猪胸膜肺炎放线杆菌的分离和鉴定

某猪场哺乳仔猪及断奶猪临床表现喘气、咳嗽、皮肤发绀以及胸膜肺炎的病理特征，从病猪体内分离出3株菌，根据细菌培养特性、CAMP试验及生化反应等鉴定为猪胸膜肺炎放线杆菌（App)。该菌对小白鼠有一定毒力，回归猪可致死猪只，对常用治疗药有一定的抵抗力，用分离菌制备自家苗可预防本病发生。     

猪胸膜肺炎放线杆菌的分离鉴定

通过实验室检验，从福建省2个送检猪场猪的肝。脾病料中分离出2株猪胸膜肺炎放线杆菌，结合临床表现，推断它们均具有较强的致病性。     

胸膜肺炎放线杆菌apxIIC-/kanr 基因缺失减毒株的构建

重组转移载体pBSKA通过电转化导入亲本菌胸膜肺炎放线杆菌血清7型（APP-7）WF83株,电转化后的产物涂布于TSB／Kan平板,2d获得突变株。卡那霉素抗性实验证实突变株有卡那霉素抗性;NAD依赖性实验证实突变株依赖NAD生长;PCR鉴定证实了卡那霉素抗性基因置换了apxlIC基因,并证实突变株中无pBSKA质粒的存在;溶血活性实验证实突变株完全失去了溶血活性;细胞毒性实验证实突变株的细胞毒性完全丧失;对小鼠的安全性实验证实突变株的毒力显著减弱,突变株对小鼠是安全的;遗传稳定性实验证实,突变株在体外连续传30代和在体内传10代均不会发生卡那霉素抗性的丢失。结果表明实验成功构建了基因缺失减毒株,为进一步以此突变株作为基因工程弱毒活疫苗株开展研究奠定了一定的基础。     

3株猪胸膜肺炎放线杆菌山东株的分离及鉴定

从山东省几个猪场的疑似猪传染性胸膜肺炎病例中,采集病猪的肺脏、气管和扁桃体等病变组织,经细菌分离得到3个分离株BZ1株、BZ2株、BZ3株。经过革兰氏染色镜检、菌落形态观察、培养特性试验、生化试验、药敏试验、动物感染试验等一系列细菌学鉴定方法,可将3株分离菌鉴定为猪胸膜肺炎放线杆菌。     

猪胸膜肺炎放线杆菌的分离与鉴定

吉林省某规模化养猪场送检疑似猪传染性胸膜肺炎的死猪病料,采用不同培养基进行病原菌分离,结果分离出3株疑似胸膜肺炎放线杆菌。通过对分离菌进行镜检、培养特性试验、生化特性试验、PCR鉴定,确定为3株均为胸膜肺炎放线杆菌,致病性试验结果表明,分离的胸膜肺炎放线杆菌具有强致病性。     

猪传染性胸膜肺炎放线杆菌的分离和鉴定

对重庆某猪场病死仔猪,经剖解从气管和肺脏上分离得到一株革兰氏阴性短杆菌,通过实验室诊断,表明该菌在血培养基上生长并呈现β溶血,生长需要NAD,尿素酶等呈阳性;CAMP试验呈阳性;能感染小白鼠并对小白鼠有很强的致病作用。通过实验室诊断结果,结合发病猪群的流行病学特征、临床症状和病理学特征,最终鉴定此菌为猪传染性胸膜肺炎放线杆菌。     

猪胸膜肺炎放线杆菌的分离鉴定

采用巧克力琼脂平板从贵州省某养猪场发病猪体分离到3株细菌,经培养特性观察、生化特征检查和血清型鉴定,确定3株分离菌均为猪胸膜肺炎放线杆菌血清7型。经药敏试验显示,分离菌对氨苄西林、硫酸庆大霉素、丙氟哌酸、氟哌酸、头孢三嗪和四环素等药物高度敏感。     

猪胸膜肺炎放线杆菌的分离与鉴定

猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae，APP)可引起猪以纤维素性肺炎为主要特征的传染性胸膜肺炎，该病具有高度传染性，是规模化养猪场猪的主要呼吸道疾病。APP自1957年由英国的Pattison首次报道以来，现已广泛存在于世界各养猪国家，且各年龄的猪均易感。在我国1990年杨旭夫正式报道了APP的存在，近年来其流行日趋严重，给养猪业造成了较大损失。     

Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes worldwide severe losses in pig farming. The virulence of the 15 serotypes of A. pleuropneumoniae is mainly determined by the three major RTX toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX toxin, ApxIV, is produced by all 15 serotypes only during infection of pigs, but not under in vitro conditions. Pigs infected with A. pleuropneumoniae show specific antibodies directed against ApxIV. In contrast, antibodies against the other three toxins ApxI, ApxII and ApxIII are also found in pigs free of A. pleuropneumoniae. The antibodies to the three latter might result from other, less pathogenic Actinobacillus species such as A. rossii and A. suis. We used a recombinant protein based on the N'-terminal part of ApxIV to serologically detect A. pleuropneumoniae infections in pigs by immunoblot analysis. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected A. pleuropneumoniae infections in the second to third week post infection. We developed an indirect ELISA based on the purified recombinant N'-terminal moiety of ApxIV. The analysis of sera from pigs that were experimentally or naturally infected by A. pleuropneumoniae, and of sera of pigs that were free of A. pleuropneumoniae, revealed that the ELISA had a specificity of 100% and a sensitivity of 93.8%. The pre-validation study of the ApxIV-ELISA revealed that the latter was able to detect A. pleuropneumoniae-positive herds, even when clinical and pathological signs of porcine pleuropneumonia were not evident. Pigs vaccinated with a subunit vaccine Porcilis App were serologically negative in the ApxIV-ELISA.     

The seroprevalence of Actinobacillus pleuropneumoniae in Swiss pig breeding herds--a study with the ApxIV ELISA

At the end of the national eradication program for Enzootic Pneumonia (EP) and Porcine Actinobacillosis (APP) in Switzerland (2003), A. pleuropneumoniae serotype 2 is considered to have been eradicated. There is no current information about the distribution of the other serotypes available. The ApxIV ELISA detects antibodies against all serotypes of A. pleuropneumoniae, without cross-reaction with other bacterial species. The aim of this study was to achieve actual data concerning the seroprevalence of A. pleuropneumoniae in breeding-herds and to validate the ApxIV ELISA under field conditions, especially for the diagnosis of latently infected breeding-herds without clinical signs, and to achieve more information about the role of herd book farms for the spread of the infectious agent. A total of 2068 serum samples from 96 pig herds in Switzerland were examinated. Over half of the examinated herd book farms showed positive results in this ELISA. 93% of the breeding herds were positive. On single animal level sensitivity was 96% and specifity 100%. Herd sensitivity ranged between 67% and 99%. Herd specifity was 100%. The results show that the ApxIV ELISA is a valuable tool for the detection of latently infected herds.     

猪胸膜肺炎放线杆菌血清7型抑制消减杂交文库的构建和分析

为了研究猪传染性胸膜肺炎放线杆菌（APP）不同血清型之间的差异表达基因,采用抑制消减杂交（SSH）法,以APP血清7型DNA作为测试方（Tester）、APP血清1型DNA作为驱动方（Driver）,进行2轮杂交和2轮选择PCR扩增,将PCR扩增产物连接pMD18-T载体,筛选阳性克隆后进行测序和序列比对.结果显示,15个阳性克隆中的13个为已知序列,其同源性为88%～100%,2个为未知序列,其结构和功能还需进一步研究.首次构建了APP7的抑制消减杂交文库,为APP感染和致病机制的研究奠定了基础.     

Molecular Identification and Antimicrobial Susceptibility Test of Porcine Contagious Actinobacillus pleuropneumoniae Serotype 3

In order to understand the serotype and drug resistance of porcine contagious Actinobacillus pleuropneumoniae(APP), a pair of primers was designed according to GenBank database to amplify specific 950 bp fragment, and the molecular identification and antimicrobial susceptibility test of APP serotype 3 were investigated.The results showed that the PCR product sequences were more than 99% homology with the APP serotype 3 published in GenBank.The isolated strains were highly resistant and multiple drug resistance.The molecular identification and antimicrobial susceptibility test of APP serotype 3 provided the basis for the identification, diagnosis and prevention of porcine contagious pleuropneumonia.     

Serotype and apx genotype profiles of Actinobacillus pleuropneumoniae field isolates in Korea.

A total of 100 field isolates of Actinobacillus pleuropneumoniae isolated from lung tissues of pigs with severe pleuropneumonia were serotyped by slide agglutination and precipitation tests. Polymerase chain reactions for apxICA, apxIICA, apxIIICA, apxIBD and apxIIIBD genes were used to determine their genotype prevalence. Serotypes 2 (56 isolates), 5 (28 isolates) and 6 (11 isolates) were the most common; only two isolates belonged to serotype 7, and three were untyped. Among the 97 isolates identified by serotype, 70 had the same apx genes as their respective serotype reference strains, but 27 did not have any of the apx genes present in the corresponding serotype reference strain. Among these 27 isolates, 10 were serotype 2, 12 were serotype 5, three were serotype 6 and two were serotype 7.     

猪接触传染性胸膜肺炎放线杆菌血清3型分子鉴定及药敏试验

为了解猪接触传染性胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)的血清型及耐药情况,本研究根据GenBank数据库设计1对引物,特异性的扩增950 bp核苷酸片段,对血清3型APP进行分子鉴定及药敏试验。结果显示,所得PCR产物经过测序,与GenBank已发表的血清3型APP的同源性达99%以上,所分离菌株耐药性较强,大多为多重耐药。通过对血清3型APP进行分子鉴定及药敏试验,为猪传染性胸膜肺炎的鉴定、诊断及防制提供了基础。     

猪胸膜肺炎放线杆菌血清3型间接血凝试验检测方法的建立

本试验用分离纯化的猪胸膜肺炎放线杆菌（APP）3型荚膜多糖（CPS）,致敏双醛化处理的绵羊血红细胞,建立了APP的血清学诊断方法,结果表明,该方法特异性强,仅与APP 3型阳性血清反应,可以用其进行APP疫苗免疫后抗体检测,该诊断方法简单、快速,适用于临床疾病的诊断。     

猪链球菌2型溶血素基因缺失菌株的构建及生物学特性的研究

溶血素(SLY)在猪链球菌2型(SS2)侵入和裂解细胞的过程中发挥重要作用,被认为是SS2的一种重要的毒力相关因子。为了探讨SLY在2005年中国四川资阳分离强毒株SS205ZY致病过程中的作用,作者利用同源重组基因敲除法成功构建了SS205ZY的sly基因敲除突变菌株(Δsly)。并比较了菌株的溶血能力以及对小鼠的致病力。结果表明sly基因敲除后可导致猪链球菌裂解红细胞的能力显著下降,对小鼠的致病力也有一定程度的减弱,但仍表现较高的致病力。本研究结果提示SLY是猪链球菌裂解细胞的重要毒力相关因子,但造成SS205ZY的高致病性很显然与多种毒力相关因子的协同作用有关。     

猪传染性胸膜肺炎放线杆菌血清型7型25-4株ApxⅡCA基因的克隆和基因缺失

猪传染性胸膜肺炎放线杆菌血清型7型25-4株ApxIICA基因用特异性引物进行PCR扩增并克隆到T载体上,构建重组质粒pMD18-ApxIICA,将pMD18-ApxIICA转化到E.coli JM83中并测序.序列分析表明血清型7型25-4株ApxII CA与其它血清型(5型和9型)核苷酸序列同源性达98%以上,氨基酸序列同源性达90%以上.利用ApxIIC基因内部单一的SpeI位点,设计特异性的引物,利用PCR技术在ApxIIC基因内部缺失165bp的核苷酸片段,PCR产物再用SpeI进行酶切,并体外连接构建得到含有ApxIIC缺失基因的重组质粒pMD18-ApxII△/CA.     

猪胸膜肺炎放线杆菌ArcA基因在低氧环境下的转录变化

为研究低氧条件对猪胸膜肺炎放线杆菌（Acfinobacillus pleuropneumaniae，APP）血清1型ArcA基因表达的影响，并探讨ArcA在低氧环境中的作用机制，作者建立了体外培养模型，在低氧环境中培养APP，采用半定量RT-PCR方法，以看家基因recF为内参，检测ArcA基因在低氧条件下的表达水平。结果表明ArcA基因在常氧条件下不转录或低转录，而在低氧环境下高转录，并呈现转录量先升高后降低的趋势。这表明ArcA基因可能是胸膜肺炎放线杆菌潜在的毒力基因，在致病过程中发挥重要作用。