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。     

两株牛源荚膜A群脂多糖3型多杀性巴氏杆菌的分离鉴定

用北京、山东两奶牛场疑似牛出败的病死牛组织感染小鼠,小鼠死亡后取组织染色镜检、接种血清TSA和麦康凯培养基,分离到2株疑似多杀性巴氏杆菌,命名为Pm1和Pm2。经细菌培养特性及形态检验、多杀性巴氏杆菌种特异性PCR、荚膜A、B血清群特异性PCR、脂多糖基因分型PCR、荚膜A群透明脂酸抑制试验鉴定其为荚膜血清A群、脂多糖3型多杀性巴氏杆菌。将Pm1和Pm2回归小鼠证明有强毒力。本试验为国内荚膜A群多杀性巴氏杆菌的流行病学研究增添了一些新数据。     

禽多杀性巴氏杆菌强毒株及其经盐类浸提胞壁荚膜抗原后电镜显微结构的观察

禽多杀性巴氏杆菌是引起鸡、鸭和鹅等多种禽类的一种急性败血性传染病病原菌。制成的菌苗在安全性或免疫性等方面还不十分理想。近年来国外不少学者曾就禽巴氏杆菌细胞壁荚膜及免疫性进行了研究,我国对该菌菌体及其荚膜抗原性本身的研究还不多。我们曾用2.5％NaCl溶液自禽多杀性巴氏杆菌C_(48-1)强毒株浸提出一种保护性荚膜抗原,已经证明这种粗提荚膜抗原对鸡、鸭安全和具有良好的免疫保护力。有关对该菌的超微结构及其提取菌体胞壁的荚膜后的形态变化,目前在国内还未见有正式报道。为了给今后免疫研究工作提供依据,我们曾将我国的一株禽多杀性巴氏杆菌C_(48-1)强毒株用     

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.     

1株鹦鹉源多杀性巴氏杆菌的鉴定

<正>多杀性巴氏杆菌(Pasteurella multocida,Pm)属于革兰阴性球杆菌,定居于家禽和野禽的鼻咽部,能引起宿主出血性败血症,发病率和死亡率都很高,但不同类型的家禽易感性不同。多杀性巴氏杆菌有5个荚膜血清型,分别为A、B、D、E和F,引起禽类动物的多杀性巴氏杆菌荚膜血清型多为A型~([1])。此外,有较多技术对多杀性巴氏杆菌进行分型,其中多位点序列分型技术(multilo-     

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。     

牛源A型多杀性巴氏杆菌hyaD基因缺失株的构建及其在小鼠上的交叉保护性分析

hyaD为A型多杀性巴氏杆菌荚膜多糖合成相关基因,为探讨该基因对多杀性巴氏杆菌毒力及其免疫保护特性的影响,本研究利用同源重组方法,构建了牛源A型多杀性巴氏杆菌CQ2株（PmCQ2）的hyaD基因缺失株（ΔhyaD）。结果发现,与野生株相比,ΔhyaD的荚膜产生量及其感染后在脏器中的细菌定殖量均显著下降,其毒力显著降低。细胞试验发现,ΔhyaD更易黏附于巨噬细胞,被吞噬数量显著多于野生株,致使巨噬细胞相关炎性因子表达显著上调。hyaD基因的缺失,可调控与荚膜合成、LPS合成转运、铁转运等相关的基因表达显著下调,促使相关保护性抗原基因表达显著上调。以制备的PmCQ2株和ΔhyaD株灭活苗免疫小鼠（加强免疫1次）,免疫后第21天分别采用同源和异源多杀性巴氏杆菌攻毒,ΔhyaD株免疫小鼠肺组织感染后24 h无明显或轻微病理损伤,对牛源A型、B型和F型多杀性巴氏杆菌的免疫保护率分别为100%、100%和80%,对兔源、猪源和禽源A型多杀性巴氏杆菌的免疫保护率分别为90%、100%、100%;而野生株PmCQ2除对牛源A型多杀性巴氏杆菌的保护率在80%以上外,对牛源B型和F型及兔、猪、禽源A型多杀性巴氏杆菌均无明显交叉保护作用。研究结果表明,hyaD基因可通过调控荚膜产生及毒力相关因子表达影响菌株毒力;hyaD基因缺失可调控相关交叉保护性抗原表达,赋予菌株交叉免疫保护特性。该研究为多杀性巴氏杆菌通用型疫苗的研发提供了参考。     

肉兔免疫程序

商品肉兔(70日龄出栏)兔病毒性出血症,多杀性巴氏杆菌病二联灭活疫苗2毫升皮下注射35～40日龄或兔病毒性出血症(兔瘟)灭活疫苗2毫升皮下注射商品肉兔(70日龄以上出栏)兔病毒性出血症,多杀性巴氏杆菌病二联灭活疫苗2毫升皮下注射兔病毒性出血症,多杀性巴氏杆菌病二联灭活疫苗1毫升皮下注射或兔病毒性出血症(兔瘟)灭活疫苗1毫升皮下注射35～40日龄60～65日龄繁殖母兔(每年2次定期免疫)兔病毒性出血症,多杀性巴氏杆菌病二联灭活疫苗2毫升皮下注射产气荚膜梭菌病(魏氏梭菌病)灭活疫苗2毫升皮下注射兔病毒性出血症,多杀性巴氏杆菌病二联灭活疫苗2…     

野生草食动物源8株多杀性巴氏杆菌的PCR鉴定和分型

从表现出血性败血症临床症状的斑马、白唇鹿、黑鹿和长颈鹿中分离出8株多杀性巴氏杆菌（Pasteurellamuhtocida,Pm）,采用Pm种特异性的KMT1/KMT2引物分别与荚膜血清群特异性的Cap A1/Cap A2、Cap B1/CapB2、Cap D1/Cap D2引物组合来鉴定分离到的菌株,并与间接血凝试验及金黄色葡萄球菌抑制试验的结果相比较,证实PCR鉴定方法与传统的生化反应鉴定结果完全一致。荚膜PCR分型结果与间接血凝试验金黄色葡萄球菌抑制试验结果完全一致,这说明多重PCR方法可用于多杀性巴氏杆菌菌种及荚膜血清型的鉴定,我国野生草食动物多杀性巴氏杆菌病中存在多个荚膜血清型。     

兔病毒性出血症、多杀性巴氏杆菌病、产气荚膜梭菌病(A)型三联灭活疫苗研制

对 5批兔病毒性出血症、多杀性巴氏杆菌病、产气荚膜梭菌病三联灭活疫苗进行了动物试验 ,结果表明该疫苗安全有效。近期效检对兔病毒性出血症的保护率为 1 0 0 % ,对兔多杀性巴氏杆菌病保护率为 92 % ,对产气荚膜梭菌病 (A)型保护率为 88%。在免疫期试验中 ,免疫 6个月后 ,对兔病毒性出血症保护率为 1 0 0 % ,对多杀性巴氏杆菌病的保护率为 79% ,对产气荚膜梭菌病 (A)型的保护率为 88%。在保存期试验中 ,4～ 8℃保存 1年仍有效。     

Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin

Gnotobiotic pig antisera to purified toxoid from a capsule type A or D strain of Pasteurella multocida contained large quantities of antitoxin but comparatively little antibody to a crude lysate of P. multocida. These sera given intraperitoneally to further pigs were almost completely protective against turbinate atrophy after intranasal inoculation of dilute acetic acid and infection with type D toxigenic P. multocida. In contrast, antisera to a crude lysate or bacterin of toxigenic P. multocida which contained large titres of antibody to P. multocida lysate, but no detectable antitoxin, were not protective. Colonisation by toxigenic P. multocida was significantly reduced in protected pigs and was similar to colonisation by nontoxigenic P. multocida in pigs untreated or treated with dilute acetic acid. These results indicated (1) that antitoxin was protective and cross protective between toxins from different capsule types; and (2) that the toxin was the main colonisation factor produced by toxigenic bacteria in the acetic acid model of infection and that immunity to it did not eliminate infection.     

Haemolytic and cytotoxic activities of the Tween 80-extracted putative haemolysin of Pasteurella multocida B:2

The objective of this study was to investigate the haemolytic and cytotoxic activity of Pasteurella multocida B:2 strains, originally from cases of haemorrhagic septicaemia in cattle. All six P. multocida B:2 strains were non-haemolytic on sheep blood agar (SBA) and horse blood agar (HBA) when grown aerobically and on SBA anaerobically but they were haemolytic on HBA when grown anaerobically. No haemolytic activity against horse red blood cells was detected in culture supernates from aerobically or anaerobically grown cultures and only very weak haemolytic activity was obtained in supernates or pellet fractions from sonicated cells. However, after repeated extraction of sonicated cells with Tween 80, haemolytic activity was found in various cell fractions, both Tween-soluble and -insoluble. The Tween-extracted putative haemolysin and other bacterial fractions were also cytotoxic for mouse macrophage-like J774.2 cells. Further characterisation of the putative haemolysin revealed it to be a heat-labile, non-pore-forming protein of molecular weight >10 kDa whose activity was completely destroyed by trypsin and greatly reduced with protease and proteinase K treatment. Congo red also reduced the haemolytic activity. Non-denaturing gel-electrophoresis and RBC agar overlay revealed clear haemolytic zones but suggested that Tween was bound to some component of the P. multocida B:2 fractions and was responsible, to some extent, for the haemolytic activity observed. However, the effect of heat and other reagents on the Tween-extracted fractions and the lack of haemolytic activity in different Tween-extracted cell fractions of organisms other than P. multocida suggested that some proteinaceous component of the organism could indeed act as a haemolysin. This putative haemolysin may be one of the virulence attributes of P. multocida, but its characterisation and role in pathogenesis require further study.     

Capsule thickness and amounts of a 39 kDa capsular protein of avian Pasteurella multocida type A strains correlate with their pathogenicity for chickens

Capsule thickness of avian Pasteurella multocida type A strains was determined by transmission electron microscopy after labeling with polycationic ferritin and compared with their pathogenicity for chickens. The capsule thickness of P. multocida strains Pm-18 and X-73 was 81.4 and 50.1 nm on average, respectively. These strains were highly virulent for chicken, whereas the less virulent strains Pm-1 and Pm-3 had a thin and irregular capsule, 21.0 and 29.8 nm on average, respectively. However, the thickest capsule was observed in strain P-1059, 101.2 nm on average, and the strain revealed moderate virulence. The noncapsulated variant P-1059B, which was derived from strain P-1059, revealed low virulence. The six P. multocida strains were examined with regard to protein content on the capsule of organisms. Amounts of total proteins of crude capsular extract (CCE) from capsulated strains were approximately twice those of the noncapsulated strains. The amount of an antigenic 39 kDa protein in the CCE were found to correlate with the capsule thickness, since heavily capsulated strains exhibited the greatest amount, whereas noncapsulated strains including noncapsulated and low virulent variant P-1059B possessed little 39 kDa protein. The results demonstrated that the capsule thickness and the quantity of a 39 kDa capsular protein of avian P. multocida type A strains correlated with their pathogenicity for chickens.     

Characterization of a 39kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies

The role of a 39kDa protein of avian Pasteurella multocida in pathogenesis of fowl cholera was investigated using monoclonal antibodies (Mabs). Mabs were prepared by immunization of BALB/c mice with a crude capsular extract (CCE) of P. multocida strain P-1059 (serovar A:3). Totally eight hybridomas producing Mab were obtained. Immunoblot analysis of the hybridomas revealed that all the Mabs recognized a 39kDa protein of CCE. Treatment of CCE antigen with proteinase K or periodic acid indicated that the epitope recognized was proteinaceous. The Mabs reacted with a major 39kDa protein of CCE from encapsulated strains but not with any protein of non-capsulated strains indicating that a direct correlation between encapsulation and the 39kDa protein. Immunoelectron microscopy on strain P-1059 and the non-capsulated derivative P-1059B (serovar -:3) reacting with the Mabs and gold-labeled anti-mouse IgG indicated that the protein is associated with the capsule. The Mabs significantly inhibited the adherence of encapsulated P. multocida strains to chicken embryo fibroblast cells, but only slightly that of non-capsulated strains. Mice passively immunized with the Mabs were protected from lethal challenge with virulent strains P-1059 and X-73 (serovar A:1). Thus the capsular 39kDa protein was determined to be an adherence factor and a cross-protective antigen of avian P. multocida type A strains.     

Cloning and characterisation of the ahpA gene of Pasteurella multocida serogroup b:2 (strain P52): short communication

Pasteurella multocida B:2 is responsible for haemorrhagic septicaemia in cattle and buffaloes, causing severe economic losses in the developing countries. In the present study, the ahpA gene of P. multocida B:2 (P52) was cloned, sequenced and compared with the previously reported ahpA gene sequence in P. multocida A:1, which is responsible for its haemolytic phenotype. E. coli DH5a cells were further transformed with recombinant plasmid carrying the ahpA gene from P. multocida B:2 (P52) but SDS-PAGE analysis failed to show the expression of haemolysin protein. Slight haemolysis was albeit observed in horse blood agar plates streaked with recombinant E. coli carrying the ahpA gene. Our study indicates that there is 99.6% similarity and 0.4% divergence between ahpA gene of P. multocida B:2 (P52) and P. multocida A: 1, while membrane topology analysis has predicted that ahpA is an inner membrane protein with two strong hydrophobic regions at the N and C terminals. The presence of significant homology in ahpA sequence in A: 1 and B:2 perhaps suggests a common mechanism of pathogenesis in different species of animals.     

Induction of pneumonia in rabbits by use of a purified protein toxin from Pasteurella multocida.

Heat-labile toxin from a cell sonicate of a virulent type-D strain of Pasteurella multocida was purified by ammonium sulfate precipitation followed by ion exchange chromatography, gel filtration chromatography, and polyacrylamide gel electrophoresis. Toxic activity was assayed during toxin purification by cytopathic effect in Vero or bovine embryonic lung cell cultures. Toxicity for cells correlated with dermonecrosis in guinea pig skin. Toxicity was accounted for by a single protein with a molecular weight of 149,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbits were inoculated intranasally with purified toxin to determine whether toxin had a role in the induction of pneumonia in rabbits infected with P multocida. Pneumonia, pleuritis, acute hepatic necrosis, and splenic lymphoid atrophy were found in 4 of 5 rabbits. One of 5 rabbits had bilateral turbinate atrophy. Western blotting with monoclonal antibodies to toxin from a P multocida isolate causing atrophic rhinitis in pigs revealed the toxin that induces pleuritis and pneumonia in rabbits to be the same or a closely related toxin.     

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.     

A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.     

Genetic organisation of the capsule biosynthetic locus of Pasteurella multocida M1404 (B:2)

Capsules from a range of bacterial species have been shown to be major virulence determinants and capsule has been implicated in virulence in Pasteurella multocida. Moreover, capsular serogroup appears to be related to disease predilection. Haemorrhagic septicaemia strains belong to serogroup B and E, fowl cholera strains to serogroup A and atrophic rhinitis strains to serogroup D. The entire capsule biosynthetic locus of P. multocida A:1 has been cloned and its nucleotide sequence determined (Chung et al., 1998. FEMS Microbiol. Lett. 166, 289-296); however, nothing is known of the P. multocida B:2 capsule locus. In this work we have determined the nucleotide sequence and genetic organisation of the P. multocida M1404 (B:2) capsule locus. By analogy with the cap loci of other bacteria, the nucleotide sequence can be divided into three functional regions. Regions 1 and 3 comprise six genes involved in transport of the polysaccharide capsule to the cell surface. The deduced products of these genes show high similarity to proteins involved in capsule export in other bacteria. Region 2 comprises nine genes which are likely involved in biosynthesis of the polysaccharide capsule. The deduced products of three of these genes (bcbA, bcbB and bcbC) show significant similarity to proteins known to be involved in polysaccharide biosynthesis while the other six show no similarity to known proteins. However, their organisation indicates they are co-transcribed with bcbA, bcbB, bcbC and the Region 1 capsule export genes, suggesting strongly that they are also involved in capsule biosynthesis.     

Occurrence of Pasteurella multocida and related species in village free ranging chickens and their animal contacts in Tanzania

Investigation was done to determine the presence of Pasteurella multocida and related species in free ranging chickens and ducks, dogs, cats and pigs in three climatic zones (cool, warm and hot) of rural Morogoro, Tanzania. A total of 153 isolates of P. multocida ssp. multocida and related species were obtained by direct culture on blood agar, selective medium and mouse inoculation. P. multocida ssp. multocida was isolated from 0.7% of chickens and 7% of ducks. In dogs and cats, P. multocida ssp. multocida was isolated from 1 and 68%, respectively. One isolate of Pasteurella gallinarum was isolated from a duck. Other species obtained were; P. multocida ssp. septica, Pasteurella stomatis and taxon 16 from dogs and cats, while Pasteurella dagmatis and Pasteurella canis were found in dogs only. Prevalence of P. multocida ssp. multocida was significantly higher (P<0.01) in ducks of the warm zone (22%) than in ducks of other zones (0%). No significant difference was observed between the prevalence of P. multocida ssp. multocida in chickens of the warm zone (2%) and chickens of the cool and hot zones (0%). Extended phenotypic characterization revealed phenotypic similarities between two isolates from chickens and the duck strains. Mouse inoculation appeared to be more sensitive in detecting P. multocida ssp. multocida than blood agar and selective medium. Direct culture on blood agar recovered most of the isolates from dogs. This study has demonstrated for the first time the presence of P. multocida and related species in the village free ranging chickens, ducks, dogs and cats in Tanzania. Other non-classified Pasteurella spp. were also observed in the study, but further characterization is required before the final classification can be made. This paper reports for the first time the isolation of unclassified Pasteurella from dogs and cats in Africa. The results implies that fowl cholera might be occurring in free ranging poultry, and dogs and cats kept in contact might serve as sources of P. multocida to chickens and ducks. Subsequent applications of molecular techniques to analyse the epidemiological relatedness of clones isolated from different host species is indicated.