Rapid virulence typing of Pasteurella multocida by multiplex PCR

The gram-negative bacterium Pasteurella multocida constitutes a heterogeneous species associated with wide range of disease in many animals. Isolates are classified into five groups based on capsular antigen (capA, B, D, E and F). Recently, a new valuable PCR-based method was introduced to determine the epidemiological correlation between P. multocida infection and existence of virulence genes including tbpA, pfhA, toxA and hgbB. However, this method is tedious and laborious. Thus, in the current study, we designed a reliable multiplex PCR method for rapid detection of virulence genes in P. multocida. Eighty seven strains of P. multocida isolated from various clinically healthy and infected hosts were examined by uniplex PCR method for each virulence associated genes. Based on our improved and simplified multiplex PCR method, rapid detection of four virulence genes was accomplished. It is proposed that its implementation may benefit the epidemiological investigations.     

猪源多杀性巴氏杆菌的生物学鉴定与荚膜PCR分型

从表现猪萎缩性鼻炎临床症状的猪群中分离出13株多杀性巴氏杆菌(Pasteurella multocida,Pm),采用Pm种特异性的KMT1-KMT2引物进行PCR扩增,结果与传统的生化反应鉴定完全一致。基于对甘露醇、卫茅醇、山梨醇、海藻糖的发酵能力和产生鸟氨酸脱羧酶的特性,Q_1、Q_3、Q_6、Q_7、Q_(10)、Q_(13)和C_(48-1)鉴定为Pm多杀亚种(Pm subsp.multocida);Q_2、Q_4、Q_5、Q_8、Q_9、Q_(11)、Q_(12)鉴定为Pm败血亚种(Pm subsp.septica)。对13株Pm分离物采用A型、B型和D型引物进行PCR扩增,8株鉴定为A血清型(61.5%);5株鉴定为D血清型(38.5%);没有发现B血清型的菌株。同时对金黄色葡萄球菌抑制试验、中性吖啶黄沉淀试验与荚膜PCR分型的相关性进行了讨论。     

利用菌落多重PCR方法进行不同动物来源多杀性巴氏杆菌鉴定的研究

参照文献报道的多杀性巴氏杆菌种和荚膜型的特异基因合成6对特异引物,建立多杀性巴氏杆菌鉴定的菌落多重PCR方法,结果5株多杀性巴氏杆菌荚膜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、胸膜肺炎放线杆菌、大肠埃希菌的扩增均为阴性。利用此多重PCR方法对48株不同动物来源的多杀性巴氏杆菌进行了鉴定和荚膜型分型,同时与间接血凝试验以及Biolog细菌快速鉴定系统进行比对,结果表明,所建立的多重PCR方法与间接血凝试验、Biolog细菌快速鉴定系统的符合率均达到100%。     

应用双重PCR检测产毒素多杀性巴氏杆菌

参照有关文献设计了2对引物。分别扩增多杀性巴氏杆菌的Kmt及toxA基因。以使同时检测并区分产毒素与非产毒素多杀性巴氏杆菌。该PCR能检出10^3cfu菌量的模板。特异性试验表明。这2对引物都不能从猪的其他6种常见病原菌扩出特异性的带。对扩增的2个PCR产物测序结果表明，2序列都有很高的保守性，从而进一步证明了该PCR方法的特异性及灵敏性。临床应用，从386份病料分离出66株多杀性巴氏杆菌，其中有8株均能同时扩出457bp和864bp的片段，而其他54株只能扩出457bp的片段。     

Characterization of Pasteurella multocida mutants of low virulence

Ten temperature-sensitive mutants of the Clemson University (CU) vaccine strain of Pasteurella multocida have been developed and were characterized by phenotypic attributes such as carbohydrate fermentation, antibiotic resistance, and membrane protein profiles. Some mutants were found to have lost the ability to utilize some substrates, notably xylose and gluconate, whereas others were able to ferment additional carbohydrates such as arabinose and rhamnose. CU was found to be resistant to sulfisoxazole, of intermediate resistance to bacitracin, and sensitive to rifampin; the sensitivity to these three antibiotics varied among the mutant strains, but 60% were resistant to rifampin. Membrane protein profiles demonstrated some changes in major bands, and there was variation in 50% of the mutants in proteins in the 31 kilodalton range. All strains were assayed for the presence of several virulence factors, and many were found to produce siderophore and to exhibit some degree of complement resistance.     

Specific identification of Pasteurella multocida serogroup-A isolates by PCR assay

A polymerase chain reaction (PCR) assay targeting the hyaC-hyaD gene was developed and used to identify strains of Pasteurella multocida belonging to serogroup-A. A set of serogroup-specific-PCR primers amplified a 564 bp product from genomic DNA prepared from bacterial cells or directly from bacterial colonies. This method detected as low as 10 ng of bacterial DNA and had a specificity of 100% for P. multocida serogroup-A. A nested PCR method yielded a single 374 bp product. All fifty isolates were also shown to be identical by restriction fragment length polymorphism (RFLP) analysis of the PCR products after digestion with BglII.     

副猪嗜血杆菌、多杀性巴氏杆菌和胸膜肺炎放线杆菌多重PCR检测方法的建立及应用

在已建立的副猪嗜血杆菌(HPS)、多杀性巴氏杆菌(Pm)、胸膜肺炎放线杆菌(APP)的单重PCR检测方法的基础上,通过对扩增条件的优化,利用一次PCR反应可同时扩增出APP的256 bp、Pm的457 bp、HPS的822 bp的特异性片段,建立了HPS、Pm、APP的多重PCR实验室诊断方法。该复合PCR可检测60 pg的HPS、120 pg的Pm、50 pg的APP。利用该方法检测猪繁殖与呼吸综合征病毒(PRRSV)阳性猪体内分离到的39株细菌,结果显示HPS、Pm、APP分别为12、16、2株,对39份病料的检测与常规细菌分离鉴定结果一致,可见该方法适用于HPS、APP、Pm的临床快速检测。     

PCR检测方法在多杀性巴氏杆菌定种中的应用

为修订行业标准《猪巴氏杆菌病诊断技术》（NY/T 564-2002）,改进多杀性巴氏杆菌定种方法,采用16S rDNA基因序列分析法对中国兽医微生 物菌种保藏管理中心（CVCC）保藏的63株猪源多杀性巴氏杆菌进行复核鉴定。63株猪源多杀性巴氏杆菌中有60株菌种与原鉴定结果一致。建立多杀性巴 氏杆菌基于kmtΙ基因的PCR定种方法,对经16S rDNA PCR方法确认为多杀性巴氏杆菌的60株菌进行定种。结果表明,60株菌均扩增出了预期片段,基于 kmtΙ基因的PCR定种方法与16S rDNA PCR方法试验结果相一致。研究结果显示,可将基于kmtΙ基因的PCR定种方法增添至行业标准中,作为原有生化鉴 定方法的补充进行多杀性巴氏杆菌的定种。     

猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌复合PCR检测方法的建立

目的建立可以同时检测猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速而可靠的PCR检测方法。方法和结果根据胸膜肺炎放线杆菌的Apx-VIA基因序列、多杀性巴氏杆菌和副猪嗜血杆菌的16SrRNA基因序列设计5条引物。猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌模板的PCR扩增产物大小分别为342bp,485bp和1258bp。复合PCR对1～12型猪胸膜肺炎放线杆菌标准株,6株多杀性巴氏杆菌标准株,1～15型副猪嗜血杆菌以及25株经生化鉴定确认为上述三种细菌的分离株的基因组DNA作为模板进行检测,均获得预期大小的扩增产物。以猪放线杆菌、吲哚放线杆菌等14种常见细菌作为阴性对照进行PCR检测,结果仅有支气管败血波氏杆菌产生了可以和上述三个特异性条带明显区分的PCR产物。复合PCR针对胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的敏感性分别为14pg、34pg和37pg。结论本研究建立的复合PCR特异性好,敏感性高,可以用于猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速检测。     

菌落多重PCR鉴定不同荚膜血清型的多杀性巴氏杆菌

参照文献报道的多杀性巴氏杆菌KMT1基因和荚膜生物合成位点hyaD-hyaC、bcbD、dcbF、ecbJf、cbD基因的序列合成了6对特异性引物,建立了多杀性巴氏杆菌种和型的菌落多重PCR方法。结果表明,本所保藏的A、B、D、E、F各型多杀性巴氏杆菌均扩增出了相应的预期片段,PCR结果与Biolog鉴定结果和Carter氏间接血球凝集试验结果相一致;而支气管败血波氏杆菌、胸膜肺炎放线杆菌、大肠埃希菌、猪链球菌和粪肠球菌的扩增均为阴性。     

野生草食动物源多杀性巴氏杆菌荚膜分型的研究

采用Carter荚膜群鉴定法对分离于我国黑鹿、白唇鹿、斑马和赤大袋鼠的56株多杀巴氏杆菌进行荚膜血清型鉴定,结果表明：B型占78．6％（44／56）,h型占14．3％（8／56）,D型占3．6％（2／56）,另有2株未能确定型,占3．6％（2／56）。其中荚膜B型是斑马、黑鹿和白唇鹿源多杀性巴氏杆菌的主要血清群,荚膜A型是赤大袋鼠源多杀性巴氏杆菌的主要血清群。黑鹿和白唇鹿源多杀性巴氏杆菌均存在2个血清群。     

Profiling of virulence associated genes of Pasteurella multocida isolated from cattle

Pasteurella multocida is a causative agent of many major diseases of which haemorrhagic septiciemia (HS) in cattle & a buffalo is responsible for significant losses to livestock sector in India and south Asia. The disease outcome is affected by various host- and pathogen-specific determinants. Several bacterial species-specific putative virulence factors including the capsular and virulence associated genes have been proposed to play a key role in this interaction. A total of 23 isolates of P. multocida were obtained from 335 cases of various clinically healthy and diseased cattle. These isolates were examined for capsule synthesis genes (capA, B, D, E and F) and eleven virulence associated genes (tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA, sodC, oma87 and ptfA) by PCR. A total of 19 P. multocida isolates belonging to capsular type B and 4 of capsular type A were isolated. All isolates of capsular type B harboured the virulence associated genes: tbpA, pfhA, hgbA, sodC and nanH, coding for transferrin binding protein, filamentous hemagglutinin, haemoglobin binding protein, superoxide dismutase and neuraminidases, respectively; while isolates belonging to capsular type A also carried tbpA, pfhA, hgbA and nanH genes. Only 50 % of capsular type A isolates contained sodC gene while 100 % of capsular type B isolates had sodC gene. The gene nanB and toxA were absent in all the 23 isolates. In capsular type A isolates, either sodA or sodC gene was present & these genes did not occur concurrently. The presence of virulence associated gene ptfA revealed a positive association with the disease outcome in cattle and could therefore be an important epidemiological marker gene for characterizing P. multocida isolates.     

Rapid detection of Escherichia coli virulence factor genes using multiplex real-time TaqMan PCR assays

Three multiplex real-time TaqMan PCR assays were developed for the detection of Escherichia coli virulence factor genes in veterinary samples. Target virulence factors chosen were the fimbriae K88 (F4), K99 (F5), F41, F17, F18 and 987p (F6) and the toxins LT, STa and CDT IV. Detection of genes coding GAD were included in each assay as an internal control. These assays allow rapid identification of virulence factor genes using identical cycling conditions on an Mx3000Ptrade mark real-time PCR machine with the capacity to test up to 20 strains for 9 virulence genes in 1h.     

猪源多杀性巴氏杆菌PCR鉴定方法的建立

为建立一种灵敏、特异的猪源多杀性巴氏杆菌PCR检测方法,根据GenBank已公布的多杀性巴氏杆菌plpE基因序列的保守片段设计合成引物,经plpE基因阳性质粒构建、反应条件的优化、特异性试验和敏感性试验,对猪源多杀性巴氏杆菌特异性PCR检测方法进行了研究;并将该PCR方法用于检测来自四川省6个规模化猪场的64份疑似多杀性巴氏杆菌肺部组织样品。结果显示,建立的PCR方法具有良好的特异性和敏感性,检测的敏感性为5×101拷贝的目的基因;多杀性巴氏杆菌(A、B、D血清型)PCR均为阳性、APP和HPS等病原均为阴性;64份临床疑似样品中检出36份样品阳性,阳性检出率为56.2%。以plpE基因初步建立的多杀性巴氏杆菌PCR检测方法具有较好的特异性、重复性、敏感性和可靠性,可用于多杀性巴氏杆菌的检测鉴定。     

Diagnostic and typing options for investigating diseases associated with Pasteurella multocida

Pasteurella multocida is responsible for major animal diseases of economic significance in both developed and developing countries whereas human infections related to this bacterium are infrequent. Significantly, development of a carrier status or latent infections plays a critical role in the epidemiology of these diseases. Aiming at increased knowledge of these infections, we examine potential diagnostic and selected typing systems for investigating diseases caused by P. multocida. Detection of P. multocida from clinical specimen by; (i) isolation and identification, (ii) polymerase chain reaction (PCR), iii) specific hybridisation probes, (iv) serological tests and (v) other alternative methods is critically evaluated. These detection systems provide a wide spectrum of options for rapid diagnosis and for detecting and understanding of latent infections in herd/flock health control programmes, though PCR methods for detecting P. multocida in clinical specimen appear increasingly preferred. For establishing the clonality of outbreak strains, we select to discuss macromolecular profiling, serotyping, biotyping, restriction enzyme analysis, ribotyping and multiplex PCR typing. Although P. multocida infections can be rapidly diagnosed with molecular and serological tests, isolation and accurate species identification are central to epidemiological tracing of outbreak strains. Our review brings together comprehensive and essential information that may be adapted for confirming diagnosis and determining the molecular epidemiology of diseases associated with P. multocida.     

A survey of potential virulence markers from avian strains of Pasteurella multocida

Twenty-four isolates of Pasteurella multocida from clinical cases of fowl cholera and the Clemson University vaccine strain were surveyed for the presence of potential virulence markers. Membrane proteins, enzymatic activity of the membrane proteins, and carbohydrate fermentation patterns were also determined to demonstrate phenotypic relationships within the groups. Few differences were found in these phenotypic characteristics among the isolates. Almost all the organisms produced siderophore and were hemolytic on turkey red blood cells. No extracellular enzyme or bacteriocin activity was detected and little antibiotic resistance was found. However, many organisms contained plasmids and demonstrated some degree of resistance to complement. Both characteristics were correlative markers in Pasteurella multocida isolated from birds with fowl cholera.     

Serum resistance as an indicator of virulence of Pasteurella multocida for turkeys

Wildlife isolates of Pasteurella multocida, whose virulence for turkeys had previously been determined by intravenous inoculation, were characterized regarding their ability to survive incubation in fresh non-immune turkey serum. The relative virulence of the isolates was significantly associated with their ability to resist the bactericidal power of the serum as determined by standard plate counts following incubation. Organisms with a high survival value were more virulent; those with a low survival value were less virulent. A statistical model was specified and was successfully used to predict relative virulence of the P. multocida isolates. This method of assaying serum resistance was rapid, repeatable, and practical and could be performed with minimal laboratory equipment. Also studied was the serum resistance of seven serotype 3, 4 isolates obtained from the lungs of M9-vaccinated turkeys from seven flocks experiencing increased mortality due to fowl cholera. These isolates were shown to be identical to the M9 vaccine by restriction endonuclease analysis of chromosomal DNA. Six of the seven isolates had higher serum survival values than the original M9 vaccine.