Characterisation of Australian isolates of Actinobacillus capsulatus, Actinobacillus equuli, Pasteurella caballi and Bisgaard Taxa 9 and 11

Objective The objective of this work was to perform a comprehensive phenotypic characterisation of 16 isolates of bacteria previously identified as Actinobacillus equuli.
Design The 16 isolates that had been obtained from Australian animals – 15 from horses and one from a rabbit – were compared with reference strains of A equuli, A capsulatus, Pasteurella caballi and Bisgaard Taxa 9 and 11.
Results The characterisation study demonstrated that only nine of the isolates were A equuli . The other isolates were identified as A capsulatus (the isolate from rabbit), P caballi (one isolate), Bisgaard Taxon 11 (two isolates) and Bisgaard Taxon 9 (one isolate). The final two isolates could not be assigned to any recognised species or taxa.
Conclusion This study has highlighted the importance of a complete characterisation of Actinobacillus -like organisms isolated from horses and rabbits. The study represents the first time that A capsulatus, P caballi and Bisgaard Taxa 9 and 11 have been recognised as being present in Australia.     

Biochemical fingerprinting and ribotyping of isolates of Actinobacillus equuli from healthy and diseased horses

A total of 112 isolates of Actinobacillus equuli, including both clinical isolates and isolates from the oral cavity of healthy horses, were included in this study. All isolates were ribotyped and 92 of the isolates were also typed biochemically, with the commercially available Pheneplate (PhP) system, which includes 48 different substrates. As expected, ribotyping was more sensitive than biochemical fingerprinting in detecting differences between the isolates. The correlation between the two methods used was poor. It was not possible to distinguish clinical isolates from normal flora isolates by either of the two methods used.     

猪传染性胸膜肺炎放线杆菌的套式PCR检测

根据猪传染性胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)apxⅣA毒素基因的序列,设计了两对特异性引物P1/P4和P6/P8,建立了检测APP全部15个血清型的套式PCR方法.对APP的15个国际标准血清型和国内的APP菌株进行了PCR检测,都能得到223 bp的特异性扩增产物;检测的灵敏度可达1.3 CFU,最低检出DNA浓度为9 fg.     

猪传染性胸膜肺炎放线杆菌的鉴定和耐药性研究

猪传染性胸膜肺炎是由猪传染性胸膜肺炎放线杆菌(Actinobacillus pleuralpneumoniae,APP)引起的一种高度传染性呼吸道疾病,临床上准确鉴定该病原菌并合理选择抗生素施药十分重要。研究比较了应用传统方法、PCR方法和MALDI TOF方法鉴定45株临床APP,并比较了三种优势血清型的毒力表型,最后测定了其对临床34种常见抗生素的MIC。结果发现MALDI TOF微生物学鉴定方法具有快速、准确的优势;临床菌株血清型主要包括3型（20株）、1型（13株）、7型（7株）,其他血清型菌株5株,其中1型毒力最强;APP对罗红霉素、头孢噻呋、恩诺沙星、氨苄西林等高度敏感,对四环素、土霉素等耐药率较高。研究为临床快速鉴定APP和合理选择用药提供了参考依据,为APP临床耐药折点的制定奠定了基础。     

胸膜肺炎放线杆菌的实时荧光PCR检测

猪传染性胸膜肺炎是由胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）引起的危害养猪业的五大疾病之一。至目前为止App共报道有2个生物型15个血清型。所有型都可能致病，但有显著差异。使用血清学方法监测猪传染性胸膜肺炎有其局限。一些猪细菌分离培养阳性，但血清反应仍为阴性，对这些猪群只能使用病原分离进行确诊。亚临床感染及处于潜伏期的动物，     

Serological characterisation of Actinobacillus pleuropneumoniae isolated from pigs in 1993 to 1996

OBJECTIVES: To clarify the serological identity of the prototype strain of a group of Actinobacillus pleuropneumoniae isolates that could not be serotyped in previous studies and to establish the serovar of 378 isolates of A pleuropneumoniae obtained from pigs in Australia over the period 1993 to 1996. DESIGN: After initial validation, QGD and IHA tests were used to characterise the prototype isolate (HS143) selected to represent the cross-reacting isolates that were found in a previous study. Next, 378 recent field isolates of A pleuropneumoniae were characterised using the existing gel diffusion serotyping technique and/or the IHA or QGD tests. RESULTS: The indirect haemagglutination test was shown to be capable of correctly recognising the reference strain for all serovars except serovar 11. While the quantitative gel diffusion test was not as effective as indirect haemagglutination, it could recognise serovar 11. When the two tests were used to examine the prototype strain (HS143) of the cross-reactive isolates, the results indicated that HS143 is serologically distinct from all 12 of the recognised serovars of A pleuropneumoniae. However, as HS143 was subsequently identified as serovar 12 by one of the leading international reference laboratories, the antiserum to isolate HS143 was used as the serovar 12 antiserum. A total of 346 of the 378 A pleuropneumoniae field isolates examined could be confidently serotyped with almost 90% of the isolates being either serovar 1 (104 isolates); serovar 7 (83 isolates) or serovar 12 (142 isolates). A range of other serovars and some cross-reactive isolates made up the remainder of the isolates. CONCLUSION: The serovar 12 antiserum produced against the international reference strain (1096) does not recognise Australian serovar 12 isolates. The antiserum raised against isolate HS143 does recognise the Australian serovar 12 isolates. The dominant serovars of A pleuropneumoniae infecting Australian pigs are (in decreasing order) serovars 12, 1 and 7.     

胸膜肺炎放线杆菌在不同培养基中生长、生物膜形成及Apx毒素分泌能力的差异研究

为比较胸膜肺炎放线杆菌(APP)血清1型～12型菌株在不同培养基中的生长、生物膜形成以及Apx毒素分泌能力的差异,选用5种细菌培养基BHI、LB、MHB、PPLO和TSB,首先测定APP不同血清型菌株在5种培养基中的生长曲线,进而测定不同血清型菌株在5种培养基中的生物膜形成能力,同时测定这些菌株对20种抗菌药物的耐药性...     

套式PCR和实时荧光PCR在检测胸膜肺炎放线杆菌上的应用

应用胸膜肺炎放线杆菌套式PCR和实时荧光PCR检测方法分别检测了不同来源的猪鼻拭子、肺、扁桃体等494份样品。套式PCR 77份阳性,实时荧光PCR 62份阳性,前者的阳性完全覆盖了后者的阳性,两者符合率为97%(479/494)。2种方法均可应用于猪传染性胸膜肺炎的诊断和监测。     

猪胸膜肺炎放线杆菌脂多糖的制备及对小鼠的免疫保护作用

采用酚-水法制备血清3型、4型、5型、7型和8型猪胸膜肺炎放线杆菌（APP）脂多糖（LPS），以该多糖免疫小鼠，进行同源攻毒保护试验，结果LPS在20斗∥只的免疫剂量下可对小鼠产生较强的保护作用，用同血清型菌株对免疫后的小鼠攻毒，仅表现肺脏轻微出血，无死亡，而对照组未经免疫直接攻毒的小白鼠全部死亡，肺脏严重出血；小鼠免疫后第2d就可以检测到抗体，并且抗体水平上升较快，到第6d抗体达到最高水平，之后，抗体水平开始下降，但下降幅度不大，可持续2个月左右；交叉保护试验结果表明血清3型LPS对血清5型和7型APP，血清4型LPS对血清5型和7型APP没有保护作用，免疫后的小鼠攻毒仍表现多数死亡；血清3型LPS对血清4型和8型APP有交叉保护作用，血清4型LPS对血清3型和8型APP有交叉保护作用，血清5型、7型、8型LPS对5个血清型的APP都有交叉保护作用，免疫后的小鼠攻毒无死亡，仅表现肺脏有不同程度的出血。上述结果表明LPS是APP的主要免疫保护性抗原之一，该研究为APP亚单位疫苗的研制及应用提供了理论依据。     

利用菌落多重PCR对传染性胸膜肺炎放线杆菌进行血清分型

参照文献报道的传染性胸膜肺炎放线杆菌的特异基因合成5对特异引物,建立传染性胸膜肺炎放线杆菌血清型分型的菌落多重PCR方法,结果为10株传染性胸膜肺炎放线杆菌血清型参考菌株均扩增出了相应的预期片段,而支气管败血波氏杆菌、多杀性巴氏杆菌、大肠埃希菌的扩增均为阴性。利用此多重PCR方法对41株传染性胸膜肺炎放线杆菌分离菌株进行血清型分型,结果所有菌株均扩增出了相应的特异片段,其中6株为1型,5株为7型,1株为5型,29株为9型。     

替米考星、红霉素体外诱导猪胸膜肺炎放线杆菌耐药性的初步研究

采用微量稀释法测定了替米考星和红霉素对5株临床分离猪胸膜肺炎放线杆菌(App)的最小抑菌浓度,并用药物浓度递增法体外诱导App对两种药物的耐药性。结果表明替米考星和红霉素对App都具有很高的体外抑菌活性;经15代诱导,App对替米考星的最高耐受浓度没发生明显变化,而对红霉素的最高耐受浓度有了较大程度的提高,表明App对替米考星不易产生耐药,而对红霉素可缓慢产生耐药。试验结果提示替米考星是治疗App感染的理想药物。     

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。     

猪放线杆菌SHLI株的分离鉴定

从某猪场病死猪肉样变肺脏和严重出血的心脏、肝脏、肾脏及肠系膜淋巴结中分离到一株革兰氏阴性球杆菌。此菌大小０３５ ～０５ ×０５ ～１５μｍ , 单在、成双或短链状。菌落与琼脂有粘连性, 绵羊血琼脂平板上呈β溶血, 能生长于麦康凯琼脂上。发酵碳水化合物产酸不产气, 不发酵甘露醇、卫茅醇和肌醇, 能水解马粟苷和马尿酸钠, 尿酶、硝酸盐还原试验阳性, 不产生靛基质。鉴定为猪放线杆菌（ Ａｃｔｉｎｏｂａｃｉｌｌｕｓｓｕｉｓ） , 并将其定名为猪放线杆菌 Ｓ Ｈ Ｌ Ｉ株。其对小鼠和仔猪有强的致病性, 对豚鼠的致病性较弱。     

猪传染性胸膜肺炎病原特性及其诊断方法研究进展

胸膜肺炎放线杆菌是当前危害集约化养猪业的主要呼吸道疾病的病原之一。近几年以来,该病在我国呈暴发性流行,造成巨大的经济损失。研究病原和建立科学、快速、准确的诊断方法对于净化本病有重要的意义。目前,国内外对该病病原学特性、血清学检测技术以及分子生物学技术的诊断方法研究比较多,文章对该病的病原学和诊断方法研究进展进行了综述。     

猪胸膜肺炎放线杆菌的分离与鉴定

从山东省泰安市采集的38份具有严重呼吸道症状的病死猪的肺脏、气管和扁桃体进行胸膜肺炎放线杆菌(APP)的分离鉴定,其中14株菌株表现出形态多样性、革兰氏染色阴性等特点,小鼠试验显示有较强的毒力,PCR电泳结果得到了650bp的预期目的片段。系统的生物学鉴定表明,这14株分离菌为胸膜肺炎放线杆菌(Actinobacillus Pleuropneaumoniae,APP)。对确诊的14株APP采用凝集试验进行血清型检测。结果从山东省泰安市分离到14株2个血清型的胸膜肺炎放线杆菌,其中,血清7型8株、血清5型6株,血清7型、5型为绝对优势血清型。     

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。     

猪胸膜肺炎放线杆菌的血清型分型及毒力因子研究进展

根据荚膜多糖和脂多糖抗原表位的不同，猪胸膜肺炎放线杆菌分为15个血清型，某些血清型问存在交叉反应，不同血清型甚至同一血清型不同菌株间的毒力大小不一样，致病性也有强弱之别。该菌的致病性与多种毒力因子密切相关，主要有外毒素、外膜蛋白、脂多糖、英膜多糖、转铁蛋白、脲酶等。本文综述了国内外对胸膜肺炎放线杆菌的几种主要毒力因子及血清型的研究进展。     

猪传染性胸膜肺炎放线杆菌外膜蛋白基因的克隆和表达

以猪传染性胸膜肺炎放线杆菌（APP）血清7型25-4株基因组DNA为模板，用PCR扩增外膜蛋白（OMP）基因特异片段，并克隆于pMD18-T中，经酶切及核苷酸序列分析鉴定后，亚克隆于原核表达栽体pGEX-6P-1，成功构建了重组表达载体pGEX-omp；以此转化大肠埃希氏菌BL21（DE3），经SDS-PAGE鉴定，表达的可溶性融合蛋白分子质量约为61 ku，命名为GST-OMP。以GST亲和层析柱纯化并利用Xa因子酶解，获得切掉标签的OMP。经ELISA检测，该OMP蛋白能够与兔抗APP的阳性血清反应，具有很好的免疫活性。GST-OMP蛋白的成功表达为APP OMP相关分子生物学功能的研制奠定了基础。     

胸膜肺炎放线杆菌生物Ⅰ型标准抗血清的制备及初步临床应用

用十三种血清型的胸膜肺炎放线杆菌标准菌株免疫家兔,制备了标准抗血清,通过玻片凝集试验,琼脂扩散试验和免疫电泳试验,对抗血清的特异性、效价和保存期进行了监测,结果表明,制备的抗血清有较好的特异性,在4℃可以保存一个月、-20℃和、-80℃下保存一年无明显变化.用抗血清对从湖南,安徽,河南等地分离的胸膜肺炎菌株进行分型鉴定,分别为血清2,3,8型;而PCR检测均为阳性,说明抗血清可用于野生株的鉴定和流行病学调查.     

Genetic diversity and toxin gene distribution among serovars of Actinobacillus pleuropneumoniae from Australian pigs

Objective

To explore the diversity among isolates of the Actinobacillus pleuropneumoniae serovars most common in Australia (serovars 1, 5, 7 and 15) and to examine the Apx toxin profiles in selected representative isolates.

Design

A total of 250 isolates selected from different farms were examined for their genotypic profiles and a subset of 122 isolates for their toxin profiles.

Methods

The isolates of serovars 1, 5, 7 and 15 selected for this study came from different farms and different Australian states and were submitted for serotyping to the reference laboratory. The overall diversity of the strains was explored with the enterobacterial repetitive intergenic consensus (ERIC) PCR and the presence of the toxin genes was investigated with a toxin PCR assay.

Results

Some degree of variation was observed in the ERIC‐PCR pattern within all four serovars, ranging from 38% to 61% genetic diversity. When looking at the toxin gene profile and, therefore, the predicted ability to produce the expected toxin pattern, one isolate each of serovars 1 (n = 20) and 7 (n = 47) and 17 isolates of serovar 15 (n = 40) showed variation to the expected gene profile.

Conclusion

The variations in toxin gene patterns, as detected by PCR, found in this study could be related to significant changes in the gene sequence or total absence of the gene. Variation in toxin gene sequences has been observed in other countries. This variation in the toxin profile could also explain possible variation in pathogenicity observed in the field.