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1.
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2.
The outer membrane protein (OMP), plasmid, and antimicrobial resistance profiles of Pasteurella haemolytica serotypes 1 through 12, a bovine isolate of P multocida, a chicken isolate of P multocida, and an unidentified Pasteurella species of bovine origin were examined. Isolates of P haemolytica serotypes belonging to the same biotype possessed similar OMP profiles. Biotype A isolates contained 2 prominent OMP of 43 kilodaltons (kD) and 29 kD, whereas biotype-T serotypes contained 3 major OMP of 43, 36, and 25 kD. The major OMP profiles of the 2 P multocida isolates and the unidentified Pasteurella species were different from each other and from P haemolytica isolates. Plasmid DNA screening indicated both plasmid-containing and plasmid-free P haemolytica and P multocida isolates. Multiple drug resistance was found in pasteurellae isolates with and without plasmids. However, a relationship between drug resistance and plasmid isolation was found in 3 of 4 haemolytica serotype 1 field isolates, all of which contained a 2.51-megadalton plasmid and had multiple drug resistance for benzylpenicillin, ampicillin, streptomycin, and tetracycline.  相似文献   

3.
Pasteurella haemolytica biotype A serotype 1 is the principal etiologic agent of bovine pneumonic pasteurellosis. A clear understanding of the pathogenesis of this disease and the mechanisms of resistance to it has been limited by a lack of information on the important antigens of the organisms. Using recombinant DNA techniques we have cloned a segment of DNA from P. haemolytica A1 that encodes three proteins of 28, 30, and 32 kDa. Two of these proteins, 30 and 28 kDa, react strongly on a Western blot with a bovine serum raised against live cells of P. haemolytica A1. The gene for the 30 kDa protein was localized to a 3.1 kbp EcoRI fragment, and expression of the 30 kDa protein was found to be independent of an E. coli promoter. The 30 kDa protein comigrated with a 30 kDa P. haemolytica protein that was susceptible to radioiodination and presumably exposed on the bacterial cell surface. The other principal radiolabeled P. haemolytica proteins were 100, 45, and 15 kDa. Antibodies against the 30 kDa protein, isolated from E. coli carrying the recombinant plasmid, recognized 30 kDa and 15 kDa proteins in P. haemolytica serotypes 1-15 and caused agglutination of whole P. haemolytica A1 cells. Cattle vaccinated with live P. haemolytica, P. haemolytica outer membrane proteins, or the cloned 30 kDa protein developed antibodies to the cloned 30 kDa protein as detected by Western blotting and densitometry. Sera were obtained from cattle vaccinated with live or killed P. haemolytica or saline and challenged with P. haemolytica. Those sera were evaluated for antibody responses to the cloned 30 kDa protein. High antibody responses to the 30 kDa protein significantly correlated (P less than 0.01) with resistance to challenge. From these studies it is concluded that the 30 kDa protein represents a surface antigen of P. haemolytica A1 that may be important in inducing immunity to P. haemolytica.  相似文献   

4.
The dynamics and duration of maternally derived antibodies as well as the onset of acquired immunity against Mannheimia haemolytica and Pasteurella multocida in range-pastured beef calves were investigated. Two groups of unvaccinated cattle were used in this study. Serum antibody responses were measured by enzyme-linked immunoassay for antibodies of the IgG1, IgG2 and IgM isotypes binding M. haemolytica whole cells (WC) or leukotoxin (LKT) and P. multocida outer membrane proteins (OMPs). Comparisons of mean antibody responses to M. haemolytica LKT and WC and P. multocida OMPs were made within each group. Maternally derived antibodies against M. haemolytica and P. multocida reached lowest levels at 30-90 days after birth. Calves began production of antibodies against M. haemolytica and P. multocida between 60 and 90 days of age in both groups. Based on the results of this study, in beef herds vaccinated against M. haemolytica and/or P. multocida, it may be best to vaccinate calves around 3 months of age. In contrast, beef calves from unvaccinated herds might benefit from vaccination at 4 months of age.  相似文献   

5.
A genomic fragment of Pasteurella haemolytica biotype A coding for a serotype 1-specific agglutinating antigen was used as a probe in a series of hybridization experiments to determine distribution of the fragment in various P. haemolytica serotypes as well as other bacteria. Results showed presence of the fragment in seven out of the 12 serotypes tested, all of which belonged to biotype A. Two other serotypes belonging to biotype A, all three serotypes belonging to biotype T, two Pasteurella multocida isolates and Escherichia coli did not have the fragment in their genome. Thus the expression of the P. haemolytica biotype A serotype 1-specific agglutinating antigen (PHA1SAA) seems to be due to serotype-specific regulation of protein expression rather than to genetic deletion. Differences in methylation of the PHA1SAA-coding fragment was also noted in DpnI and Sau3AI genomic DNA digests from the various serotypes analyzed by Southern blot. However, no apparent correlation was observed between methylation and PHA1SAA expression. E. coli with a recombinant plasmid containing a homologous genomic fragment derived from P. haemolytica serotype 2 also expressed PHA1SAA.  相似文献   

6.
Polypeptides from whole cell preparations of Pasteurella multocida serotypes A:12 and A:3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Antigens were detected by immunoblot analysis, using sera from 3 groups of rabbits. Sera were obtained from rabbits inoculated intranasally with P multocida serotype A:12 or A:3, from rabbits maintained in a rabbitry with enzootic P multocida A:12 infection, and from rabbits maintained in a rabbitry with enzootic P multocida A:3 infection. Immunoblot analyses of pre- and postinoculation sera from experimentally infected rabbits, using serotype A:12 antigen, revealed 3 polypeptides with approximate molecular mass of 28, 30, and 37 kDa that consistently detected antibodies after P multocida-induced infection. Sera from rabbits naturally infected with either serotype, tested against serotype A:12 and A:3 antigens, detected the same polypeptides in both serotypes. Thus, immunologic reactivity to these polypeptides may be useful for serologic detection of P multocida infection.  相似文献   

7.
A protein from Pasteurella haemolytica that was highly immunogenic and toxic toward bovine alveolar macrophages was partially purified. When isolated from culture supernatants of P haemolytica serotype 1 or serotype 6, the protein reacted on Ouchterlony immunodiffusion tests with antisera from 12 serotypes of P haemolytica, but did not cross-react with antisera to serotypes of P multocida. This indicated that the protein may be specific for P haemolytica. Bacteria were grown in dialysis culture in a brain-heart infusion and calf-serum growth medium. The protein was isolated from the medium by ultrafiltration and size-exclusion chromatography and has a molecular weight of approximately 150,000 daltons. The protein, which is highly immunogenic and has the characteristics of a virulence factor, is common to all serotypes of P haemolytica, and may be an effective agent for immunization against P haemolytica in cattle.  相似文献   

8.
Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.  相似文献   

9.
Membrane associated proteins from 8 untypeable Pasteurella haemolytica strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with those of P haemolytica serotypes 1 and 2. Cattle antisera obtained from P haemolytica serotype 1 vaccine trials were used in immunoblotting assays to compare the membrane proteins from the 8 untypeable strains with those from P haemolytica serotypes 1 and 2. Densitometry was used to identify bands, and using linear regression analyses, the peak area optical densities (measuring antibody response) were correlated to lesion scores from the vaccinated calves. Significant antibody responses to proteins of 99, 69, 60, 55, 47, 45, 39, 33, 30, 16, and 14.5 kDa were detected for 4 or more of the 8 P haemolytica untypeable strains. Serotypes 1 and 2 of P haemolytica contained a comigrating 30-kDa protein. Antibody responses to proteins of 39, 33, and 32.5 kDa were significant for 3 of the untypeable strains and had significant correlation to lesion scores. Antibody responses to various other proteins were significant for 2 untypeable strains each.  相似文献   

10.
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   

11.
Mannheimia haemolytica serotype 1 (S1), S6 and S2 are the most common bacterial isolates found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. Recombinant PlpE (rPlpE) is highly immunogenic in cattle, and the acquired immunity markedly enhanced resistance to experimental challenge. We previously demonstrated that the immunodominant epitope (R2) is located between residues 26 and 76 on the N-terminus of PlpE from a reference S1 strain (). This region consists of eight hexapeptide repeats. The potential of this epitope as a vaccine or supplement to commercial vaccines is dependant on its state of conservation amongst isolates of the three serotypes. To determine this, we sequenced plpE genes from 32 isolates. The sequences from S1 and S6 were identical with one exception. Substantial variation was observed among sequences from S2 strains, particularly in the R2 region of the protein. These variations in S2 isolates range from 3 to 28 hexapeptide repeats. Calculated molecular weight of PlpE from S1 and S6 isolates was 37 kDa, where as PlpE from S2 strains ranged from 30 to 50 kDa. These similarities and differences were demonstrated by western blot. Competitive binding assay was used to determine that antibody against rPlpE from S1 binds native PlpE on surfaces of both S1 and S2 cells.  相似文献   

12.
Hybridoma-derived monoclonal antibodies (MAB) against the cell surface antigens of Pasteurella haemolytica serotype 1 were obtained by the fusion of murine myeloma cells (P3 X 63 - Ag 8.653) with splenocytes of BALB/c mice immunized with crude logarithmic growth-phase culture supernatant. Initial screening was performed, using an ELISA, with the same bacterial growth culture supernatant as coating antigens. Further selection was done, using a panel of purified antigens--either capsular polysaccharide or lipopolysaccharide--as the coating antigen in an ELISA, and then performing a leukotoxin-neutralization assay. Two MAB, designated IIB-6 and H-2, reacted specifically with the capsular polysaccharide and the other 3, designated IVG-3, IH-3, and IIC-2, reacted with the lipopolysaccharide. One MAB, designated IH-6, did not react with leukotoxin, capsular polysaccharide, or lipopolysaccharide. The MAB to the capsular polysaccharide (IIB-6 and H-2) were characterized further; both antibodies belonged to the IgM class and were agglutinating. In addition, they promoted neutrophil-mediated opsonophagocytosis and complement-mediated immune bacteriolysis of P haemolytica serotype 1. Results from 3 studies indicated that the MAB IIB-6 and H-2 were specific only to the capsular polysaccharide of serotype 1 of P haemolytica. The MAB to the lipopolysaccharide (IVG-3, IH-3, and IIC-2) were of the IgG1, IgG3, and IgM classes, respectively and were not characterized further. The availability of a MAB identifying a serotype-specific, surface-exposed determinant on the capsule of P haemolytica serotype 1 should facilitate and expand studies concerning the role of the capsular material and lipopolysaccharide in the pathogenicity of P haemolytica infection in cattle.  相似文献   

13.
The outer membrane proteins (OMP) were extracted from the P. haemolytica A2, A7 and A9 to determine their potential as immunogens and their capability for cross-protection. Sixty lambs of approximately 9 months old were divided into four main groups. Animals in Group 1 were vaccinated with 2ml vaccine containing 100microg/ml of the outer membrane proteins of P. haemolytica A2. Animals in Group 2 were similarly vaccinated with the OMPs of P. haemolytica A7 while Group 3 with OMPs of P. haemolytica A9. Animals in Group 4 were unvaccinated control. During the course of the study, serum was collected to evaluate the antibody levels toward each OMP. There appeared to be good immune responses. However, high antibody levels did not necessarily result in good protection of the animals, particularly against cross-infection with P. haemolytica A9 in animals vaccinated with the OMPs of P. haemolytica A2. It seemed that the antibody responses were more specific toward the homologous challenge but generally did not cross-protect against heterologous serotype challenge. However, the OMPs of P. haemolytica A7 produced good in vivo cross-protection and excellent correlations when good antibody responses against all serotypes led to successful reductions of the extent of lung lesions following homologous and heterologous challenge exposures. Thus, the OMPs of P. haemolytica A7 was effective in protecting animals against homologous and heterologous infection by live P. haemolytica A2, A7 and A9.  相似文献   

14.
Mouse monoclonal antibodies were produced to Haemophilus pleuropneumoniae bacteria of the most common serotype 2. 11 hybridoma cultures were recovered that produced antibodies with moderate to strong reactivity to the antigen. 10 of these antibodies were specific to isolated capsular antigens from H. pleuropneumoniae of serotype 2, while one antibody reacted with capsular antigens from bacteria of all 8 serotypes. One hybridoma producing antibodies with a titre of 1:1000 was cloned and the antibody specificity studied further. The binding of this antibody (1F3) to whole bacteria, and capsular extracts isolated at different temperatures indicate that the antibody is specific for a thermostable polysaccharide antigen present in the cellular capsule of H. pleuropneumoniae of serotype 2.  相似文献   

15.
A panel of six monoclonal antibodies (MAbs) produced from mice immunized with Pasteurella multocida (M1404) (Heddleston serotype 2) reacted with homologous lipopolysaccharide, as indicated by enzyme immunoassay and immunoblotting. All six MAbs reacted with serotypes 2 and 5 of the 16 Heddleston serotypes. The reactive epitopes were localized on the bacterial cell surface by immunogold labelling. The antibodies could agglutinate P. multocida only if cells were first treated with 1 N HCl. All six MAbs opsonized P. multocida for phagocytosis by mouse macrophages but were not bactericidal in the presence of complement. They afforded only partial protection against infection in mice. The results, together with those of active immunization experiments with LPS, suggest a subordinate role for LPS in protection from experimental infection in mice.  相似文献   

16.
Gu HW  Lu CP 《Veterinary microbiology》2006,115(4):339-348
A random 12-peptide library was used to screen immunodominant mimics of 99kDa iron-regulated outer membrane protein (IROMP-99) of rabbit Pasteurella multocida. In the present study, expression of IROMPs of rabbit P. multocida strain C51-12 were analyzed by SDS-PAGE, and Western blot to determine the specificity of rat antiserum antibodies against IROMP-99. Only IROMP-99 whose expression was induced under iron-restricted conditions was detected on nitrocellulose paper. The phage display library was screened with rat normal and IROMP-99-specific antiserum. The positive phage clones were identified using enzyme-linked immunoadsorbent assay (ELISA) and inhibition assays for their reactivity to the antiserum. Out of the 18 randomly selected positive clones that showed higher reactivity to rat antiserum, only ten clones efficaciously inhibited binding of rat antisera to IROMPs and their displayed peptides were determined. Alignment using DNAStar-MegAlign software, results showed that motif WHxTxP was highly conserved among nine clones, only clone A7 had no obvious linear homology with either. Our findings suggest that the motif WHxTxP could be an immunodominant mimic epitope of IROMP-99 of rabbit P. multocida strain C51-12.  相似文献   

17.
Pasteurella haemolytica biotype A, serotype 1 (P haemolytica A1) was the most commonly isolated Pasteurella species from 80 calves examined at necropsy from 40 outbreaks of respiratory disease, the majority of which were pathologically confirmed as bovine pneumonic pasteurellosis (transit fever; shipping fever). Similarly, nasopharyngeal swabs from in-contact and apparently healthy calves indicated the widespread presence of P haemolytica A1. Pasteurella multocida and other serotypes of P haemolytica A1 were found including six isolations of P haemolytica T10, a fairly common pathogen in sheep. Approximately two-thirds of the isolates were tested for their antimicrobial sensitivity patterns and the degree of sensitivity for P haemolytica A1, the most frequently isolated serotype, was chloramphenicol (100 per cent), sulphamethoxazole trimethoprim (98 per cent), oxytetracycline (80 per cent), ampicillin (85 per cent), penicillin (82 per cent), streptomycin (3 per cent) and lincomycin (1 per cent).  相似文献   

18.
The antibody response to various combined polyvalent Pasteurella haemolytica vaccines was studied in sheep and cattle. In sheep, certain oil adjuvant vaccines gave rise to a better antibody response to P. haemolytica than an A1(OH)3-adsorbed vaccine. This finding, however, was not consistent for all serotypes, and with respect to P. multocida, oil adjuvants had no advantage. Furthermore, it was found that the removal of all the culture supernatant fluid during the production process had no deleterious effect on the antigenicity of the product. In cattle, good responses were obtained with both alum-precipitated and A1(OH)3-adsorbed vaccine where all culture supernatant fluid was not removed during the production process. No advantage was gained with oil emulsion vaccines. The degree of immunity afforded to mice and the antibody response to different serotypes of P. haemolytica varied considerably. Further detailed studies with respect to specific serotypes of P. haemolytica are therefore required.  相似文献   

19.
The goal of this study was to characterize Pasteurella multocida isolated from rabbits. Five hundred and fifty-three apparently healthy rabbits were sampled for this study. Nasal swabs were collected from each rabbit for P. multocida isolation and identification. Isolates were further characterized by capsular and somatic antigens and genomic DNA fingerprinting. Thirty-nine P. multocida isolates were recovered from 553 rabbits (7%). Capsular typing was done by depolymerization of P. multocida capsule by Staphylococcus aureus hyaluronidase and by disc diffusion with mucopolysaccharidase enzymes (heparinase III, chondroitinase AC, and hyaluronidase). Thirty-one (79%) of the isolates were capsular type A, and 8 isolates (21%) had untypable (UT) capsules. The gel-diffusion precipitin test was used to determine the somatic type of P. multocida isolates. Nineteen isolates were somatic serotype 3 (49%), 12 were serotype 1 (31%), 1 was serotype 2, 2 were serotype 5, 2 were serotype 12 with a weak reaction to antiserum raised against serotype 7 (5%), and 1 was serotype 4. Two of the isolates (5%) were UT. Restriction endonuclease analysis of the DNA of the isolates revealed 7 distinct profiles by digestion with HindIII, and 12 profiles were obtained with HpaII, whereas digestion with EcoRI did not differentiate between any of the P. multocida DNA isolates studied. The DNA restriction endonuclease enzyme HpaII was found more useful for differentiating between DNA fingerprints of P. multocida rabbit isolates. However, no correlation between capsular type, somatic serotypes, and DNA fingerprints was seen in this study.  相似文献   

20.
When Actinobacillus pleuropneumoniae (A. pp) is grown under iron-restricted conditions in vitro, transferrin binding proteins (Tbps) are induced. The functional transferrin receptor of A. pp is composed of two outer membrane proteins (Tbp1 and Tbp2) and shows an exquisite specificity for porcine transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60-70kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron-restricted culture. It was found that Tbp2 was not expressed in iron replete medium by any serotype except serotypes 5a, 5b and 6 where a weak expression was seen. There was a weak expression of related antigens in Actinobacillus indolicus and Actinobacillus suis under iron-depleted conditions while no similar antigens were detected with the Mab in iron-starved Actinobacillus lignieresii, Actinobacillus porcinus, Actinobacillus minor, Haemophilus influenzae, and Haemophilus parasuis.Using an enzyme-linked immunosorbent assay (ELISA) based on the Mab 1.48, Tbp2 could be detected in both recombinant E. coli expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found both in the cytosol and on the outer membrane. These results indicate that the Mab 1.48-reactive epitope of Tbp2 is surface exposed when it is expressed without Tbp1 in E. coli while the inaccessibility of this epitope of Tbp2 in A. pp could be due to shading by the association between Tbp2 and Tbp1.  相似文献   

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