的一种常见兔呼吸道疾病。主要引起哺乳仔兔和断乳仔兔的急性死亡,成年兔的鼻炎、支气管炎和脓疱性肺炎等,对养兔业造成很大的危害。2006年以来,山东省某一大型兔场(饲养新西兰肉种兔2400余只),所产仔兔(共计465只),均在20～30日龄发病,表现为精神不振,多数病兔鼻腔流出脓性鼻液,周围的被毛沾有粘液脓性分泌物,后期出现结痂,随着病程的延长,逐渐表现为呼吸困难,并出现鼻鼾声,体况瘦弱,下痢拉稀。为了进一步探讨引起该规模化兔场呼吸道疾病的原因,本研究通过病原学检查,对主要病原进行了分离鉴定和分子生物学特性的研究,同时对分离到主要病原菌的外膜蛋白提取后进行了免疫原性研究,为规模化种兔场呼吸道疾病的防治提供了理论依据。
本研究共分2部分:
一、兔支气管败血波氏杆菌的分离鉴定及其分子生物学特性的检测
2006年,山东省某一大型养兔场的20～30日龄的幼兔突然大批发病死亡。病兔早期精神不振,多数病兔鼻腔流出脓性鼻液,随着病程的延长逐渐表现为呼吸困难,并出现鼻鼾声,以体况瘦弱,下痢拉稀为主要症状。从中主要分离到4株细菌,根据其形态学特性、培养特性、生化特性、血清学反应特性等可将分离菌鉴定为兔支气管败血波氏杆菌。然后对所分离到的兔支气管败血波氏杆菌P13菌株进行(G+C)mol%含量的测定以及16SrRNA的扩增,结果(G+C)mol%含量为61.7～62.4%,与Kersters K结果相符(61.6～62.6%);而该菌16SrRNA核苷酸序列与Genbank中收录的AF177666株禽波氏杆菌菌株核苷酸序列同源性为82.7%,与本实验室保存的兔支气管败血波氏杆菌菌株P11菌株及参考菌株同源性高,分别为99.7%和99.9%。最后利用所分离到的兔支气管败血波氏杆菌菌株制备免疫原,通过免疫仔兔,使仔兔获得抗体,而使幼仔兔获得保护,并且效果较好。
二、兔支气管败血波氏杆菌外膜蛋白的提取及其免疫效果的检测
本研究以小鼠为实验动物模型,研究支气管败血波氏杆菌的外膜蛋白(OMP)和其中的有效保护抗原成分(OMP68)的免疫原性及免疫保护作用。本研究利用改进的Wooldridge的方法提取了支气管败血波氏杆菌P11和P13的OMP,利用SDS-PAGE电泳比较了两株之间差异,采用Western- blotting进行了分析并确定了P13菌株OMP(P13-OMP)中的有效保护性抗原成分,采用电洗脱方法获得分子量为68 KD的P13-OMP中的有效保护抗原成分(OMP68),然后制备了油乳剂P13的全菌、P13-OMP和OMP68免疫抗原。抗体动态变化:将清洁级小鼠70只随机分成7组,10只/组,用油乳剂P13的全菌和不同剂量的P13-OMP和OMP68免疫抗原分别免疫,采用间接ELISA检测免疫小鼠的相应抗体水平并分析抗体动态变化;主动免疫保护试验:将清洁级小鼠80只随机分成8组,10只/组,分别采用制备的油乳剂P13-OMP(OMP25μg/只)、OMP68(25μg/只)和P13菌体免疫抗原在免疫10d后,分别用100 LD50的P11和P13腹腔攻毒,然后统计保护率并分析免疫原所提供的免疫保护力。免疫P13-OMP和OMP68油乳剂抗原后,7d时,抗体水平开始逐渐上升,42d时,抗体水平分别达到2~(15.3)和2~(14.8),然后逐渐下降,70d时,抗体水平分别达到2~(9.1)和2~(10.2)。主动免疫时,P11和P13攻毒的P13-OMP免疫组分别均得到9/10和9/10的保护,OMP68免疫组分别均得到9/10和10/10的保护,而P13全菌免疫组分别得到4/10和6/10保护,对照组全部死亡。P13-OMP和OMP68抗原均具有良好的免疫原性和免疫保护作用,为支气管败血波氏杆菌OMP亚单位疫苗的研制奠定了良好的理论基础。
Bordetellosis in rabbit caused by Bordetella bronchiseptica is a serious respiratory tract diseases. It can cause lactation rabbit and adult rabbit to be dead. Rhinitis, bronchitis and pustular pneumonia can be caused by Bordetella. From 2006, all of rabbits(from 20to30 days old) of a livestock farm appeared low sprit. we can also find lots of purulent secretion in clothing hair and nasal. At last, The clinical symptoms incuding weediness, washy and snoring could be appeared.In order to study on the reason of causing respiratory tract diseases in scale rabbit farm, we isolated the main pathogen and studied the molecularbiology characterisitcs. At the same time we extracted the outer membrane protein of main pathogen and studied on immunogenicity of it. So, the results of this study lay good foundation for controlling serious respiratory tract diseases of rabbits in scale rabbit farm.
The research divide two parts:
Part One: Isolation, Identification And Molecularbiology Characterisitcs of Bordetella bronchiseptica from Rabbit
In 2006, the 20, 30-day-old young rabbits of a large-scale farm in Shandong were diseased suddenly and were caused to death. The rabbits were deressed with most outfolw of purulent nasal in early phase, and with the process of disease, the rabbits showed the diffculties in respiration, the snore, feebleness, and diarrhea. Four bacteria were isolated, and through morphological, cultural, biochemical characteristics and seral reactional traits, the bacteria were identified as Rabbit bordetella Bronchiseptica. The G+C mol% of isolated bacteria was from 61.7% to 62.4%, which accorded to the results of Kereters(61.6-62.6%); the homology of gene sequnence of 16SrRNA from the isolated strains with the corrensponding parts of Avian bordetella Bronchiseptica recored in Genebank was 82.7%; and the homology of gene sequnence of 16SrRNA with the corrensponding parts of Avian strian and Rabbit bordetella Bronchiseptica Reference strian AF177666 was higher, which was 99.7% and 99.9%, respectively. The immunogens were prepared using the isolated bacteria, and the female rabbits were vaccinated before pregnancy. The new-born rabbits showed the high titer abtibody and the good protection against Rabbit Bordetella Bronchiseptica.
Part Two: Extraction and Detect on Immune Effect of Outer protein of Bordetella bronchiseptica
In order to evaluate the immunogenicity and their protection of outer membrane protein of Bordetella bronchiseptica and effective antibody(OMP68, we established a mice model.The research improved on the method of Wooldridge in order to extract OMP of Bordetella bronchiseptica(P13, P11),and the differences of OMP were analyzed by SDS-PAGE. The P13-OMP was analyzed with Western blotting and OMP68 was gained by the way of electrophoretic elution .then we maked the effective antibodies of P13-OMP be known.Then P13-OMP, OMP68 and whole-cell bacterin (WCB)immunizing antigen was prepared 70 mice were equally assigned to 7 groups at random. They were immunized by P13-OMP, OMP68 and P13-WCB of different dose respectively. The indirect ELISA was used to detect antibody responses.80 mice were equally assigned to 8 groups at random.Group a and b were immunized by P13-OMP (25μg) respectively. Groups c and d were immunized by OMP68(25μg) ; Groups e and f were immunized by WCB(P13). Ten days after the immunizations, group a, c, e and f were challenged with P13. The other groups were challenged with P11. Then we registered the rate of protective potency and evaluated protection given by P13-OMP, OMP68 and P13-WCB[Result] Results showed that at 42th day, the titer of serum reach the top: P13-OMP(2~(15.3))> OMP68(2~(14.8)). Then, the titer of serum started the slow-moving descent. At 70th day, the titer of serum reach the OMP68 (2~(10.2))> P13-OMP (2~(9.1)). P13-OMP and OMP68 group challenged with P13 and P11 can be efectivly protected; P13-WCB group challenged with P13 and P11 can not be efectivly protected; the control group was died out. The P13-OMP and OMP68 of Bordetella bronchiseptica has good immunogenicity and protection, so the results of this study lay good theoretical foundation for OMP subunit vaccine.
本研究共分2部分:
一、兔支气管败血波氏杆菌的分离鉴定及其分子生物学特性的检测
2006年,山东省某一大型养兔场的20～30日龄的幼兔突然大批发病死亡。病兔早期精神不振,多数病兔鼻腔流出脓性鼻液,随着病程的延长逐渐表现为呼吸困难,并出现鼻鼾声,以体况瘦弱,下痢拉稀为主要症状。从中主要分离到4株细菌,根据其形态学特性、培养特性、生化特性、血清学反应特性等可将分离菌鉴定为兔支气管败血波氏杆菌。然后对所分离到的兔支气管败血波氏杆菌P13菌株进行(G+C)mol%含量的测定以及16SrRNA的扩增,结果(G+C)mol%含量为61.7～62.4%,与Kersters K结果相符(61.6～62.6%);而该菌16SrRNA核苷酸序列与Genbank中收录的AF177666株禽波氏杆菌菌株核苷酸序列同源性为82.7%,与本实验室保存的兔支气管败血波氏杆菌菌株P11菌株及参考菌株同源性高,分别为99.7%和99.9%。最后利用所分离到的兔支气管败血波氏杆菌菌株制备免疫原,通过免疫仔兔,使仔兔获得抗体,而使幼仔兔获得保护,并且效果较好。
二、兔支气管败血波氏杆菌外膜蛋白的提取及其免疫效果的检测
本研究以小鼠为实验动物模型,研究支气管败血波氏杆菌的外膜蛋白(OMP)和其中的有效保护抗原成分(OMP68)的免疫原性及免疫保护作用。本研究利用改进的Wooldridge的方法提取了支气管败血波氏杆菌P11和P13的OMP,利用SDS-PAGE电泳比较了两株之间差异,采用Western- blotting进行了分析并确定了P13菌株OMP(P13-OMP)中的有效保护性抗原成分,采用电洗脱方法获得分子量为68 KD的P13-OMP中的有效保护抗原成分(OMP68),然后制备了油乳剂P13的全菌、P13-OMP和OMP68免疫抗原。抗体动态变化:将清洁级小鼠70只随机分成7组,10只/组,用油乳剂P13的全菌和不同剂量的P13-OMP和OMP68免疫抗原分别免疫,采用间接ELISA检测免疫小鼠的相应抗体水平并分析抗体动态变化;主动免疫保护试验:将清洁级小鼠80只随机分成8组,10只/组,分别采用制备的油乳剂P13-OMP(OMP25μg/只)、OMP68(25μg/只)和P13菌体免疫抗原在免疫10d后,分别用100 LD50的P11和P13腹腔攻毒,然后统计保护率并分析免疫原所提供的免疫保护力。免疫P13-OMP和OMP68油乳剂抗原后,7d时,抗体水平开始逐渐上升,42d时,抗体水平分别达到2~(15.3)和2~(14.8),然后逐渐下降,70d时,抗体水平分别达到2~(9.1)和2~(10.2)。主动免疫时,P11和P13攻毒的P13-OMP免疫组分别均得到9/10和9/10的保护,OMP68免疫组分别均得到9/10和10/10的保护,而P13全菌免疫组分别得到4/10和6/10保护,对照组全部死亡。P13-OMP和OMP68抗原均具有良好的免疫原性和免疫保护作用,为支气管败血波氏杆菌OMP亚单位疫苗的研制奠定了良好的理论基础。
Bordetellosis in rabbit caused by Bordetella bronchiseptica is a serious respiratory tract diseases. It can cause lactation rabbit and adult rabbit to be dead. Rhinitis, bronchitis and pustular pneumonia can be caused by Bordetella. From 2006, all of rabbits(from 20to30 days old) of a livestock farm appeared low sprit. we can also find lots of purulent secretion in clothing hair and nasal. At last, The clinical symptoms incuding weediness, washy and snoring could be appeared.In order to study on the reason of causing respiratory tract diseases in scale rabbit farm, we isolated the main pathogen and studied the molecularbiology characterisitcs. At the same time we extracted the outer membrane protein of main pathogen and studied on immunogenicity of it. So, the results of this study lay good foundation for controlling serious respiratory tract diseases of rabbits in scale rabbit farm.
The research divide two parts:
Part One: Isolation, Identification And Molecularbiology Characterisitcs of Bordetella bronchiseptica from Rabbit
In 2006, the 20, 30-day-old young rabbits of a large-scale farm in Shandong were diseased suddenly and were caused to death. The rabbits were deressed with most outfolw of purulent nasal in early phase, and with the process of disease, the rabbits showed the diffculties in respiration, the snore, feebleness, and diarrhea. Four bacteria were isolated, and through morphological, cultural, biochemical characteristics and seral reactional traits, the bacteria were identified as Rabbit bordetella Bronchiseptica. The G+C mol% of isolated bacteria was from 61.7% to 62.4%, which accorded to the results of Kereters(61.6-62.6%); the homology of gene sequnence of 16SrRNA from the isolated strains with the corrensponding parts of Avian bordetella Bronchiseptica recored in Genebank was 82.7%; and the homology of gene sequnence of 16SrRNA with the corrensponding parts of Avian strian and Rabbit bordetella Bronchiseptica Reference strian AF177666 was higher, which was 99.7% and 99.9%, respectively. The immunogens were prepared using the isolated bacteria, and the female rabbits were vaccinated before pregnancy. The new-born rabbits showed the high titer abtibody and the good protection against Rabbit Bordetella Bronchiseptica.
Part Two: Extraction and Detect on Immune Effect of Outer protein of Bordetella bronchiseptica
In order to evaluate the immunogenicity and their protection of outer membrane protein of Bordetella bronchiseptica and effective antibody(OMP68, we established a mice model.The research improved on the method of Wooldridge in order to extract OMP of Bordetella bronchiseptica(P13, P11),and the differences of OMP were analyzed by SDS-PAGE. The P13-OMP was analyzed with Western blotting and OMP68 was gained by the way of electrophoretic elution .then we maked the effective antibodies of P13-OMP be known.Then P13-OMP, OMP68 and whole-cell bacterin (WCB)immunizing antigen was prepared 70 mice were equally assigned to 7 groups at random. They were immunized by P13-OMP, OMP68 and P13-WCB of different dose respectively. The indirect ELISA was used to detect antibody responses.80 mice were equally assigned to 8 groups at random.Group a and b were immunized by P13-OMP (25μg) respectively. Groups c and d were immunized by OMP68(25μg) ; Groups e and f were immunized by WCB(P13). Ten days after the immunizations, group a, c, e and f were challenged with P13. The other groups were challenged with P11. Then we registered the rate of protective potency and evaluated protection given by P13-OMP, OMP68 and P13-WCB[Result] Results showed that at 42th day, the titer of serum reach the top: P13-OMP(2~(15.3))> OMP68(2~(14.8)). Then, the titer of serum started the slow-moving descent. At 70th day, the titer of serum reach the OMP68 (2~(10.2))> P13-OMP (2~(9.1)). P13-OMP and OMP68 group challenged with P13 and P11 can be efectivly protected; P13-WCB group challenged with P13 and P11 can not be efectivly protected; the control group was died out. The P13-OMP and OMP68 of Bordetella bronchiseptica has good immunogenicity and protection, so the results of this study lay good theoretical foundation for OMP subunit vaccine.
引文
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