禽多杀性巴氏杆菌强毒株与弱毒株制备的蜂胶灭活疫苗免疫原性的比较试验

用禽多杀性巴氏杆菌强毒株(C48-1)与弱毒株(G190E40和B26-T1200)制备的蜂胶灭活疫苗的免疫原性对比试验结果表明,强毒株C48-1制备的禽霍乱蜂胶灭活疫苗的免疫效果显著高于弱毒株G190E40和B26-T1200,近期保护率相差20%～40%,3个月后相差40%～80%.强毒株C48-1的免疫原性显著高于弱毒株G190E40和B26-T1200,说明禽多杀性巴氏杆菌荚膜上的毒力蛋白可能具有免疫原性.     

干粉培养基在禽多杀性巴氏杆菌病活疫苗培养中的应用

正商品化干粉培养基在培养中应用越来越广泛,给细菌培养提供充分营养需求,简化培养基制备过程,并可使批间次差异减少。1菌种及原材料1.1菌种禽多杀性巴氏杆菌G190E40弱毒株,效检用禽多杀性巴氏杆菌C48-1强毒株。1.2培养基青岛海博公司,巴氏杆菌干粉培养基,批号:2015121.3试验动物3~4月龄的健康易感鸡2菌种制备2.1基础种子G190E40株冻干菌种用灭菌2~5mL pH7.4     

不同动物源多杀性巴氏杆菌荚膜分子分型及其致病性研究

为了确定西南民族大学动物医学实验室分离的鸭源、猪源、牛源和山羊源多杀性巴氏杆菌(Pm),即Q1、Z1、N1、Y1株的荚膜血清型及其致病性,试验采用PCR方法对分离的这4株不同动物源多杀性巴氏杆菌的种属和荚膜血清型进行鉴定,对目的基因测序分析,并用Balb/c小鼠对分离菌的致病性进行研究。结果表明:4株菌均为多杀性巴氏杆菌;鸭源Q1株为荚膜血清A型、猪源Z1株和牛源N1株为荚膜血清B型、山羊源Y1株为荚膜血清D型;对目的基因测序后与GenBank上已公布的相应荚膜血清型比对,同源性为98%～100%;不同动物源多杀性巴氏杆菌对Balb/c小鼠的致病性研究显示,猪源荚膜血清B型多杀性巴氏杆菌对Balb/c小鼠有较强的致病力。     

新疆石河子规模化牛场多杀性巴氏杆菌的分离鉴定及药敏试验

为了确定石河子地区某规模化牛场出现呼吸困难、咳嗽、病牛消瘦甚至死亡的病因,本研究以病牛病变组织为研究对象,采用常规细菌分离鉴定和细菌16SrRNA序列分析来鉴定菌种,以及多杀性巴氏杆菌种特异性基因Kmt-1和各个荚膜血清型特异性基因(hyaD-hyaC、bcbD、dcbF、ecbJ、fcbD)PCR扩增来确定细菌的血清型,同时应用纸片扩散法对分离细菌进行药物敏感性试验和小鼠感染试验。结果表明,从病变的肺组织中分离到1株菌落为灰白色、露珠状、不溶血,染色为革兰阴性球杆状细菌,生化鉴定结果符合巴氏杆菌特征,同时16SrRNA序列分析与NCBI上已公布的多杀性巴氏杆菌16SrRNA序列同源性在99%以上;对多杀性巴氏杆菌特异性基因Kmt-1以及各血清型特异性基因PCR扩增只扩增到Kmt-1和hyaD-hyaC特异性基因片段;分离菌株对链霉素、庆大霉素、卡那霉素耐药,对其他30种药物敏感,同时感染小鼠全部死亡。结果显示从病牛体内分离到1株毒力较强的血清A型多杀性巴氏杆菌。     

牛源荚膜血清型A型多杀性巴氏杆菌的分离鉴定及其种特异性基因的序列分析

从病死牛组织中分离到4株致病菌,编号分别为P1、P2、P3、P4。经菌落形态学观察、培养特性、生化反应和小鼠毒力试验,初步鉴定为多杀性巴氏杆菌。参考多杀性巴氏杆菌16S rRNA和种特异性kmt1基因以及荚膜血清型基因A(capA)、B(capB)、D(capD)、E(capE)和F(capF)合成引物,利用多重PCR扩增16S rRNA、kmt1和荚膜血清型基因,对PCR产物测序并进行同源性分析。结果显示,P1～P4都扩增出了16S rRNA、kmt1和荚膜血清型A型基因的特异性目的条带;P1～P4的16S rRNA、kmt1、A型基因序列与Genbank数据库中的多杀性巴氏杆菌相应序列具有高度同源性(99.0%);P2、P3、P4间的16S rRNA基因同源性较高(99.0%),而P1与其它3株(P2～P4)同源性较低,P1～P4间的kmt1基因、荚膜血清型A型的同源性较高(99.0%)。说明本次分的4株病原菌P1～P4均为荚膜血清型A型多杀性巴氏杆菌。     

牛源荚膜血清A型多杀性巴氏杆菌的分离鉴定

从采集于黑龙江、天津的病牛肺组织中分离到2株病原菌,经菌落形态学观察、培养特性、生化反应和小鼠毒力试验,初步鉴定为多杀性巴氏杆菌,分别命名为Pm-HLJ和Pm-TJ.参考多杀性巴氏杆菌种特异性基因kmt1和荚膜血清型特异性基因hyaD-hyaC、bcbD、dcbF、ecbJ和fcbD,合成引物,通过多重PCR扩增2株菌的种特异性基因和荚膜血清型特异性基因.选取Pm-HLJ的目标PCR产物进行克隆、序列测定、Blast搜索同源序列并且比较分析.结果显示,Pm-HLJ的kmt1基因片段全长460 bp,与GenBank中各血清型kmt1基因同源性均在96.6%以上;荚膜血清型A菌株特异性基因同源性为99.9%;而与其他荚膜血清型B、D、E、F的型特异性基因的同源性均低于50%.由此确认,分离的2株多杀性巴氏杆菌均为荚膜血清A型,这是我国A型多杀性巴氏杆菌引发牛出血性败血症的首例报道.     

6株山羊源多杀性巴氏杆菌血清型、耐药性及致病性分析

为了研究6株分离自四川省内江市2个养殖场患病山羊支气管的多杀性巴氏杆菌的主要生物学特性,试验采用特异性PCR方法鉴定多杀性巴氏杆菌血清型,采用纸片扩散法(Kirby-Bauer)评价了临床分离菌株对14种抗菌药物的敏感性,采用Reed-Muench法测定分离菌株对昆明小鼠的半数致死量(LD_(50)),分析其致病性。结果表明:在6株多杀性巴氏杆菌中,4株为A型,2株为B型。分离菌株对青霉素和林可霉素100%耐药,而对复方新诺明和氟苯尼考具有较高的敏感性。分离菌株对昆明小鼠的半数致死量均大于5×10~(8.0) cfu/0.5 mL,致病性较弱。说明本次分离的山羊源多杀性巴氏杆菌菌株为弱毒株,丰富了我国山羊源多杀性巴氏杆菌的生物学特征,为其感染的防控提供了有用的信息。     

兔多杀性巴氏杆菌16S rRNA PCR检测方法的建立和应用

以兔多杀性巴氏杆菌的高度保守的16S rRNA为靶基因,参考已公布的多杀性巴氏杆菌的16SrRNA基因设计1对特异性引物,优化PCR反应条件,建立了兔多杀性巴氏杆菌PCR快速检测方法。该PCR方法的敏感性达到60cfu/mL,使用建立的PCR方法扩增兔多杀性巴氏杆菌标准株和分离株均能扩增出643bp的目的片段,扩增兔大肠杆菌、支气管败血波氏杆菌结果为阴性,证明本试验所建立的兔多杀性巴氏杆菌病原PCR检测方法敏感、特异、可靠。     

羊源D型多杀性巴氏杆菌的分离鉴定及毒力基因检测

为了确定石河子地区规模化羊场出现呼吸道症状死亡羊的细菌性病原,采用常规细菌分离鉴定方法、细菌16SrRNA序列分析以及多杀性巴氏杆菌特异性基因kmt,从病变肺组织分离鉴定细菌,利用5个荚膜血清型特异性基因确定其血清型,扩增分离株的16个毒力相关基因,分析分离菌致病性和对常用抗菌药物的耐药性。结果表明,从病羊的病变肺组织中分离鉴定到一株血清D型多杀性巴氏杆菌,具有较强的致病性;携带8个毒力相关基因,其片段序列与NCBI上己公布的多杀性巴氏杆菌参考株同源性在99%以上;分离株对青霉素、林可霉素、庆大霉素、复方新诺明耐药,对其他23种药物敏感。     

猪源多杀性巴氏杆菌的分离鉴定及其荚膜血清型鉴定

从病死母猪肺脏中分离到一株革兰氏阴性小杆菌,用生理生化鉴定、药敏试验、致病性试验和PCR鉴定方法对分离菌株进行鉴定,并用多杀性巴氏杆菌荚膜分型引物对分离株的荚膜血清型进行鉴定。结果表明:本菌为猪多杀性巴氏杆菌,对多种抗生素高度敏感,对小白鼠有强致病性;PCR扩增16SrDNA基因获得1415bp片段,分离株的16SrDNA核苷酸序列与多杀性巴氏杆菌(AY078999)的同源性为99%,因此该分离菌株被鉴定为致病性巴氏杆菌,命名为YN20110122株;本菌分离株为荚膜A型血清型多杀性巴氏杆菌。     

DNA fingerprinting for differentiation of field isolates from reference vaccine strains of Pasteurella multocida in turkeys

The genomes from field isolates of Pasteurella multocida in turkeys and those of P multocida reference CU and M9 vaccine strains were analyzed and compared after cleavage with restriction endonucleases. The electrophoretic profiles obtained with DNA fragments from field isolates and vaccine strains of the same serotype were characteristic and reproducible. These features indicated the existence of differences among the isolates of the same serotype that cannot currently be detected, using available serotyping methods. However, several field isolates had electrophoretic profiles similar to those of either CU or M9 vaccine strain. It was concluded that restriction endonuclease analysis of DNA genomes from P multocida isolated from turkeys provides the information for differentiation of field isolates from vaccine strains of the same serotype.     

DNA fingerprinting of plasmid-containing serotype A: 3,4 Pasteurella multocida isolated from cases of fowl cholera in chickens and turkeys

The live, attenuated vaccine strains of Pasteurella multocida have been hypothesized to be responsible for homologous serotype outbreaks of fowl cholera on farms that use the commercial vaccines. We have further hypothesized that the naturally occurring Clemson University (CU) vaccine strain may be transformed to virulence by the acquisition of plasmid DNA. To test this hypothesis, we obtained seven homologous serotype (A:3,4) P. multocida isolates, all plasmid bearing, that were cultured from fowl cholera cases in vaccinated flocks and compared the isolates with the CU reference vaccine by molecular methods. Restriction fragment length polymorphisms (RFLPs) were detected by DNA/DNA hybridization with labeled probes specific for the cya, aroA, and rrn genes of P. multocida. The RFLPs obtained from BglII-digested genomic DNA probed with cya demonstrated no differences among the isolates. Although three isolates probed with aroA showed a RFLP identical to the vaccine strain, five isolates were distinctly different. Isolates probed with rrn grouped into three different restriction patterns that were dissimilar from that of the vaccine strain. Therefore, we have shown that these fowl cholera isolates are different from the CU vaccine strain and that these outbreaks were not vaccine related.     

禽多杀性巴氏杆菌血清型与外膜蛋白型相关性研究

为了解禽多杀性巴氏杆菌(Pasteurella multocida,Pm)分离菌株的荚膜血清型、菌体血清型与外膜蛋白型之间的相关性,首先对自行分离的10个菌株采用间接血凝试验和琼脂扩散试验进行鉴定(均为A:1);然后采用超声波破碎、高速离心和十二烷基肌氨酸钠提取外膜蛋白,通过SDS-PAGE电泳的方法对上述10个菌株与C48-1(A:1)、X73(A:1)、P1059(A:3)、CU(A:3,4)等一起进行外膜蛋白(Outer membrane proteins,OMP)分型研究。结果表明:14个禽多杀性巴氏杆菌菌株以2个主要蛋白OmpH和OmpA的差异为依据分为3种主要OMP型;依据次要蛋白的差异,OMP-1,3菌株进一步分为OMP型1.1,1.2和3.1,3.2,其中OMP型1.1有6个菌株,OMP型1.2有5个菌株;OMP型2有1株(YZ7031);P1059为OMP型3.1;CU为OMP型3.2;另外,血清型为A:1的12个菌株中有11个菌株外膜蛋白型均为OMP-1型,血清型为A:3、A:3,4的菌株外膜蛋白型属于3型。说明禽多杀性巴氏杆菌血清型与特定的外膜蛋白型具有很强的相关性。     

Attenuated live fowl cholera vaccine. I. Development of vaccine strain M3G of Pasteurella multocida

A live cholera vaccine was developed from a virulent avian septicemia strain of Pasteurella multocida serotype 1. The virulent parental strain was mutagenized with N-methyl-N'-nitro-N-nitroso guanidine. Mutants were selected that had either smaller colonies at 37 C or temperature sensitivity for growth at 41 C. Four small-colony mutants and 2 temperature-sensitive mutants were studied. All the mutants were avirulent for turkeys. Sixteen days after turkeys were vaccinated with each mutant, both the vaccinates and unvaccinated controls were challenge-exposed to virulent P. multocida of the homologous serotype and the heterologous serotype 3. Two of the small-colony mutant strains protected against both homologous and heterologous challenge. Suggested for a live cholera vaccine is P. multocida M3G, a small-colony-forming mutant, innocuous for both mice and turkeys and stable against reversion.     

Molecular epidemiology of Pasteurella multocida in dairy and beef calves

The molecular epidemiology of Pasteurella multocida has rarely been studied at the farm level in cattle. The aim of this study was to determine whether single or multiple strains of P. multocida tend to exist within farms. Molecular characterisation was carried out on isolates obtained from nasal swabs from 105 calves from 32 randomly selected beef and dairy farms located throughout Scotland, and from 131 calves from 20 farms in the Mayenne region of France, where sampling occurred in response to respiratory disease outbreaks. P. multocida isolates were characterised by random-amplified polymorphic DNA (RAPD) typing and pulsed-field gel electrophoresis (PFGE) using restriction enzyme ApaI. In addition, isolates representative of each farm/RAPD profile combination were typed by multilocus sequence typing (MLST). Among 105 Scottish isolates, 15 RAPD profiles were distinguished. The majority of farms (27/32) had indistinguishable profiles in all positive animals. Five farms had two profiles. Among 140 French isolates, 23 RAPD profiles were distinguished. More within-farm heterogeneity was observed although 10/20 farms had just one profile (E4) in sampled calves. Profile E4 accounted for 60% (84/140) of French isolates. PFGE was more discriminatory than RAPD but confirmed results with respect to within farm homogeneity or heterogeneity of strains, whereas MLST was not discriminatory enough for farm level epidemiology. As in other host species, either several strains or one dominant strain of P. multocida may exist within farms, with evidence for a role of management factors such as movements onto the farm in the number of strains detected.     

Transmission of Pasteurella multocida on California turkey premises in 1988-89.

Restriction endonuclease analysis (REA) of whole-cell DNA was used to determine possible sources of Pasteurella multocida for each outbreak of fowl cholera occurring in turkey flocks in eight commercial poultry companies in California from October 1988 to September 1989. Over this period, 179 isolates of P. multocida were obtained from dead turkeys in 80 meat and breeder flocks on 43 premises. P. multocida was isolated from wildlife on five premises. Isolates were characterized by subspecies, serotype, presence of plasmid DNA, and REA type. In 52 (65%) flocks, all isolates of P. multocida had the same REA pattern as the M9 live vaccine strain following digestion of DNA with the restriction enzyme SmaI. Field strains of P. multocida were obtained from 27 (34%) flocks, and one flock (1%) yielded both M9 and a field strain of the organism. REA of field strains of P. multocida revealed 17 different SmaI REA types. Based on matching SmaI REA types, potential sources of P. multocida were identified for 15 of the 28 flocks infected with field strains of the organism, and transmission between turkey premises was a possibility in only seven flocks.     

Random amplification of polymorphic DNA and phenotypic typing of Zimbabwean isolates of Pasteurella multocida

Eighty-one isolates presumptively identified as Pasteurella multocida from a variety of diseases in animals in Zimbabwe were subjected to biochemical characterization, capsular typing and RAPD analysis. The majority of isolates (over 80%) were assigned into named taxa and were predominantly P. multocida subsp. multocida and P. multocida subsp. septica, whilst the remainder were unassigned. Serogroup A was predominant among the three capsular types (A, B and D) of P. multocida detected. Three main RAPD clusters and three subclusters were observed among the majority of isolates (93.8%), whilst the remainder was found to be weakly related. Nine different groups of strains with similar RAPD profiles (100% similarity) were also observed. The reference strain of capsular serogroup F clustered with the reference strain of P. multocida subsp. septica, whilst all other serogroups clustered with reference strains of subsp. multocida and gallicida. Notably, serogroups A and D were observed to be closely related to the reference strain of subsp. multocida. The relationship between biotype, capsular type, host origin and disease manifestation was not clear-cut. However, most pig isolates of subsp. multocida clustered together as did most cattle isolates of subsp. multocida. RAPD tended to separate subsp. multocida from septica.     

Taxonomy of pasteurella anatipestifer. 1. DNA base composition and DNA-DNA hybridization analysis

DNA was isolated from 15 strains of Pasteurella anatipestifer and from one strain each of Moraxella nonliquefaciens, M. bovis, Pasteurella multocida, P. haemolytica, P. gallinarum, P. pneumotropica, and P. ureae. The guanine-plus-cytosine contents of P. anatipestifer ranged from 32 to 35 mole %, whereas those of Moraxella and Pasteurella spp. were much higher, ranging from 40 to 45 mole %. DNA-DNA hybridization analysis revealed that homology of nine P. anatipestifer strains to strains ATCC 11845 and PA 15 was 52 to 100%, whereas homology of Moraxella and Pasteurella strains to these strains was only 3 to 17%. Similarly, homology of P. anatipestifer strains, Moraxella, and Pasteurella species other than P. multocida to P. multocida reference strain P-2192 was low. These results strongly suggest that P. anatipestifer is genetically unrelated to either Pasteurella or Moraxella.     

禽多杀性巴氏杆菌C48—1成熟外膜蛋白H重组亚单位疫苗免疫效果的研究

将融合表达禽多杀性巴氏杆菌成熟外膜蛋白H（OmpmH）的重组菌pGEX—ompmH／BL21大量培养，在最佳诱导条件下诱导表达，表达产物经蛋白酶剪切及亲和层析纯化，得到OmpmH基因的原核表达产物，将其与弗氏完全佐剂混合制成油乳剂亚单位疫苗，用该疫苗肌肉注射接种5周龄鸡，首免后每周采血检测抗体，二免后第2周用10LD50禽多杀性巴氏杆菌强毒菌株C48-1进行攻击。结果显示，OmpmH具有良好的免疫原性，能诱导鸡体产生特异性抗体，可抵抗强毒菌株C48-1的致死性攻击，免疫效果优于禽多杀性巴氏杆菌弱毒疫苗。     

Characterization of nine Pasteurella multocida isolates from avian cholera outbreaks in Indonesia

Avian cholera outbreaks have been identified in Indonesia in recent years. Despite vaccination programs, outbreaks continue to occur. To date, there has been a lack of information on the characteristics of Pasteurella multocida isolates involved in these outbreaks. Hence, the objective of this study was to characterize Indonesian P. multocida isolates in poultry. During 1998-99, 20 field outbreaks were reported in Indonesia. Nine isolates of P. multocida were recovered from these field outbreaks. The isolates were compared with four vaccine strains that were used in Indonesia and designated PM-V1, PM-V2, PM-V3, and PM-V4. The isolates were characterized by biotype, capsular type, somatic serotype, restriction endonuclease analysis, plasmid presence, and antimicrobial susceptibility patterns. Of the nine Indonesian isolates, three were of capsular type A (A:1,3,13; A:1,3; and A:8). One isolate was of type B:2,3 and one isolate was of capsular type F. For three isolates, the capsular serogroup could not be identified. Plasmids the size of 2.3 kbp were present in three of the field isolates and two of the vaccine strains. One plasmid less than 2 kbp was isolated from the vaccine strain PM-V4. Eight distinct DNA profiles were obtained from digestion with the restriction endonuclease EcoRI, and seven distinct DNA profiles were obtained from digestion with the restriction endonuclease HindIII. All of the isolates were resistant to lincomycin and sulfadiazine and were susceptible to ampicillin and trimethoprim. Of the nine isolates, seven (78%) were susceptible to doxycycline and gentamicin and six (67%) were susceptible to enrofloxacin.