【作者】 罗勇；

【导师】 吴斌；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 猪瘟(classical swine fever;CSF)是由猪瘟病毒(classical swine fever virus;CSFV)引起的猪的一种高度接触性传染病,死亡率非常高。目前,繁殖母猪往往呈现猪瘟病毒持续性感染状态,作为传染源而长期存在于猪群中,这是规模化猪场猪瘟持续发生的一个重要原因。如何准确的检测出猪瘟带毒阳性种猪并及时进行淘汰,对于规模化猪场控制甚至消灭猪瘟具有非常重要的意义。在开展种猪群净化工作的同时,针对仔猪群,应当采取有效的免疫方案,提高仔猪群的猪瘟抗体水平,以抵御猪瘟病毒对仔猪的感染。因此本研究围绕着建立能灵敏准确检测猪瘟带毒猪的诊断方法和提高仔猪群的免疫抗体水平开展了以下工作:1.猪瘟病毒套式RT-PCR检测方法的建立及其在种猪群猪瘟净化中的运用利用序列分析软件(VectorNTI8)对NCBI数据库中不同国家和地区发布的29株猪瘟野毒及6株疫苗毒株的E2基因序列及BVDV基因序列的相应位置进行了比较分析,设计了两对引物,外套引物扩增E2基因中目的片段大小为806bp,内套引物扩增大小为512bp。经各种反应条件的优化,建立了猪瘟病毒套式RT-PCR检测方法。结果表明:该方法的灵敏度与real-time RT-PCR相当,比普通RT-PCR提高了100倍;运用该方法对中国兽药监察所提供的19株猪瘟病毒的核酸及6种猪源常见病原的核酸样品进行检测,结果显示,19株猪瘟病毒的核酸样品检测均呈阳性,其它样品则为阴性。运用本方法对种猪猪瘟疫苗免疫后的扁桃体疫苗毒株带毒规律的研究,结果表明:当种猪免疫5头份猪瘟兔化弱毒细胞疫苗(750RID)25d之前均存在种猪扁桃体携带疫苗毒,免疫30d后进行扁桃体活体取样检测全部呈阴性。为临床如何有效的避免疫苗免疫对野毒检测的影响提供试验依据。运用该方法在4个规模化猪场开展猪瘟的净化工作,共检测了种猪扁桃体样品1491份,阳性数为66份,阳性率为4.43%,并将阳性猪只实行了淘汰。同时对部分阳性样本进行了核酸序列分析,显示扩增的阳性样本均为猪瘟野毒株而非疫苗毒株。并对其中某场2008年不同时间送检的阳性样本的PCR产物进行核酸序列分析。结果表明,不同时间检测到的猪瘟阳性样本属于同一猪瘟野毒株。以上研究表明,本研究建立的套式RT-PCR方法适用于规模化猪场种猪群猪瘟野毒阳性猪只的剔除,为控制和净化种猪群猪瘟提供技术支撑。2.规模化猪场猪瘟免疫状况调查及仔猪的免疫防控为了调查规模化猪场猪瘟的免疫方案,比较及评价其免疫效果,本研究将湖北省7个规模化种猪场作为研究对象,将猪群分为经产母猪、种公猪、后备母猪、哺乳仔猪(0～30日龄)、保育仔猪(3～60日龄)和生长猪(61日龄以上)6个群体,并按各猪群5%～8%的比例采血,共采集了1650份血样,检测猪瘟抗体。结果表明:各猪群猪瘟免疫合格率分别为99.55%、96.43%、99.22%、89.88%、81.68%及78.64%,不同免疫方案下,各场猪群表现出不同免疫效果。其中保育仔猪的猪瘟免疫是各猪群中最为薄弱的环节。因此开展了某规模化猪场的仔猪猪瘟母源抗体消退规律的摸索及仔猪猪瘟最佳首免日龄的优化试验,结果显示:仔猪猪瘟母源抗体水平随仔猪日龄的增长而逐渐降低,14日龄至28日龄抗体水平下降最快,35日龄时已有70%的猪只在保护界限之下,该场猪瘟最佳首免日龄为28日龄左右,并根据结果进行了调整。本研究的开展对规模化猪场制定合理的猪瘟免疫方案具有较好的参考价值。更多还原

【Abstract】 Classical swine fever (CSF) is a highly contagious viral disease of pigs caused by Classical swine fever virus (CSFV), and the mortality rate is often very high. Now, brood sow usually take CSFV which become infective agent for long, this is a significant reason that CSF always happen in scale swine field.How to select the brood sow taking CSFV in time that is significance for controlling and eliminating CSF in scale swine field.However, for controlling and preventing piglet away from CSFV,we should take well arranged measure and get effective the immunity program.thus, In order to search for eradication measure of CSF in breeding swine herd and boost antibody level of piglet herd,the study carry out following work.1. Establishment and clinical application of RT-nested PCR detection for classical swine fever virusUtilizing VectorNTI8 to contrast and analyze the E2 sequence of 29 wild CSFV strains , 6 vaccine strains and BVDV published by Different country and area, Devised two couple primer,Amplification target size of outer and inner primer is 806bp and 512bp. Test result indicate that the Sensitivity of RT-nested PCR and real-time RT-PCR is corresponding and raise 100 time contrasting general RT-PCR , the result of detecting 25 standard samples indicate the RT-nested PCR is specificity for amplificating CSFV sequence.And applied the RT-nested PCR to search for rule that brood sow’s tonsils take vaccine virus after immunization,the result show that when brood sow immunize 750RID of cell cultures vaccine , brood sow’s tonsils taking vaccine virus could last 25d.Thus,maybe we can say the RT-nested PCR can distinguish wild virus infection when brood sow immunize 750RID of cell cultures vaccine 30d later.Accomplished above studies later, we engaged in eradication measure of CSF in 4 breeding swine fields with the RT-nested PCR.and detect 1498 brood sow’s tonsils,the result show that wild virus positive number is 66, positive rate is 4.43%.moreover,PCR product of positive sample and vaccine strains coming from a scale swine field during 2008 were carried out nucleotide sequencing,the result indicated that it just exist a wild virus strains in the field. And other nucleotide sequencing of positive sample show that the RT-nested PCR is just applied to amplificate wild CSFV strains.All clinical trial indicated that the RT-nested PCR refer to detect wild CSFV strains existing persistently in brood sow herd at scale swine field. Development of RT-nested PCR detection for classical swine fever virus that offered technology support to control and eliminate CSF of brood sow herd. 2.Survey of Classical Swine Fever Immunization level in Large-scale Pig Farms and Optimization of Piglets’ ImmunizationIn order to investigate immunization schedules of classical swine fever(CSF) in large-scale pig farms, and to evaluate the immune effects, seven large-scale pig farms in Hubei province were studied. Swine herds were divided into the sows, the boars, the prepubertal gilts, the breast-feeding piglets (about 0～30 days), the nursing piglets (about 31-60 days old) and the growing pigs (above 61 days old). In each swine herd, 5%～8% of pigs’ blood were collected and antibody titers were tested; Results showed that the passing rate of all swine herds’ CSF Immunization are 99.55%、96.43%、99.22%、89.88%、81.68% or 78.64%,and different immunization schedule induced different immune effects. Overall, the nursing piglets and the growing pigs is the weakest in all pig groups .Therefore, piglet’s CSF maternal antibody retrogression rule and first Immunity time of Piglet was studied based on the antibody investigation.In the study, CSF maternal antibody subsided following day-age of Piglets and these of 70% is under protection line.Results indicated that 28 days after birth was the best time of first immunization.This study has certain reference value to the pig farm’s formulation of reasonable CSF immunity schedule.更多还原