【作者】 林荔雯；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2007， 博士

【摘要】 猪传染性胸膜肺炎（Porcine Contagious Pleuropneumonia，PCP）是由胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）引起的猪的一种高度接触传染性呼吸道疾病，给世界各地的养猪业造成了巨大的经济损失。预防和控制该病的主要措施是利用本地主要流行的血清型进行免疫接种。目前商品化的全菌灭活疫苗和亚单位疫苗能够减轻同源血清型菌引起的临床症状并降低死亡率，但不能降低发病率、慢性感染和阻止肺部病变，对异源血清型菌的感染也不能提供完全的交叉保护。可以说，采用灭活苗和亚单位疫苗免疫不是最好的选择，迫切需要更安全、高效的新型疫苗来预防和控制该传染病的发生与流行。与灭活疫苗和亚单位疫苗不同，APP的自然感染或试验感染能够诱导抗任何异源血清型的保护。而且弱毒活疫苗与传统疫苗相比有能够重复提呈抗原和持续性刺激免疫应答的优点。因此通过缺失毒力因子构建弱毒活疫苗已成为国内外疫苗研究的热点。构建APP突变株的传统方法存在很多缺陷。利用化学或转座子介导的突变方法存在随机性，遗传背景不明晰；而通过传统的同源重组方法导致靶基因突变而获得的突变株最后含有抗性标记，由于不符合生物安全性要求不能作为疫苗株用于疫苗生产。鉴于上述背景，本研究旨在以本地分离APP-1型菌株SLW01为亲本菌，缺失ApxI和ApxⅡ的激活因子apxⅠC和apxⅡC，构建不含抗性标记的APP双基因缺失株，从而发展一个能提供有效的交叉保护效率的无外源基因的安全、廉价的APP弱毒活疫苗菌株来预防和控制猪传染性胸膜肺炎的发生与流行，主要的研究内容包括：1．基因的克隆与重组自杀性质粒的构建参照GenBank中apxⅠapxⅡ基因的序列，从SLW01中扩增了apxⅠC和apxⅡC基因的上下游片段。将得到的上下游片段，连接到携带蔗糖敏感基因（SacB）的自杀性质粒pEMOC2上，构建缺失了385 bp的apxⅠC基因的重组自杀性质粒pEM△ⅠC和缺失了340 bp的apxⅡC基因的重组自杀性质粒pEM△ⅡC。2．接合转移与缺失株SLW02、SLW03的筛选鉴定以转化了重组自杀性质粒pEM△ⅠC的大肠杆菌β2155为供体菌，APP-1型菌株SLW01为受体菌进行接合转移。涂布氯霉素（Cm）抗性平皿并转印到含10％蔗糖的平皿上，筛选Cm抗性（Cm^R）蔗糖敏感（Sur^s）的接合子，进一步用PCR鉴定重组自杀性质粒已经整合到染色体中。鉴定正确的接合子在不含NaCl的培养基中培养促使第二次同源重组的发生。涂布含10％蔗糖的平皿，并转印到Cm平皿上，筛选出Cm敏感（Cms）蔗糖抗性（Sur^R）的克隆，用进行PCR鉴定，以确定发生了第二次交换，重组自杀性质粒已经丢失。从而构建单基因缺失株SLW02。在单基因缺失株SLW02（△apxⅠC）的基础上，用转化了质粒pEM△ⅡC的大肠杆菌β2155作为供体菌，单基因缺失株SLW02（△apxⅠC）作为受体菌进行接合转移，用同样的方法构建双基因缺失株SLW03（△apxⅠC/△apxⅡC）。3．缺失株生物学特性的研究在绵羊血平皿上测定SLW01、SLW02和SLW03的溶血活性，结果显示SLW01具有极强的溶血活性，单基因缺失株SLW02（△apxⅠC）具有较弱的溶血活性，而双基因缺失株SLW03（△apxⅠC/AapxⅡC）则失去了溶血活性。用Western blot检测双缺失株SLW03和亲本菌SLW01中毒素的分泌情况，结果表明SLW01和SLW03都能够分泌毒素Ⅰ和毒素Ⅱ，说明apxⅠC和apxⅡC基因缺失后，SLW03仍然可以分泌无毒力的毒素。将缺失株和亲本株都进行活菌计数，绘制生长曲线，结果表明，缺失株SLW02和SLW03与亲本菌比较，生长速度没有明显的变化，说明apxⅠC和apxⅡC的缺失对SLW01的生长无影响。测定亲本菌和缺失菌在Balb/C小鼠体内的毒力，计算LD₅₀的值。结果显示，与亲本菌SLW01比较，单基因缺失株SLW02（△apxⅠC）的毒力降低了约200倍，双基因缺失株SLW03（△apxⅠC/AapxⅡC）的毒力降低了约2000倍。4．双基因缺失株SLW03（△apxⅠC/AapxⅡC）对小鼠的免疫试验及攻毒保护试验将SLW03和1、2、7型三价灭活疫苗分别免疫Balb/C雌鼠，免疫2次，间隔2周。于免疫前、一免后2周和二免后2周分别检测了APP-1 IHA抗体和毒素ELISA抗体水平。结果表明，未免疫组的小鼠在整个试验过程血清抗体均呈阴性。结果显示，SLW03免疫小鼠后不但能够刺激机体产生较高的菌体抗体，而且能够产生较高的毒素抗体。灭活苗免疫组只能产生IHA抗体，不能产生毒素抗体。二免两周后，分别用低剂量(10 LD₅₀)和高剂量(100 LD₅₀)的同源血清型1型（SLW01）和异源血清型7型（XFNZ）攻毒。对于低剂量的攻毒，SLW03免疫组和灭活疫苗免疫组的小鼠都得到了100％的保护。对于高剂量的攻毒，SLW03对1型的保护率是100％，7型是75％。而灭活疫苗免疫组对1型和7型的高剂量的攻毒的保护率分别为25％和37.5％。所以弱毒菌株SLW03在小鼠中的免疫效力优于传统灭活疫苗。5．SLW03（△apxⅠC/△apxⅡC）对断奶仔猪的免疫试验及攻毒保护试验将SLW03分别通过肌肉免疫和鼻内免疫两种途径免疫断奶仔猪，免疫2次，间隔2周。于免疫前、一免后2周和二免后2周分别检测了APP-1 IHA抗体和毒素ELISA抗体水平。结果表明．两个免疫组的IHA抗体和ELISA抗体都显著高于对照组。但第28天，鼻内免疫组的ELISA抗体水平显著高于肌肉免疫组。二免两周后分别用同源血清型（1型）和异源血清型（9型）进行攻毒。结果表明，所有免疫组的猪都得到了100％的保护，对照组的猪并没有产生任何保护。免疫组的猪肺损伤指数和临床症状指数都显著低于未免疫组，相同血清型攻毒的鼻内免疫组的肺损伤指数显著低于肌肉免疫组。所以鼻内免疫途径比肌肉免疫途径好。更多还原

【Abstract】 Porcine contagious pleuropneumonia （PCP） caused by Actinobacilluspleuropneumoniae （APP） is a severe, contagious pulmonary disease of pigs that causesimportant economic losses in industrialized pigs production worldwide.The first observation of this disease was made by Pattison et al. （1957）, now occursin most pig-keeping countries. Immunization is the main method to prevent and control it.The current dominant commercial vaccines are still killed whole cell bacterins andsubunit vaccines, which generally reduce the mortality of APP infection. However thosevaccines frequently failed to prevent severer morbidity and economic losses, due to thechronic effects of the disease, on growth rate and feed efficiency.In contrast, natural and experimental infection can induce protection against anyheterologous serotype. The attenuated live vaccines could re-present protective antigensand stimulate a lasting immune response that may be more efficacious in preventing thedisease. Therefore, studies of A. pleuropneumoniae vaccines tend to focus on liveattenuated vaccines constructed by inactivating virulence-associated genes. The methodsdescribed previously for the construction of mutant strains of A. pleuropneuminiae havesome disadvantage. For example, mutagenesis using a chemical or a transposon mediatedapproaches present a random approach and allow for the selection of mutants with adistinct phenotype only; muttions not resulting in a distinct phenotype can not beconstructed by these means. All these mutants, which are achieved by systems allowingtargeted mutagenesis by homologous recombination, however, carry a permanentantibiotic resistance marker and, therefore, are not suitable as vaccine strains. Because theApx toxins are confirmed to be particularly important in the virulence and induction ofcross-protective immunity, a live attenuated vaccine strain with avirulent RTX toxinsmust be the optimal candidate for an A. pleuropneumoniae vaccine.Based on above considerations, we developed an attenuated A. pleuropneumoniaeserovar 1 strain, SLW03, which expressed and secreted inactive ApxⅠA and ApxⅡA, bydeleting the apxⅠC and apxⅡC genes of the SLW01 strain in this study. A potentialvaccine candidate was produced without an antibiotic resistance marker by a sucrosecounter-selection strategy. Actinobacillus pleuropneumoniae serovar 1 is the predominantserovar in China, and a strain that produces ApxⅠand ApxⅡtoxin was chosen. Here wedescribe the construction and characterization of the double mutant, SLW03, testing of itsvirulence in mice, and demonstration of its cross-protective efficacy against challengewith virulent A. pleuropneumoniae in pigs. The main research was described as follows:1. Cloning of genes and construction of recombination suicide plasmids According to the GenBank sequences, the two flanks of both apxⅠC and apxⅡCgenes were amplified and cloned from SLW01 genome. Those genes were highlyconserved compared with the sequences published. The upstream and downstream ofapxⅠC and apxⅡC gene were respectively subcloned into suicide plasmid pEMOC2,which contained a sucrose sensitive （sacB） gene. The recombination suicide plasmidswere designated as pEM△ⅠC and pEM△ⅡC which contained 385bp-deleted apxⅠC and340 bp-deleted apxⅡC respectively.2. Transconjugation and identification of the mutants SLW02 and SLW03The single mutant strain SLW02, from which only apxⅠC was deleted, wasconstructed first. The E. coil donor strainβ2155 transformed with plasmid, pEM△ⅠC, wasconjugated with the recipient, SLW01. After transconjugation, Chloramphenicol-resistant（Cm^R） transconjugants were analyzed for the presence of a first crossover event by PCR.This first step selected for clones in which the whole plasmid had been incorporated intothe recipient chromosome. Colonies with the correct PCR profile were incubated in TNB.Aliquots were then plated on to TNA plates containing 10% （w/v） sucrose. Afterincubation for 24 h, sucrose-resistant （Sue^R） colonies exhibiting a non-mucoid phenotypewere tested for the chloramphenicol sensitivity （Cm^s） phenotype, which was indicative ofloss of plasmid vector sequences. Following this, Sue^R and Cm^s colonies were identifiedusing PCR to determine the presence of the second crossover. The double mutant strainSLW03 was constructed using the same method based on the single mutant strain SLW02.The single mutant strain SLW02 was used as the recipient strain, and the E. coilβ2155transformed with plasmid pEM△ⅡC was used as the donor strain.3. Characterization of the mutantsTo investigate hemolytic activity, colonies of the mutant or parent strains wereinoculated on to TSA plates containing fresh, defibrinated sheep erythrocytes and 10μg/ml NAD. Plates were incubated for 24 h at 37℃and hemolysis was assessed visually.Clear zones around the colony indicated hemolysis activity. After 24 h culture, the parentstrain SLW01 showed strong hemolytic activity in an approximately 2-3 mm zone. Thesingle mutant strain, SLW02, showed weak hemolytic activity, giving a 0.5-1 mm zone,but the double mutant strain SLW03 produced no hemolysis.The expression of ApxⅠand ApxⅡproteins in the double mutant strain SLW03 wasexamined by two separate Westem blot analyses （using the anti-ApxⅠA monoclonalantibody and using the anti-ApxⅡA monoclonal antibody） with TE buffer as the negativecontrol. In the Western blot using the anti-ApxlA monoclonal antibody, a unique proteinband with a molecular mass of 110 kDa was shown in both the supematant of the double mutant strain SLW03 and the supernatant of the parent strain SLW01. In the Western blotusing the anti-ApxⅡA monoclonal antibody, a band of the expected 105 kDa was seen inthe supernatants of both the double mutant strain SLW03 and the parent strain SLW01.The results of the Western blot analysis indicate that the double mutant strain SLW03 isstill capable of secreting ApxⅠand ApxⅡafter deletion of apxⅠC and apxⅡC, as is theparent strain, SLW01.For growth of SLW03, cultures of the parent strain SLW01 and mutant strain SLW03were grown with shaking at 37℃in TSB （supplemented with NAD and sterile bovineserum） overnight. The cultures were sub-inoculated into fresh supplemented TSB at a1:1,000 dilution. The OD₆₀₀ of the bacterial cultures was determined at intervals of 1 h.No obvious difference was observed in the in vitro growth curves of SLW01 and SLW03,indicating that deletion of the apxⅠC and apxⅡC genes had no significant influence on thegrowth ofA. pleuropneumoniae SLW01.4. The immune and protective assay of the double mutant strain SLW03 in miceEighty female Balb/C mice were divided into three big groups. Group 1 of 32 micewere vaccinated 2x10⁸ CFU SLW03. Group 2 of 32 mice were injected with 0.2 mLkilled vaccine. Group 3 of 16 mice were injected with TSB as control. Two weeks afterthe first vaccination, mice were vaccinated again at the same dose. Two weeks otter thebooster immunization, mice were challenged with difference dose of SLW01 （serovar 1）and XFNZ （serovar 7）.Mice in control group were all death within 12 h after challenge. Mice in bothSLW03 vaccinated group and killed cell vaccinated group were gained 100% protectionagainst the low dose challenge. With the hige dose challeng, SLW01 still offered mice100% protection against homologous serovar and 75% protection against heterogenousserovar. While the killed cell vaccine only offered respectively 25%and 37.5%protection against challenge with homologous and heterogenous serover.Sera from unvaccinated control mice did not display ELISA or IHA antibodiesthroughout the experiment. A significant difference was observed between the SLW03vaccinated group and the control group （P＜0.01）. There was no significant differences inELISA-ApxⅠor -ApxⅡtiters were observed between killed cell vaccinated group and thecontrol group （P＞0.05）, the antibody level in the IN immunization group was higher thanthat in the IM group on day 28 and the difference was statistically significant （P＜0.05）.The results suggest that the attenuated live vaccine SLW03 is better than the killed cellvaccine in mice.5. The immune and protective assay of the double mutant strain SLW03 in pigs Thirty-six 6-week-old pigs were randomly divided into six experimental groups ofsix. Groups 1 and 4 were vaccinated intramuscularly （IM） twice with 1x10⁹ CFU ofSLW03 in 1 ml TSB. Groups 2 and 5 were inoculated twice intranasally （IN） with thesame dose of SLW03 at an interval of 2 weeks. Groups 3 and 6 served as unvaccinatedcontrol groups inoculated with TSB. Two weeks after the booster immunization, groups 1,2 and 3 were challenged intratracheally with SLW01 （serovar 1）. Groups 4, 5 and 6 werechallenged with SHB09 （serovar 9）. Groups 3 and 6 therefore served asunvaccinated/challenged control groups.One hundred percent （6/6） protection against challenge with homologous （SLW01）and heterologous （SHB09） A. pleuropneumoniae was conferred on the pigs immunizedwith SLW03 by the IM and IN routines, compared with no protection in unvaccinatedpigs. After challenge, pigs in the IM and IN vaccinated groups showed slight depressionand decreased appetite for 2 days. These signs were not observed after the third daypost-challenge. All pigs in unvaccinated groups 3 and 6, challenged with serovars 1 and 9,respectively, showed severe respiratory distress and were euthanized （except one in group6） within 72 h after challenge. Postmortem examination showed severe lung lesions,pleural effusion and adhesive pleuritis in the unvaccinated/challenged pigs. Both the IMvaccinated/challenged group （1 and 4） and the IN vaccinated/challenged groupindividually showed significantly lower clinical scores compared with theunvaccinated/challenged group （P＜0.01）. However, there was no significant differencebetween the IM vaccinated/challenged group and the IN vaccinated/challenged group（P＞0.05）.All unvaccinated pigs challenged with serovars 1 and 9 showed adhesive pleuritisand focal lung abscesses. In group 1, one of the six pigs showed disseminated pulmonaryhemorrhage, and two pigs showed localized hemorrhagic foci in the lungs; the other threepigs had no detectable lung lesions. In group 2, only one pig showed localized pulmonaryhemorrhage; the other five had no observable lung lesions. In group 4, two of six pigswere detected disseminated pulmonary hemorrhage, and three pigs showed localizedhemorrhagic foci in the lungs. In group 5, two pigs were observed localized pulmonaryhemorrhage; the others had no detectable lung lesions. The lung lesion scores in the IMand IN vaccinated/challenged groups （groups 1, 4 and groups 2, 5） were significantlylower than those of the unvaccinated/challenge pigs （groups 3 and 6） （P＜0.01）. The lunglesion scores of the IN vaccinated/challenged groups, 2 and 5, was lower than those of theIM vaccinated/challenge groups, 1 and 4, and the difference was statistically significant（P＜0.05）. The result indicates that IN immunization is more effective than the IM route. A large increase in IHA and ELISA antibody titers in pigs given IM or INimmunization was observed alter the initial （day 14） or second （day 28） vaccination withlive vaccine SLW03. A significant difference was observed between IM or IN vaccinatedgroups and unvaccinated groups （P＜0.01）. Although no significant differences inELISA-ApxⅠor -ApxⅡtiters were observed between IN-and IM-immunized groups onday 14 （P＞0.05）, the antibody level in the IN immunization group was higher than that inthe IM group on day 28 and the difference was statistically significant （P＜0.05）. Inaddition, no significant difference in IHA titers was observed between the IN and IMimmunization groups （P＞0.05） following either the initial or second vaccination. Serafrom unvaccinated control pigs did not display ELISA or IHA antibodies throughout theexperiment.更多还原