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1.
A 39 kDa protein of avian Pasteurella multocida strain P-1059 (serovar A:3) was purified from a crude capsular extract by immunoaffinity chromatography by using a ligand of purified mouse monoclonal antibody to the 39 kDa capsular protein of the strain. Protective activity of the purified 39 kDa protein antigen was determined by inoculation of ddY mice twice with 25 or 125 mug of the protein and challenge-exposure with 10 or 50 LD(50) of strains P-1059 or X-73 (serovar A:1). The results showed that the antigen gave high protection (60 to 100%). These results indicated that the 39 kDa protein of avian P. multocida is a cross-protective antigen over serovars A:1 and A:3.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
The outer membrane protein (OMP) profiles of two strains of capsular type A Pasteurella multocida isolated from the lungs of pigs with enzootic pneumonia were studied. Sarkosyl extracted OMPs from P. multocida grown under iron-restricted and iron-replete conditions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 74 kDa, 94 kDa, 99 kDa and 109 kDa were expressed by strain A52, while 74 kDa, 82 kDa, 94 kDa and 99 kDa IROMPs were expressed by strain B80. Swine immune sera, obtained from pigs which were first immunized with a polyvalent P. multocida type A and type D bacterin and subsequently challenged with type A strain of P. multocida, contained antibodies against the IROMPs. These antibodies cross-reacted with the IROMPs expressed by avian strain P1059 of P. multocida. Convalescent-phase serum obtained from turkeys which survived fowl cholera, also cross-reacted with the IROMPs from porcine strains of P. multocida. These results suggested that IROMPs from porcine and avian strains of P. multocida may share common epitopes that were recognized by swine immune serum as well as turkey convalescent-phase serum.  相似文献   

5.
A mutant strain, PBA322, was constructed by electroporation of a phagemid containing the coding region of antisense RNA of the ompH gene, encoding 39 kDa capsular protein or OmpH, into the parental strain P-1059 (serovar A:3) of Pasteurella multocida, and the pathogenicity was determined in mice and chickens. Grayish colonies of the mutant, indicating loss of capsule synthesis, were observed under a stereomicroscope using obliquely transmitted light, while iridescent colonies were observed for the parental strain. Moreover, strain PBA322 showed a low amount of OmpH compared with the parental strain on SDS-PAGE. Additionally, the capsule of strain PBA322 was thinner than that of the parental strain according to electron microscopy, correlating to the attenuation against chickens. In conclusion, strain PBA322, the mutant of P. multocida strain P-1059, was completely attenuated for chickens.  相似文献   

6.
The avian strain P-1059 of Pasteurella multocida was grown on blood agar (BA), on dextrose-starch agar (DSA), or in Heddleston's hydrogen sulfide test broth. Cells were examined for the presence of pili using electron microscopy after staining with phosphotungstic acid, and they were examined for capsule after ruthenium red staining. Pili were found on the capsulated iridescent type, P-1059I, and on two non-capsulated variants, the blue, P-1059B, and the gray, P-1059G. Many cells grown on BA were heavily piliated. In contrast, fewer cells grown on DSA had pili, and piliation was only slight to moderate. The P-1059I, P-1059B, and P-1059G produced pellicles when grown on broth medium. Pili were found on the circumference of the cells grown on either agar or broth medium. Occasionally a pilus connecting two cells was seen on cells cultured in broth. Cultivation of the P-1059I on DSA containing the iron-chelating agent alpha,alpha'-bipyridyl produced a non-capsulated blue variant. The non-capsulated variant reverted to P-1059I when grown on BA but did not revert when grown on DSA.  相似文献   

7.
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.  相似文献   

8.
Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.  相似文献   

9.
One hundred avian Pasteurella multocida isolates recovered from cases of fowl cholera and related infections in England and Wales over a 13-year period were characterised by capsular PCR typing and analysis of outer membrane protein (OMP) profiles. Sixty-eight percent of the strains were of capsular type A, 14% were type F, 5% were type D, 4% were type B and 9% were untypable. Nineteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. Fifty-six percent of the isolates were represented by 15 OMP-types, whereas 44% of the isolates were associated with four OMP-types. The extensive molecular mass heterogeneity of the OmpA and OmpH proteins supports previous findings that avian P. multocida strains are very diverse. Furthermore, the isolates studied were associated with different clinical symptoms and were recovered from a wide range of lesions and tissues. The high degree of strain diversity together with the wide variety of clinical symptoms suggest that certain avian strains of P. multocida are opportunistic pathogens of relatively low virulence. Strains of capsular types B, D and F, as well as the untypable isolates, were associated exclusively with specific OMP-types and represent distinct and widely disseminated clonal groups. These observations support the view that avian strains of P. multocida have a clonal population structure. Based on previous studies, the molecular mass heterogeneity of the OmpA and OmpH proteins might provide a selective advantage to P. multocida by generating antigenic variation.  相似文献   

10.
In vitro experiments were undertaken to study the adhesion and colonization to tracheal mucosa, lung and aorta explants from freshly killed rabbits of two different strains of Pasteurella multocida. Serotype A:3 (capsulated, fimbriae +, haemagglutination -, dermonecrotic toxin -) isolated from a rabbit with rhinitis, and serotype D:1 (non-capsulated, fimbriae +, haemagglutination +, dermonecrotic toxin +) isolated from a dead rabbit with septicaemia, were used. When the explants were observed under the scanning electron microscope, the type D strain was highly adherent to trachea and aorta explants compared to the type A strain. Adhesion to lung explants was best achieved by the type A strain after 45 min incubation, but after 2 h incubation no significant difference was observed between the strains. Our data indicate that the presence of fimbriae and the absence of capsule seem to enhance the adherence of P. multocida type D strain to tracheal tissue. The capsular material of P. multocida type A strain and the toxin of the type D strain seem to influence the adherence to lung tissue in rabbit. Adhesion of strain D to aorta may indicate the expression of receptors on the endothelium to that strain and may also explain the ability of certain strains to cause septicaemia.  相似文献   

11.
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.  相似文献   

12.
One hundred and fifty-three bovine Pasteurella multocida strains recovered primarily from cases of pneumonia and mastitis in England and Wales over an 11-year period were characterised by capsular PCR typing, comparison of outer membrane protein (OMP) profiles, and multilocus sequence analysis. All of the strains were of capsular type A with the exception of a single capsular type F isolate. Thirteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. However, 85% of the isolates were represented by just five OMP-types and 39% of the strains were of a single OMP-type. Multilocus sequence analysis revealed a limited degree of genetic diversity among bovine P. multocida isolates; strains of the same OMP-type have identical genetic backgrounds and represent distinct clones. Analysis of OMP variation was more discriminating than multilocus sequence analysis because strains of different OMP-types had the same, or similar, genetic backgrounds. The association of a small number of clones with the majority of cases of bovine pneumonia suggests that these clones have an increased capacity to cause disease compared to less frequently recovered clones. Molecular mass heterogeneity of OmpA and OmpH, in strains of the same or similar genetic background, suggests that these proteins are subject to diversifying selection within the host and might play important roles in host-pathogen interactions. Comparison of the OMP profiles of bovine isolates with those of avian, ovine and porcine strains showed that a high proportion of the respiratory tract infections in each of these species are caused by different strains of P. multocida. However, the presence of small numbers of closely related strains in more than one host species suggests that transmission of bacteria between different host species is also a factor in the population biology of P. multocida.  相似文献   

13.
Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar 1a) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.  相似文献   

14.
Mouse monoclonal antibodies (MAbs), raised against the NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain Kyoto (serovar 2), recognized two different epitopes on a single protein of molecular weight 67 kDa. The MAbs were classified as protective or non-protective against strain Fujisawa (serovar 1). In immunoblotting analysis using the MAbs, fifteen wild strains were shown to contain different amounts of 67 kDa protective antigen. Each formalin-killed whole cell vaccine (bacterin) prepared from the fifteen wild strains conferred different levels of protection against strain Fujisawa in mice. Bacterins prepared from wild strains with larger amounts of 67 kDa protective antigen tended to give high levels of antigen-specific antibody and better protection to mice. These results indicate that the amount of 67 kDa protective antigen which influences the induction of protective immune responses may vary substantially among the strains of E. rhusiopathiae (serovar 2).  相似文献   

15.
79 strains of P. multocida were investigated, mostly isolated from porcine nasal cavities, and a mannose-resistant hemagglutination with guinea pig and human group 0, but not with porcine erythrocytes was found. Fimbriae as adhesins were demonstrated only on 2 strains. A correlation between capsular type, hemagglutination, fimbriation and toxigenicity on the examined P. multocida strains was not observed. Three strains were investigated for the adherence to the nasal mucosa of neonatal pigs; aggregates shown by scanning electron microscopy indicate colonization; a correlation of adherence with surface proteins and slime production of P. multocida is discussed.  相似文献   

16.
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。  相似文献   

17.
猪多杀性巴氏杆菌对HeLa细胞附着能力的研究   总被引：5，自引：0，他引：5  
本研究通过猪肺疫的活菌疫苗和死菌疫苗多杀性巴氏杆菌菌株(Pasteurella multocida,Pm)对小鼠的毒力试验测定它们的毒力性。结果表明,死菌疫苗Pm的毒力性比活菌疫苗Pm的强,即死亡率分别为10 0 %和0 %。通过两菌株对He L a细胞的附着试验测定它们的附着能力,结果证明强毒菌的附着能力明显地比弱毒菌强(P<0 .0 1) ,平均附着数分别为11.96和2 .4 4 ;从上述菌株细胞荚膜中分别提取荚膜蛋白,用SDS- PAGE分离测定两菌株荚膜蛋白质结构,结果表明39k Da荚膜蛋白是强毒菌的特异性蛋白。以上研究结果证明Pm的毒力与He L a细胞的附着能力是密切相关的,同时暗示本菌39k Da荚膜蛋白可能与它们的毒力和He L a细胞的附着能力有关  相似文献   

18.
Encapsulated avian strains of Pasteurella multocida possessing an A-type capsule were shown to be resistant to the bactericidal action of turkey serum, whereas unencapsulated variants as well as other unencapsulated strains were not. Removal of the capsule from serum-resistant strain P1059-1 resulted in this strain becoming susceptible to the bactericidal effects of turkey serum. Since complement was consumed when encapsulated or unencapsulated strain P1059-1 was incubated in turkey serum, we conclude that the capsule acts to shield the outer membrane rather than prohibiting the generation of an effective membrane attack complex.  相似文献   

19.
Thirteen mouse monoclonal antibodies (Mabs) against the protective protein antigen (P64) of Erysipelothrix rhusiopathiae were prepared and partially characterized. The titres of the Mabs varied from 200 to 1,638,400 as determined by enzyme-linked immunosorbent assay (ELISA). Of the 13 Mabs 10, two and one belonged to the IgG2a, IgG1 and IgM subclasses, respectively. All Mabs reacted strongly with the 64 kDa protein and weakly with the 43 kDa protein upon Western blotting of the alkaline extract (AE) of E. rhusiopathiae. The protective activity (PD50/ml) of the 13 Mabs against E. rhusiopathiae infection in mice varied from < 50 to > 50,000. These Mabs were classified into three groups, highly protective Mabs, moderately protective Mabs and Mabs which did not possess protective activity, based on the protective index (ratio of the PD50/ml to the antibody titre). These results suggest that the 64 kDa protein is an effective protective antigen, which is easily cleaved into many small proteins, including the 43 kDa protein, and possesses at least two epitopes related to its protective activity and at least one epitope which is not related to protection of mice against E. rhusiopathiae infection.  相似文献   

20.
采用菌落多重PCR方法对分离保存的28株多杀性巴氏杆菌进行种型和毒素基因的检测,结果表明,菌株C51-6、M-4和P-2237为产毒素多杀性巴氏杆菌,菌株C51-6和P-2237为荚膜血清D型,菌株M-4为荚膜血清A型。同时用金黄色葡萄球菌抑制试验、中性吖啶黄沉淀试验和豚鼠皮肤坏死试验对PCR方法进行了验证。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,3株菌株鉴定为多杀性巴氏杆菌多杀亚种。  相似文献   

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