【作者】 崔立；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 兽医， 2005， 博士

【摘要】 我国缺乏标准化的实验用猪和系统的生物安全控制体系成为制约实验猪用于病原致病性研究、人类疾病模型的培育，作为生物反应器参与人类重要蛋白的生产、以及作为人类异种器官／组织／细胞移植供体等的研究开发、应用工作。因此，开展我国特有实验用小型猪生物安全控制技术研究，培育小型猪净化种群，提出我国实验用小型猪微生物学质量标准，对于完善我国实验用小型猪的理论与技术体系，满足生物医学研究对标准化实验用小型猪的需求具有重要意义。本研究对巴马小型猪进行系统的微生物学质量控制技术研究，用获得的SPF小型猪进行了猪的疾病人工感染模型和人肠道菌群相关性仔猪模型的建立和应用研究。1、实验用小型猪微生物学质量控制技术研究实验用小型猪安全控制方法的研究试验群体为20头母猪规模，年存栏350头左右，规定控制的疾病为慢性传染性、疫苗不易控制的疾病，主要包括：猪喘气病(猪肺炎支原体)、猪传染性萎缩性鼻炎(支气管败血波氏杆菌)、猪伪狂犬病(猪伪狂犬病病毒)、猪传染性胃肠炎(猪传染性胃肠炎病毒)、猪痢疾(猪痢疾密螺旋体)、猪流行性腹泻(猪流行性腹泻病毒)、猪蓝耳病(猪繁殖与呼吸综合症病毒)、猪细小病毒病(猪细小病毒)、猪瘟(猪瘟病毒)、体外寄生虫等，控制的主要方法是种源控制(剖腹产引种)、环境控制、人员、车辆、物品控制、鼠鸟蚊蝇的控制、饲料饮水控制以及化验监测等，形成了一个人工屏障，实现适宜实验用小型猪繁殖生长的生物安全环境。试验期3年，实验猪1016头，成活965头，总成活率95.0％，接近世界先进水平，除未发生规定控制的七种特定病原外，也杜绝了其它二十几种急慢性传染病的发生。试验表明，只要认真坚持小型猪的安全防疫措施，实验用小型猪的安全保护是完全可以做到的。2、实验用小型猪病原微生物、寄生虫等级及监测标准的制定2001年8月29日国家技监局批准的2个国家标准《实验动物微生物学等级及监测》(GB14922.2 2001)和《实验动物寄生虫学等级及监测》(GB14922.1-2001)主要适用于小鼠、大鼠、兔、豚鼠、地鼠等。不适用于小型猪的微生物学等级划分和监测。鉴于目前国内多家小型猪生产和研究单位需要申报实验动物许可证，同时需要向生农医药各研究领域提供标准的实验用小型猪，而苦于没有国家标准可以参照。甚至找不到一个地方标准。已不适应目前实验用小型猪研究和应用的蓬勃发展，亟需制定小型猪微生物、寄生虫等级及监测国家或行业标准，以适应目前小型猪研究和应用的飞速发展。本人在查阅相关国家标准库和调研各地小型猪研发情况后，向国家技监局递交了相关提案，并完成标准草案。在本标准中，规定了实验用小型猪的定义、等级划分以及各级实验用小型猪的病原微生物、寄生虫监测规程等。3、SPF级小型猪全人工哺育技术研究实验1：小型猪剖腹取胎手术的研究：猪的剖腹取胎手术是获得无菌仔猪的途径。无菌仔猪的获得可采用子宫摘除手术或剖腹产手术两种方法。猪剖腹产手术效果与麻醉方法密切相关。实验2：SPF级小型猪的全人工哺育试验：为检验所研制的人工乳的的饲养效果，在隔离器饲养环境中探索小型猪的全人工哺育技术，并与自然哺乳的仔猪的生长性能、存活率、采食量、饲料转化率、腹泻率和血清生化指标进行比较；结果表明：用自行研制的人工乳配合科学管理，完全可以生产出合格的SPF级实验用小型猪。4、SPF仔猪肠道菌群结构分析目的：对SPF(Specific Pathogen Free)仔猪断奶前后的肠道菌群组成进行动态监测，并以普通饲养仔猪(自然分娩)为对照作相关分子微生物生态学分析。方法：利用剖腹取胎的方式将仔猪从母体子宫中取出放于屏障系统中饲养，于7日龄时进行补饲，14日龄断奶，分别于10日龄、16日龄和22日龄以及32日龄采集仔猪新鲜粪便样品。提取粪便样品中细菌总DNA，获得其ERIC-PCR指纹图谱，再将其中一个样品的ERIC-PCR产物以地高辛标记为混合探针通过杂交对指纹图谱上DNA条带序列的异同进一步比较。结果：SPF仔猪和普通仔猪在断奶后肠道菌群的组成发生较大变化，而且SPF仔猪的肠道菌群和普通仔猪有显著差异。5、SPF小型猪生长发育规律和血液生理生化指标变化选用50头10～150日龄的SPF巴马小型猪，从前腔静脉采血，测定血液常规指标、血浆激素水平和生化指标。结果表明，随着SPF巴马小型猪的生长和发育，血液中的激素水平及各项生理生化指标也发生相应的变化。具体如下：Ⅰ生长：SPF巴马小型猪早期的生长速度较快，后期较慢。Ⅱ激素水平：血浆中GH以10日龄最低，120日龄最高；T3前期较高，后期较低，20日龄最高；T4以60日龄最低，90日龄最高。TSH以80日龄、150日龄最高；Ins以30日龄、100日龄最高，40日龄最低；Glu在前期较高，之后逐渐下降，70日龄波动之后继续下降，100日龄达最低；Cor浓度上下波动。Ⅲ生化指标：血糖前期较高，后期较低，70日龄极显著高于10日龄、80～150日龄；BUN在30日龄时最高；GPT活性在70日龄、80日龄达到峰值。Ⅳ血液常规指标：RBC、Hb、HCT均在90日龄达最高值；40日龄的WBC极显著高于其它日龄；EOF最大抗力10～80日龄较为稳定，140日龄、150日龄高于其它日龄(P＜0.01)，10日龄、20日龄、140日龄和150日龄的最小抗力较大，均高于其它日龄(P＜0.01)；ESR速度后期比前期快。6、TGEV不同途径感染SPF仔猪后病毒消长规律的研究为确立TGEV的传染途径，定植部位以及猪感染后，病毒在猪各个脏器内的消长规律，将TGEV分别通过滴眼、气管注射和口服的方式对25日龄的SPF仔猪进行攻毒，至攻毒后24h起每天颈静脉放血致死仔猪一头，采取血液及胃、十二指肠、空肠、回肠、肺脏、肺淋巴结、肠道淋巴结、心脏、气管、扁桃体、肝脏和脾脏等组织，将组织制成冰冻切片进行免疫荧光抗体染色，在荧光显微镜下观测结果。同时取所有被检组织提取RNA，用RT-PCR方法检测病毒。另外，还用ELISA方法检测仔猪血液内抗体效价的变化。主要结果如下：Ⅰ经眼粘膜攻毒：攻毒后仔猪表现正常，剖检除肺脏表现苍白外，其他组织器官均无肉眼可见的病理变化，免疫荧光试验结果均为阴性；RT-PCR只在第30天的肺和空肠检测到病毒，其它组织则均为阴性；ELISA检测攻毒后前20天仔猪血液抗体效价一直为可疑，OD值最低为0.21，最高为0.30，但到第22天抗体效价开始升高，第30天升至0.82。Ⅱ经呼吸道攻毒：攻毒后24h实验猪出现呼吸道症状，三天后耐过，但均未出现消化道症状。剖检可见不同程度的肺部病理变化。攻毒后24h气管(持续8天)和肺脏样品(持续30天)的IFA呈阳性，肺淋巴结(5-30天)和脾脏(2-5天))样品也为阳性。RT-PCR结果与IFA结果基本一致。抗体效价至第4天(0.40)开始随着时间的延长而增高，最高升至1.41。IFA和RT-PCR在消化道系统中始终未检出TGEV。Ⅲ经消化道攻毒：攻毒3天后四头仔猪即开始发生水样腹泻，粪便呈黄色并夹有未消化的凝乳块；分别于第6、9、17天死亡三头。剖检可见明显的肠道病变，其中3头有不同程度的肺脏病变。攻毒后48小时十二指肠、空肠、回肠以及肠系膜淋巴结样品的IFA即呈阳性，随着时间的延长，阳性反应程度增加，并持续存在。在扁桃体(2-15天)、脾脏(3-5天)、直肠(4-30天)、腹股沟淋巴结和颌下淋巴结(6-30天)、气管(15-18天)和肺脏(25-30天)中都可检测到阳性。RT-PCR检测结果与IFA结果一致。抗体效价至第4天(0.33)开始随着时间的延长而增高。最高升至1.54。结果显示口服TGEV后，病毒可在肠道、呼吸道、扁桃体中增殖。7、HFA仔猪模型建立研究建立人肠道菌群相关(HFA)动物模型，对研究肠道微生物和消化道健康及疾病关系、食品和药物安全评价等均有很重要意义。本研究以5窝SPF巴马小型猪为研究对象，初步探索了人肠道微生态相关仔猪模型(Human Flora-Associated miniature piglet model)的建立技术。对5头临产母猪进行无菌剖腹产手术，将新生仔猪放入屏障系统全人工哺育，手术成功率100％；新生仔猪前三天每日每头口服接种1ml供体菌悬液，第四天到第十天每隔一天接一次，于仔猪5日龄、9日龄、12日龄时收集仔猪粪便，-70℃冻存待检测。通过ERIC-PCR指纹图谱技术和微生物群落分子杂交实验表明，在屏障系统内，人肠道菌群能够部分定植于小型猪仔猪肠道内，并且在两周内保持相对稳定，个体间差异极小。更多还原

【Abstract】 1. The study on the safety-control technology of SPF mini-pigThe study on the safety-control techniques of SPF mini-pig was carried out on a herd of the scale of 20 sows, with the amount of livestock on hand 350 a year. We basically aim to control the chronic infectious disease on which the vaccines have low effect, including: Mycoplasma Pneumonia of Swine, Atrophicrhintis, Swine dysentery, Pseudorabies, Transmissible gastroenteritis of pigs, etc. The main control method is through the control of the source （by Caesarean birth）, environment, personnel, vehicles, articles, rodents, birds, flies and other insects, feeds and water, and the assay monitors. We realized the favourable environment of bio-security for the SPF mini-pig through setting up an artificial barrier. The experiment continued on three years with 1016 pigs, among which the total survival amount was 965 （the total survival rate is 95%）. Moreover, we have eliminated two decades of acute or chronic infectious disease besides the 7 aimed specific pathogenies. The experiment indicates that as long as we strictly adhere to the safety epidemic preventive measures, the protection of SPF mini-pig is definitely reachable.2. Grade of pathogenic microorganism and parasite for experimental mini-pigs and its monitoring StandardsThe two national standards《The microorganism grading and monitoring for experimental animals》（GB14922.2 2001 ）,and《The parasite grading and monitoring for experimental animals》（GB14922.1 - 2001） authorized by the National Technical Supervise Bureau on Aug, 29th, 2001 are mainly used with the aspect of rodents. However, the standards could not serve to the microorganism classification or monitoring for the mini-pig. Given that at present more and more domestic mini-pig producers or institutions require the experimental animal license to supply the standard experimental pigs but have no national standard to refer to, it is high time that we should constitute the specific standards or criterion for the mini-pig with the aspect of microorganism and parasite grading and monitoring. After looking up to the relevant national standards and making sound investigation of the research and development status of the mini-pig, we submit National Technical Supervise Bureau the overture and make the standard draft. In this standard, we prescribe the definition, grading of the experimental mini-pig and the monitoring regulation of the pathogenic microorganism and parasite for them.3. The entirely artificial nursing experiment of SPF mini-pigThe caesarean operation is the route to obtain the sterile piglets. The effect of the operation is largely relevant to the anaesthesia methods.To verify the feeding effect of the artificial milk, we adopted an entirely artificial nursing technology for the mini-pig in isolated environment. We compared its growth performance, livability, feed intake and conversion efficiency, blood physiological and biochemical parameters with conventional natural delivery piglets. The result indicated that we could produce qualified experimental SPF mini-pig by using the artificial milk in the light of scientific managemen..4. Molecular Analysis of Intestinal Mieroflora of SPF PigletsObjective: To monitor and characterize the structural features of intestinal microbial communities in fecal samples of SPF（ specific pathogen free） piglets and conventional piglets. Methods: The newborn piglets were transferred from the womb into a barrier system immediately after Caesarean operation.The genomic DNA of bacteria was extracted from each sample and ERIC-PCR amplification was then carried out. The PCR fingerprints were southern-blotted with a mixed dig-tagged probe from the labeled ERIC-PCR product of one sample. Results: The banding-patterns of samples of SPF piglets and conventional piglets elucidate poor similarities before, during and after weaning respectively. Simultaneously there really existed much difference in the aspect of the structural features of intestinal microflora between SPF piglets and conventional piglets on the DNA level.5. Growth, blood physiological and blochemical parameters of SPF mini-pigTo investigate the growth and blood physiological and biochemical parameters in different age of BAMA SPF mini-pig, 50 pigs aging from 10～150 days were used in this experiment. The blood samples were collected via the superior vena cava, while the blood parameters, hormone contents and biochemical parameters in plasma were analyzed.Growth: BAMA mini-pig grew faster during the first 10～50 day. The RGR at day 20 was the highest, and it declined to the slowest on day 40. Hormone contents: The GH level was the lowest on day 10, and it reached its highest on day 120. The T₃ level during day 10～90 was higher than those on the other days, it was significantly the highest on day 20. The T₄ level was lowest at day 60. The TSH level on day 80 and day 150 were evidently higher than those on the other days; The Ins level climbed to its highest on day 30 and day 100, and dropped to the lowest on day 40; The Glu level was higher during day 10～60, it increased on day 70, then decreased gradually, and finally reached the lowest level on day 100, it was only 25％of day 10（p＜0.05）. The Cor level fiuctated during the whole experiment.Biochemical parameters: The Blood sugar level on day 10～80 was the highest. On day 70 the blood sugar was higher than those on day 10 and day 80～150. it reached its highest on day 30; The GPT level were significantly higher on day 70 and day 80 than those on the other days.Blood parameters: RBC, The Hb and HCT levels were the highest on day 90. The WBC level was higher on day 40 than those on the other days. The largest resistance of EOF on day 10～80 were stable, and they were observably greater on day 140 and day 150 than those on the other days. The smallest resistance of EOF on day 10, day 20, day 140 and day 150 were remarkably the higher. ESR on day 10～90 were lower than those on the rest days. The results indicated that hormone contents, physiological and biochemical parameters in blood are changed with the growth of the piglets.6. Profiling of In-vivo Waning and Waxing of Porcine Transmissible Gastroenteritis Virus Inoculated Via Different PassagesThis experimentation studied the waning and waxing of TGEV inoculated by ocular passage, tracheal passage and oral passage respectively.TGEV rejuvenated on the ST cell layer before inoculating via different passages in order to pinpoint viral replicative sites and in-vivo waning and waxing. 25 days old specific pathogen-free （SPF） piglets were inoculated via ocular, tracheal or oral passages respectively. One SPF piglet was killed every 24 hours after inoculation, and samples of tissues and blood were collected and detected by immunofluoescent antibody assay （IFA） and reverse-transcription PCR （RT-PCR）. The tissues collected included stomach, duodenum, jejunum, ileum, lung, lymph node, heart, trachea, liver, spleen and tonsil. TGEV antibody titers of all piglets were measured by ELISA. The results are as follows: Ocular passage: None of the fifteen SPF piglets showed any clinical signs and lesions. The IFA results of all samples were negative, but the RT-PCR results of lung and intestine were positive 30 days after inoculation. OD value of antibody didn’t increase on post inoculation days （PID） 1-20, of which the lowest OD value was 0.21 and the highest was 0.30, but suddenly boosted on PID 22. The result showed that TGEV couldn’t replicate in vivo after inoculating ocularly. The virus exuded with tears, which caused the spread of TGEV in the environment after uptaking it. This study confirmed that TGEV could not be infected by ocular passage firstly.Tracheal passage: All 25-day old piglets developed respiratory symptoms 24 hours after inoculation, but eventually recovered. There were different lesions of the lungs. TGEV Antibody titers of all pigs were measured by ELISA. Therefore, there was age-dependent resistance to TGEV infection. IFA and RT-PCR results of samples of trachea （PID 2-10）, lung （PID 2-30）, and spleen （PID 2-5） were positive, while all samples of intestinal tract were negative. The result confirmed viral waning and waxing in the respiratory system.Oral passage: Four of thirty 25-day old piglets developed diarrhea on PID 3, and three died on PID 6, 9 and 17 respectively. There were significant lesions of intestines, three of which had the different lesions of lungs. IFA results of samples collected from intestinal tract and lymphatic node were positive 48 hours after inoculation, and the number of positive cells continuously increased. The IFA results of tonsil, spleen and systemic lymphatic nodes were also positive. The results of RT-PCR showed that spleen of 3-5 days was positive. The OD value of antibody boosted from 0.07 to 1.54. There is age-dependent resistance to TGEV infection. The result confirmed viral waning and waxing in the digestive and respiratory system, and oral passage was the main way resulting this diseases.TGEV in spleen could be detected on PID 2-5 by both tracheal passage and oral passage, which showed that TGEV could not replicate in spleen.7. Study on establishment of Human flora associated mini-piglet model and molecular analysis of SPF piglet’s intestinal microbiotaTo investigate the availability of HFA piglet model, we selected five litters of Bama Xiang mini-pigs that were delivered by caesarean section. This study included two parts. On one hand, man-rearing procedures of caesarean piglets in barrier facility were optimized and colonizing status of donor’ intestinal microbiota in the piglets’ gut was evaluated by molecular methods as ERIC-PCR and microbial community molecular hybridization. On the other hand, the structural features of SPF piglets’ intestinal microbiota were analyzed by the above techniques.Five litters of Bama mini-piglets were delivered by caesarean section and then man-reared in barrier facility. During the first three days, donor’ fecal suspension was inoculated orally into piglets daily and then geared to once every other day until on 11^th day. On the 5^th, 9^th, 12^th day piglet’s fecal samples were collected and frozed at -70℃. The results showed that human microbiota could be partly colonized and stabilized relatively in the piglets’ gut by ERIC-PCR and microbial community molecular hybridization analysis while the difference between the piglets were negligible.更多还原