Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.     

应用16S rRNA基因测序法鉴定禽多杀性巴氏杆菌的研究

应用 16SrRNA基因测序法对以疫苗标准强毒株C4 8_1和弱毒疫苗代表株G190E4 0为阳性对照株和分离鉴定的 2株禽源多杀性巴氏杆菌Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1株 ,进行 16SrRNA基因序列分析。结果表明 ,2株分离菌与对照的强弱毒株之间的同源性为 10 0 %。后经Blastn分析Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong /2 0 0 2_1、C4 8_1、G190E4 0株与已发表 11株多杀性巴氏杆菌同源性高达 10 0 % ,与已发表的 34株多杀性巴氏杆菌同源性均超过 98% ,同时与其它巴氏杆菌种如溶血巴氏杆菌等同源性均低于 96 % ,进一步证实Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1分离株均为多杀性巴氏杆菌。生化实验鉴定表明Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1、C4 8_1、G190E4 0菌株均为subsp .Multocida亚种。     

兔多杀性巴氏杆菌16S rRNA PCR检测方法的建立

为建立一种快速准确检测兔多杀性巴氏杆菌(P.multocida)的PCR方法,本研究以P.multocida的高度保守的16S rRNA为靶基因,参考已公布的P.multocida的16S rRNA基因设计1对特异性引物,优化PCR反应条件,建立了P.multocida PCR快速检测方法。该PCR方法的敏感性达到60 cfu/mL,采用该PCR方法扩增P.multocida标准株和分离株均能扩增出643 bp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,证明本实验所建立的P.multocida PCR检测方法快速、敏感、特异、可靠,可用于P.multocida的快速鉴定与诊断。同时用建立的PCR方法对临床疑似病兔的脏器分离菌进行扩增,可扩增出目的条带,与细菌的分离结果相一致。     

Evaluation of different API systems for identification of porcine Pasteurella multocida isolates

An exhaustive biochemical characterisation of 60 porcine Pasteurella multocida clinical isolates recovered from lesions indicative of pneumonia, previously confirmed by PCR and all belonging to the capsular serogroup A, was performed by means of four commercial systems. The API 20NE correctly identified almost all isolates (95%), but only 60% could be ascribed to this species by the API 20E method. The high diversity exhibited by the API 50CHB/E system, with six different patterns, does not advise its use as additional system for a definitive identification at the species level, but this method could be a potential tool for characterising P. multocida isolates below this level. The more uniform reactions yielded by the API ZYM test make this system helpful in the confirmatory identification of this organism. The high variability (20 profiles) obtained when the four systems are taken together also suggests their usefulness for epidemiological purposes in order to sub-type P. multocida isolates.     

Ribotyping of Indian Isolates of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> Based on 16S and 23S rRNA Genes

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.     

Phylogenetic analysis of the genus Anaplasma in Southwestern China based on 16S rRNA sequence

To identify the species within the genus Anaplasma circulating among ruminants in the Southwest of China, we performed the phylogenetic analysis of the 16S rRNA gene of two Anaplasma isolates from cattle and seven from goats. The two sequences obtained from cattle strains belonged to the A. marginale cluster, whereas the other seven sequences from caprine strains formed two Anaplasma spp. clusters, which diverged earlier than the clusters of A. marginale, A. centrale and A. ovis. These results indicate that there are at least two Anaplasma species circulating among ruminants in Southwestern China.     

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.     

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.     

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。     

Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays

The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n = 94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes 460, 1044, 657 and 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.     

Comparative Analysis of the Outer Membrane Protein Profiles of Isolates of the Pasteurella multocida (B:2) Associated with Haemorrhagic Septicaemia

Outer membrane proteins (OMP) of P. multocida (serotype B:2) field isolates (n = 6) and a vaccine strain (P-52) were extracted by a sarkosyl method and characterized using SDS-PAGE and immunoblotting. About 20 polypeptide bands were observed in the profile of the vaccine strain with MW ranging from 16 to 90 kDa and, based on band thickness and intensity of staining, three polypeptides of MW 31, 33 and 37 kDa were considered to be the major OMPs. The profiles of the field isolates showed minor differences when compared with that of the vaccine strain. The OMP of 33 kDa was only expressed by the vaccine strain. Four field isolates expressed an OMP of 39 kDa, which did not appear in the profiles of the remaining two field isolates and the P-52 strain. Similarly, an OMP of 25 kDa was exclusively seen in the profile of a single isolate. By immunoblotting studies, using anti-P. multocida (P-52) whole-cell hyperimmune serum raised in rabbits as well as buffalo immune sera, it became evident that the polypeptide of 37 kDa was the most antigenic OMP in the profiles of all the isolates, including the P-52 strain. Other polypeptides were either weakly antigenic or visible in the profile of only a few of the isolates. The study thus identified the major OMP of P. multocida (B:2) and suggested that this highly antigenic 37 kDa OMP has potential for further protective and immunodiagnostic studies.     

鸡奇异变形杆菌的分离鉴定和16S rRNA基因序列测定与系统进化分析

从山东泰安一鸡场发病雏鸡眼中分离到1株致病菌(编号为QY),通过细菌形态学等常规鉴定符合奇异变形杆菌(Proteus mirabilis)特性。用奇异变形杆菌阳性血清诊断结果呈阳性,人工感染证明该菌株是造成该鸡场雏鸡大批发病死亡的致病菌。药敏试验结果显示对头孢类、恩诺沙星等高度敏感,而对青霉素和复合磺胺等不敏感。以细菌16SrRNA基因通用引物进行PCR扩增,得到QY的16SrRNA基因序列,长约1 453bp(GenBank,登录号为GU477712)。将该序列与GenBank中序列进行Blast比对,发现与其匹配度最高的均是奇异变形杆菌各株系的16SrRNA序列,均高达98%以上。运用DNAStar软件与其中10株奇异变形杆菌分离株构建系统进化树,结果表明,分离株(QY菌株)与10个代表菌株的同源性均为98.9%～99.9%,其中与AB272366同源性最高为99.9%。从分子水平证明该菌是奇异变形杆菌并分析了其遗传进化规律,为鸡奇异变形杆菌的鉴定及其引起的疾病的诊断与治疗提供了参考。     

Identification of Pasteurella multocida isolates of ruminant origin using polymerase chain reaction and their antibiogram study

A total of 100 isolates of Pasteurella multocida from various ruminant species (cattle, buffalo and sheep) belonging to different parts of country were identified using Pasteurella multocida-PCR (PM-PCR) and capsular PCR assays. PM-PCR revealed an amplicon of approximately 460 bp in all the isolates tested. As regards capsular PCR, 36 of 38 cattle isolates and 30 of 34 buffalo isolates were found to belong to capsular serogroup B whereas rest of the cattle and buffalo isolates belonged to serogroup A of P. multocida. In case of sheep, a total of 26 out of 28 isolates were positive for serogroup A specific PCR while remaining 2 amplified a PCR product specific for serogroup F of P. multocida. All the isolates were subjected to antibiotic sensitivity testing using 17 different antibiotics. Enrofloxacin was found to be most potent antibiotic as it was effective against 94% of the isolates followed by ofloxacin (93%), chloramphenicol (93%), doxycycline (89%), tetracycline (86%) and ciprofloxacin (84%). Vancomycin, bacitracin and sulfadiazine were ineffective against P. multocida isolates showing 84%, 75% and 82% resistance, respectively. Further, the antibiogram also revealed the development of resistance against multiple drugs among various isolates of the organism.     

Antimicrobial activity of cathelicidins BMAP28, SMAP28, SMAP29, and PMAP23 against Pasteurella multocida is more broad-spectrum than host species specific

The antimicrobial activity of linear, cationic alpha-helical peptides from cattle (BMAP28), sheep (SMAP28 and SMAP29), and pigs (PMAP23) were assessed to determine if activity was selective for Pasteurella multocida from a particular animal species or broad-spectrum against all P. multocida tested. The antimicrobial activities of synthetic peptides were determined for P. multocida isolated from cattle (10 isolates), sheep (10 isolates), and pigs (10 isolates) in a broth microdilution assay. All thirty isolates of P. multocida were susceptible to BMAP28 (MICs and MBCs, 1.0-1.9 microM); SMAP28 and SMAP29 (MICs and MBCs, 0.2-0.7 microM); and PMAP23 (MICs and MBCs, 4.3 to > or = 6.8 microM). Overall, the results of this study suggest that synthesized cathelicidins from cattle, sheep, and pigs had broad-spectrum antimicrobial activity against all P. multocida.     

不同牛源多杀性巴氏杆菌HasR基因的克隆及序列分析

为了克隆从患肺炎犊牛和健康犊牛上分离的多杀性巴氏杆菌血红素结合受体(heme acquisition system receptor,HasR)基因并进行序列分析,分别以致犊牛肺炎和健康犊牛携带的多杀性巴氏杆菌DNA为模板,通过PCR反应扩增出血红素结合受体基因的全部序列,PCR产物纯化后克隆到pMD19-T载体上,经菌液PCR和酶切鉴定后进行序列测定。各菌株HasR基因序列比对结果发现,Pm-BS-a、Pm142-x-4、Pm142-x-3、Pm149-x、Pm-BS-d的序列之间亲源关系较近,而Pm149-xby与参考菌株Pm70的同源性较高。健康牛源的多杀性巴氏杆菌与致犊牛肺炎多杀性巴氏杆菌之间HasR基因的同源性非常高,说明致犊牛肺炎多杀性巴氏杆菌是一种条件性致病菌,其引起的犊牛肺炎可能属内源性感染。     

Protective effect following intranasal exposure of goats to live Pasteurella multocida B:2

This study aimed to determine the effect of intranasal exposure to low doses of Pasteurella multocida B:2 on survival of goats challenged with high doses of the same organism. Eighteen goats were selected and divided into three groups. Goats of group 1 were exposed intranasally twice, with a two-week interval, to 7× 10⁶ cfu/ml of live P. multocida B:2. Goats of group 2 were not exposed to P. multocida B:2 but were kept together with the exposed group 1. Goats of group 3 remained as unexposed controls and were kept separated from the other two groups. Serum samples were collected at weekly intervals to determine the antibody levels. At week 5 post exposure, all goats were challenged subcutaneously with 3.7× 10¹⁰ cfu/ml of live P. multocida B:2. Following challenge exposure, 8 (67%) goats (4 goats from each of groups 1 and 2) were killed owing to haemorrhagic septicaemia. Four goats were killed peracutely within 48 h post challenge, while the other four goats were killed acutely between 2 and 4 days post challenge. None of the goats of group 3 were killed for haemorrhagic septicaemia. Goats of groups 1 and 2 showed significantly (p<0.05) higher antibody levels following the first intranasal exposure to P. multocida B:2. However, only group 1 retained the significantly (p<0.05) high antibody levels following a second intranasal exposure, and remained significantly (p<0.05) higher than groups 2 and 3 at the time of challenge. P. multocida B:2 was successfully isolated from various organs of goats that were killed between 1 and 4 days post challenge.     

云南鸭疫里默氏杆菌外膜蛋白A 及16S rRNA序列分析

为了探讨鸭疫里默氏杆菌(Riemerella anatipestifer, RA)云南流行株的外膜蛋白A (OmpA)的基因序列差异及其与16S rRNA序列的相关性,PCR扩增18株云南流行株鸭疫里默氏杆菌OmpA基因及16S rRNA核苷酸序列,分别构建其系统进化树,分析其系统进化关系。结果表明,18株鸭疫里默氏杆菌OmpA基因分为2个群,其同源性分别为86%~99.2%和92.6%~100%。18株鸭疫里默氏杆菌16S rRNA基因同属1个群,同源性高达96.1%~100%。 RA-1、RA-2、RA-11和RA-39 4株分离株的OmpA基因位于进化树的同一个亚群,其16S rRNA基因也位于进化树的同一亚群,两者呈现出明显的相关关系,其他14株分离株的OmpA基因系统进化树与16S rRNA基因系统进化树无明显的相关关系。     

禽大肠杆菌的分离与16S rRNA的鉴定

从疑似患有大肠杆菌病的病死鸡群中采取粪便样品,分离病原进行生化鉴定,从8份样品中分离鉴定出6株大肠杆菌。根据细菌16S rRNA 基因的高度保守性,设计合成大肠杆菌的共同引物,对随机选取的1株细菌进行PCR扩增,并与GenBank中的E.coli 16S rRNA进行序列比对,确定这株细菌与大肠杆菌的同源性达99%以上。本方法特异性好,为实验室鉴定大肠杆菌提供了一种简单、容易操作的手段。     

The 23S rRNA gene PCR-RFLP used for characterization of porcine intestinal spirochete isolates

Using three reference strains of Brachyspira hyodysenteriae (B204, B234, B169), one B. pilosicoli (P43/6/78), one B. murdochii (56-150), one B. intermedia (PWS/A), one B. innocens (B256) and ten Korean isolates, PCR-RFLP analysis of DNA encoding 23S rRNA was performed to establish a rapid and accurate method for characterizing porcine intestinal spirochetes. Consequently, B. hyodysenteriae and B. pilosicoli revealed different restriction patterns; however, the other three species shared the same pattern. These findings are not consistent with a prior report. Differences in 23S rRNA gene sequences, between two B. murdochii strains, 56-150 and 155-20, were observed. These results indicate that 23S rRNA PCR-RFLP could be used as an identification method for pathogenic Brachyspira spp. (B. hyodysenteriae and B. pilosicoli) as well as an epidemiological tool for characterizing spirochetes isolated from swine.