Characterization of outer membrane proteins participating in iron transport in Pasteurella multocida serotype A3

Iron-regulated outer membrane proteins (IROMPs) of P. multocida serotype A3, which function as receptors for complexes containing iron ions, are induced by iron deficiency in the bacterial growth environment. Analysis of an electrophoresis image of proteins isolated from bacteria grown on medium supplemented with 2,2'-dipyridyl revealed expression of 16 new proteins that were not noted in the case of the bacteria grown in standard conditions, with molecular weights from 30 to 160 kDa. Induction of IROMP expression occurred within 30 minutes after restricted iron conditions were established. In immunoblotting, distinct reactions were noted for proteins of molecular weight ranges of 25-49 kDa, 61-95 kDa, and 108-214 kDa. Proteins of the molecular weight of 68, 75 and 86 kDa were analysed using mass spectrometry and matched with the highest probability to proteins in the NCBI data base. Several dozen different proteins with similar amino acid sequences were matched to each sample.     

Immunization with outer membrane proteins of Pasteurella multocida (6:B) provides protection in mice

The immunoprotective efficacy of Pasteurella multocida (6:B) outer membrane proteins (OMPs) was examined in the mouse model. Bacterial OMPs were extracted using sarkosyl method and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. Prototype vaccines were prepared using OMPs with adjuvants including dioleoyl phosphatidyl choline-based liposome and Montanide ISA206 water-in oil-in water emulsion. Antibody response to the vaccine was monitored using indirect enzyme linked immunosorbent assay. The results of the study showed that immunized mice had high titre with both the formulations. The vaccinated mice were able to survive a live virulent bacterial challenge. Based on the findings of the study it can be inferred that OMPs are important determinants of immunoprotection hence can serve as vaccine candidates against haemorrhagic septicaemia.     

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。     

Investigation of outer membrane proteins of Pasteurella. 2: Iron-regulated outer membrane proteins of Pasteurella multocida and Pasteurella haemolytica

Pasteurella multocida and Pasteurella haemolytica produce specific proteins in the outer membrane under iron-depleted conditions. Pasteurella multocida serovar A expresses these proteins of molecular masses of 76 and 96 kDa as determined by electrophoresis. The analogous serovar D produces a further iron-regulated protein of 85 kDa. The Pasteurella haemolytica strains of serovar A1, A6 and T contain iron-regulated outer membrane proteins of molecular masses of 71, 77 and 100 kDa. These proteins possess binding positions for iron ions. Both Pasteurella multocida and Pasteurella haemolytica strains utilize iron from porcine and bovine transferrin, but not from haemin and haemoglobin.     

Overexpression and immunogenicity of the Oma87 outer membrane protein of Pasteurella multocida

The outer membrane protein of Oma87 from Pasteurella multocida A:1 has significant similarity to the D15 protective antigen of Haemophilus influenzae (Ruffolo and Adler, 1996). Four fragments of Oma87 from a P. multocida serotype D strain were cloned into a pGEX expression vector and transformed into E. coli JM105. Western blot analysis revealed that convalescent chicken sera reacted with only GST-F1 fusion protein which contained amino acids 18 through to 130 of Oma87 fused to the GST protein. Vaccination with the GST-F1 protein failed to protect chickens against challenge with a virulent P. multocida serotype A.     

Characterization of outer membrane protein-enriched extracts from Pasteurella multocida isolated from turkeys

Outer membrane protein (OMP)-enriched extracts of avian strains of Pasteurella multocida were examined by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Culture medium did not have a significant effect on the OMP profiles of strains of P multocida examined; however, in vivo propagation had an appreciable effect on the OMP profile composition of the reference strain P-1059. Such bacteria, expressed several additional OMP in the 27-kD, 48-kD, 56-kD, 60-kD, 80-kD, and 94-kD molecular mass regions. These OMP were not detected in the electrophorogram of strain P-1059 grown in vitro. The OMP profiles of reference strains of the 16 serotypes of P multocida did not identify any serotype-specific protein markers. Field strains of serotype A:3 had variation in OMP profiles and did not express OMP that all were identical to that expressed by the reference strain P-1059. The live attenuated CU and M9 bacterial vaccine strains expressed strain-specific OMP markers of 48-kD and 45-kD molecular masses, respectively. These strain-specific OMP markers may be used to differentiate these strains from virulent field strains that are of the same serotype and isolated from turkeys that have succumbed to pasteurellosis as a result of vaccine-related reactions or breakdown in immunity.     

Characterization of toxin from different strains of Pasteurella multocida serotype A and D

Chromosomal DNA from 13 different selected Pasteurella multocida spp. multocida strains of serotypes A and D were isolated and compared. All 10 toxigenic strains were recognized by a DNA probe which included the toxA gene coding for the Pasteurella multocida toxin (PMT). None of the three nontoxigenic strains reacted with the DNA probe. Toxin from the 10 toxigenic strains were isolated and compared. All were found to possess the biological characteristics previously described for the PMT isolated from P. multocida ssp. multocida NCTC 12178, including molecular mass of approx. 143 kDa and reactivity with a series of monoclonal antibodies. Toxin prepared from different toxigenic strains could not be differentiated immunologically by tandem crossed immunoelectrophoresis, Toxin, which was affinity purified from four of the strains and subsequently inactivated by formaldehyde, was cross-protective when used for vaccination of mice before challenge with PMT. It is concluded that the toxin from toxigenic strains of P. multocida ssp. multocida must be very similar, if not identical.     

Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida A:3 in cattle: molecular characterization of the immunodominant heme acquisition system receptor (HasR) protein

The iron-regulated outer membrane proteins (IROMPs) of Pasteurella multocida A:3 strain 232 (Pm232), a bovine isolate, were investigated as potential immunogens in cattle. We addressed the ability of P. multocida IROMP-enriched fractions to induce antibody responses in cattle by different vaccination strategies and the protective efficacy of these antibodies using a P. multocida-induced pneumonia challenge model. Vaccination of cattle with outer membrane-enriched fractions derived from Pm232 grown on either iron-depleted (IROMPs) or iron-sufficient (OMPs) conditions induced significant antibody responses; however, the correlation with lung lesion scores was not significant (P = 0.01 and P < 0.07, respectively). SDS-PAGE, Western blots and densitometric analyses of Pm232 grown under iron-deficient conditions revealed five major IROMPs including an immunodominant 96 kDa protein band. Mass spectrometry analysis of the 96kDa protein band suggested homology with the heme acquisition system receptor (HasR) of avian P. multocida (strain Pm70) and was confirmed by DNA sequence analysis of the cloned Pm232 hasR gene. Further analyses indicated that Pm232 HasR is a surface-exposed OMP and conserved among most P. multocida isolates investigated. In addition, cattle vaccinated with live Pm232 or IROMPs had significantly higher antibody responses to the 96 kDa protein band and the correlation with lung lesion scores approached significance (P = 0.056). These results indicate that antibody responses in cattle are induced by P. multocida IROMPs, and that the 96 kDa HasR protein is an immunodominant IROMP.     

Turkey heterophil chemotaxis to Pasteurella multocida (serotype 3,4)-generated chemotactic factors

The purpose of this study was to determine the ability of three strains or isolates of Pasteurella multocida (serotype 3,4) to generate chemotactic factors for heterophils when exposed to pooled turkey serum. Results indicated that each bacterial strain or isolate (M-9, CU, and 86-1913) was associated with the production of chemotactic factors, but the more pathogenic bacterial isolate (86-1913) elicited greater heterophil migration in chemotaxis studies.     

Cytokine profiles,apoptosis and pathology of experimental Pasteurella multocida serotype A1 infection in mice

Mice were experimentally infected with Pasteurella multocida serotype A1 to study the cytokine profiles, host cell apoptosis and sequential pathology at different hours of post-infection. Infected mice were dull, anorectic and depressed. A transient leukocytopenia followed by progressive leukocytosis was observed in the course of infection. Serum cytokine profiles showed significantly (P < 0.01) higher amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and mouse KC) in the infected mice when compared to control mice. The circulating lymphocytes were apoptotic on annexin V staining. Apoptotic nuclei were detected in splenocytes, hepatocytes and infiltrating leukocytes of the lungs on TUNEL staining. The lungs were grossly congested and hemorrhagic, and showed infiltration with polymorphonuclear cells at early and mononuclear cells in the late hours of infection. Alveolar epithelia, inter-alveolar septa and capillary endothelium of the lungs showed ultrastructural changes. Liver had degenerative changes in histological and ultrathin sections.     

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida

The SDS-PAGE patterns of the outer membrane protein (OMP) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa, 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with th three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.     

禽多杀性巴氏杆菌外膜蛋白和脂多糖的免疫保护效果

为确定禽多杀性巴氏杆菌(P.multocida)的保护性抗原外膜蛋白(OMPs)和脂多糖(LPS)在抵抗感染中的作用,本研究提取了禽P.multocida CVCC 474的OMPs和LPS成分,将该两种成分分别与弗氏佐剂混合制备免疫原,进行动物免疫。小鼠分为4组:OMPs组、LPS组、禽P.multocida弱毒活疫苗组和PBS对照组,每组16只。各组均免疫3次,每次间隔两周。间接ELISA检测免疫后小鼠血清特异性抗体水平,MTT法检测小鼠脾淋巴细胞增殖情况。以禽P.multocida强毒株CVCC 474进行攻毒,计算小鼠死亡数及保护率。实验结果表明,动物免疫后,OMPs组与LPS组免疫小鼠特异性血清抗体水平持续升高,与弱毒活疫苗组水平相近,与PBS组相比差异极显著(p<0.01)。脾淋巴细胞增殖试验表明,OMPs组、LPS组和弱毒活疫苗组的SI值极显著高于PBS对照组(p<0.01),但3个免疫组之间则无明显差异(p>0.05)。攻毒保护试验结果显示,OMPs免疫组的保护率与弱毒活疫苗相当,为8/10,高于LPS免疫组的保护率(7/10),表明OMPs作为研制禽P.multocida亚单位疫苗具有良好的...     

鸭多杀性巴氏杆菌外膜蛋白H基因的表达及抗原性鉴定

为了对多杀性巴氏杆菌(Pm)外膜蛋白基因H(OmpH)的免疫学活性进行鉴定,本研究根据已发表的Pm70株序列(AE004439)设计了两对引物,用PCR方法扩增了鸭多杀性巴氏杆菌C48-102株的Omp H,扩增片段为1180bp(ORF为1056bp)。Omp H与GenBank中登录的C44-1、P-1059、P52、X-73、PM-70、serogroup D的序列比对结果表明:Omp H在核苷酸水平上同源性为82.8%～99.2%;在氨基酸水平上同源性为82.1%～99.1%。扩增去信号肽的Omp H基因,构建了原核重组表达质粒pHT-Omp H,转化BL21并诱导表达,SDS-PAGE结果表明:表达蛋白约为41ku,与预期的分子量大小相符;western blot结果表明具有良好的免疫学活性,这为以重组OmpH蛋白建立针对鸭Pm的血清学检测方法奠定了基础。     

多杀性巴氏杆菌外膜蛋白免疫作用研究进展

外膜蛋白是细胞膜中的一种重要成分之一,在免疫方面的作用越来越受到关注.随着对多杀性巴氏杆菌外膜蛋白免疫原性及保护性的深入研究,开发有效的多杀性巴氏杆菌外膜蛋白疫苗将成为可能.笔者等就具有免疫作用的多杀性巴氏杆菌外膜蛋白的研究进展情况进行综述.     

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。     

Passive protection of mice with antiserum to neuraminidase fromPasteurella multocida serotype A:3

Antiserum to a partially purified neuraminidase fromPasteurella multocida, type A:3, was adsorbed with protease-digestedP. multocida type 3 lipopolysaccharide (LPS) to remove LPS immunoreactivity. The LPS-adsorbed antineuraminidase caused a 77% reduction in the neuraminidase activity of homologousP. multocida in anin vitro enzyme neutralization test. All 14 mice passively immunized with the adsorbed antineuraminidase were protected against challenge infection with homologousP. multocida in a mouse protection test. Ten out of 14 mice in one group that received antisera containing antibodies to both neuraminidase and LPS were protected. In contrast, only 1 out of 14 mice that were immunized with pre-immune serum survived the challenge. These results suggest that antiserum toP. multocida neuraminidase was, at least partly, responsible for the protection observed in this study. Neuraminidase may be one of the immunogenic protective proteins present in aqueous extracts ofPasteurella multocida.     

嗜水气单胞菌J-1株外膜蛋白Omp38的抗原性分析

根据GenBank已发表的嗜水气单胞菌(Aeromonas hydrophila)外膜蛋白Omp38的基因序列设计引物,以Ah J-1株基因组为模板,扩增omp38基因片段,定向克隆至表达质粒pET32 a中,将重组原核表达质粒pET32 a-omp38转化至大肠杆菌BL21,诱导表达出分子量约35 ku的重组蛋白。用纯化的重组蛋白免疫新西兰兔制备抗血清,ELISA检测抗体效价为1∶25 600,表明该蛋白有良好的免疫原性。PCR检测表明,omp38基因可在73%(22/30)的嗜水气单胞菌中发现。重组蛋白抗血清与30株嗜水气单胞菌分离株进行凝集试验,所有分离株均可在不同程度上发生反应,表明该重组蛋白是一种潜在的共同保护性抗原,可作为基因工程亚单位疫苗的候选成分。     

Characterization of a Pasteurella multocida (serotype B) bovine pneumonic pasteurellosis model and the effect of antimicrobials during peracute infection

A method to produce bovine pneumonic pasteurellosis for experimental purposes was studied and the clinical response of experimentally infected calves to selected antimicrobials was characterized. Male Holstein calves stressed with multiple hot and cold water applications followed by intratracheal inoculation of broth cultures of Pasteurella multocida serotype B developed acute clinical illness consistent with pneumonia. Infected, untreated calves consistently developed classic pneumonic pasteurellosis, infected calves treated with either oxytetracycline or sulfadimethoxine recovered from acute clinical disease, and the uninfected controls remained healthy. This disease model offers potential for use in pharmacokinetic and target tissue drug concentration studies and for dosage titration of drugs intended for treatment of bacterial pneumonias.