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1.
Pasteurella multocida serotype B:2 is the causative agent of haemorrhagic septicaemia (HS), a fatal disease of cattle and buffaloes. As a step towards the identification of individual antigens that may protect against HS, proteins present in a sonicated cell extract (SCE) and outer-membrane protein (OMP) preparation of a wild-type P. multocida serotype B:2 were investigated by immunoblotting with sera from calves which had been protected against challenge with a virulent strain of P. multocida B:2 by vaccination with a live-attenuated aroA derivative of the challenge strain. Five proteins in SCE, of approximately 50, 37, 30, 26 and 16 kDa, were recognised by the sera. In an OMP preparation, two bands, at 37 and 50 kDa, were recognised as strongly immunogenic. Mass spectrometry analysis of proteins corresponding in size to those detected by immunoblotting identified the 37 kDa band as OmpA, but the band at 50 kDa was not identified with certainty. A major 30 kDa OMP, identified as OmpH, was not strongly immunogenic.  相似文献   

2.
Abstract

Outer membrane proteins (OMP) of 10 isolates of Edwardsiella tarda were compared by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The OMP profile of the type strain E. tarda ATCC 15947 cultured at 25°C had five major protein bands of 40, 36.5, 34, 28.5, and 25 kDa and a large number of minor proteins ranging in size from approximately 10 to 120 kDa. Differences between the OMP profiles of the isolates of E. tarda included the inconsistent presence of the 34- or 36.5-kDa proteins in five isolates of E. tarda and two major bands of 47 and 44 kDa that were present in only two isolates of E. tarda. There were no differences in the outer membrane protein profiles of 9 out of 10 isolates of E. tarda incubated at a temperature of 25°C compared with those at 35°C. To evaluate the effect of salinity, 10 isolates of E. tarda were cultured in brain heart infusion broth containing 0.5, 1.5, and 3.0% sodium chloride. Reactions of isolates of E. tarda to the different salinity levels were placed into three groups. The first group expressed more or fewer protein bands at 1.5% sodium chloride. The second group lost major bands at 3% salinity, whereas the third group had no change in the OMP profile with salinity. The OMP profile differences and the different reactions to salinity levels suggest that the isolates are heterogeneous.  相似文献   

3.
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   

4.
The antigenic polypeptides in Salmonella Enteritidis (SE) were chronologically recognized by the chicken immune system, using Western immunoblotting. Broiler chicks challenged at three days of age with SE strain carrying the most prevalent plasmid profile of 14.1 and about (approximately) 50 kb were bled at 17, 24, 31, 38 and 45 days of age. Pooled sera of blood collected at each age was reacted by Western immunoblotting with banded polypeptides of three predominant SE strains that acquired the following respective plasmid profiles: 14.1 kb; 14.1 and approximately 50.0 kb; and 1.8, 14.1 and approximately 50.0 kb. The immunoblots of each pooled sera collected at a specific age against the three SE strains were similar. More specifically, the early immune response at 17 days of age had antibodies recognizing only one polypeptide in the three SE strains namely, the 35.8 kDa. At 24 or 31 days of age, the acquired immunity to infection had antibodies recognizing five similar polypeptides in the three SE strains namely, the 14.4 (fimbriae protein), 21.5 (fimbriae protein), 30.5, 35.8, and 66.2 kDa. At 38 and 45 days of age, the antibodies recognized additional polypeptides namely, the 41.5 and 55.6 kDa, respectively. The recognition of the 41.5 and 55.6 kDa polypeptides at 45 days of age was associated with higher invasiveness of SE to spleens and livers (15.6%) and in higher cecal colonization (59.4%) in comparison to absence of recognition to the two polypeptides at 31 days of age associated with low infectivity to spleens (0%), livers (3.1%), and ceca (9.4%).  相似文献   

5.
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   

6.
OBJECTIVE: To characterize strain-dependent and growth condition-dependent variability in outer membrane protein (OMP) expression of Bordetella bronchiseptica isolates from dogs and evaluate the systemic immune response to OMP of B bronchiseptica among infected dogs. SAMPLE POPULATION: 8 strains of B bronchiseptica isolated from dogs, including a historic reference strain, 2 commercially available vaccine strains, and 5 field strains, and serum samples collected from 3 specific-pathogen-free (SPF) dogs before and 1 month after infection with B bronchiseptica. PROCEDURE: OMP were isolated from cultures in the late exponential phase of growth and compared among strains and, within strains, among growth conditions by means of polyacrylamide gel electrophoresis and immunoblotting. Serum samples were probed with OMP from 1 of the field strains. RESULTS: Strain-dependent variability in OMP profiles and growth condition-dependent and strain-dependent variability in expression of filamentous hemagglutinin (FHA) and pertactin was found, along with heterogeneity of the pertactin proteins produced by these B bronchiseptica strains. All 3 SPF dogs seroconverted to proteins with estimated molecular masses of 200 and 66 kDa, suggesting that FHA and pertactin were involved in the immunologic response of these dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there is growth condition and strain variability in expression of OMP, FHA, and pertactin proteins produced by B bronchiseptica. This information could be useful in the improvement of vaccines for prevention of bordetellosis in dogs.  相似文献   

7.
Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was used to characterize 67 field isolates of Pasteurella multocida originating from different animal species and geographical regions of India. REP-PCR was found to be rapid and reproducible (three repeats were done). These isolates yielded different 23 profiles which were clustered into eight groups. The discrimination index was moderate (D value 0.83). Somatic and antigenic typing of the isolates did not reveal any correlation with REP-PCR profiles. There was no host-specific, type-specific, region-specific or pathenogenicity-specific pattern. The REP profiles of isolates obtained from wild animals were similar to those obtained from domestic animals. Two common bands were present in all the isolates irrespective of somatic or antigenic types. The results were not comparable with earlier findings, which had shown high discrimination index and correlation with disease presentation. Saxena, M.K., Singh, V.P., Kumar, A.A., Chaudhuri, P., Singh, V.P., Shivachandra, S.B., Biswas, A. and Sharma, B., 2006. REP–PCR analysis of Pasteurella multocida isolates from wild and domestic animals in India. Veterinary Research Communications, 30(8), 851–861  相似文献   

8.
The polypeptides of three fowl adenovirus-4 (FAV-4) field isolates of hydropericardium syndrome from various geographical areas of the country and the standard FAV-1 (CELO virus) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by protein immunoblotting with polyclonal antibodies to FAV-4 and FAV-1. Protein profile analysis of FAV-4 isolates revealed similarity of all the eight polypeptides with molecular weight ranging from 20 to 107 kDa but differed from CELO, particularly in their 24.2 kDa protein. Subsequent immunoblotting showed relatedness of at least five protein fractions of FAV-4 to CELO virus.  相似文献   

9.
Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.  相似文献   

10.
Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

11.
Comparison of the polypeptide patterns of 22 isolates of M. ovipneumoniae by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a marked degree of heterogeneity with only limited groupings identifiable. Of the 50 major polypeptides identified in one strain (956/2), 35 were shown to be antigenic using immunoblotting with a homologous polyclonal serum. Radioimmune precipitation of 125I-surface-labelled proteins and phase partition using Triton X-114 detergent indicated that these were membrane associated. Cross-reactivity between the isolates was examined by immunoblotting using one polyclonal serum and four monoclonal antibodies (MAbs), all raised against strain 956/2. The polyclonal serum revealed considerable antigenic heterogeneity, but at least nine major antigens were conserved across all isolates. Two MAbs cross-reacted with all 22 strains, but the other two MAbs allowed some differentiation of the strains. One (MO/3) divided the isolates into groups of 16 and 6 based on the presence of absence of a 26-kDa antigen. All strains isolated from sheep with pulmonary adenomatosis fell into the smaller group and did not possess the 26-kDa antigen.  相似文献   

12.
Three foot-and-mouth disease virus type A isolates recovered from field outbreaks in the Department of San Martin, Peru, during the period 1975 to 1981 were compared with each other, and the South American vaccine strains A24 and A27, by complement fixation (CF), virus neutralization (VN) and polyacrylamide gel electrophoresis (PAGE). Complement fixation and VN tests gave comparable results distinguishing the field isolates from each other and from the vaccine strains. Analysis of the structural polypeptides by PAGE also showed clear differences between all the viruses examined. Samples from tissue culture passaged and mouse adapted strains of one of the field isolates gave identical patterns in PAGE, but differences were observed in the polypeptide pattern of the A24/BRA/55 strain and the Peru vaccine strain, which were serologically indistinguishable. Results illustrate a continued antigenic variation in an endemic area where vaccination has been used; however, asymmetric serological reactions between the A24 vaccine strain and the most recent field isolate indicated that a vaccine incorporating A24 should still give adequate protection.  相似文献   

13.
The cytoplasmic protein antigens (CPAg) of Brucella canis were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysis of 35S-labeled polypeptides. Approximate molecular weights of the immunoreactive polypeptides were determined by migration patterns of the immunoprecipitated polypeptides after SDS-PAGE or Western immunoblotting of sera collected at various times after experimental infection of dogs. Polypeptides were specifically precipitated by sera of infected dogs, but not from the sera of normal or false-positive (seropositive, non-infected) animals. During the initial month after infection, proteins with molecular weight masses (MW) of approximately 18, 22, 31, 42 and 54 kDa were commonly recognized. A 20-kDa polypeptide was first recognized at 8-10 weeks after infection, but it was detected inconsistently after 6 months. Additional polypeptides detected from 2 to 12 months post-infection had MW of 22, 66-68 and, less regularly, 42, 60, 82, 100 and greater than 200 kDa. The polypeptides most consistently recognized in sera from B. canis-infected dogs had MW of 18, 22 and 68 kDa.  相似文献   

14.
The polypeptides of serologically related viruses of hemorrhagic enteritis (HE) in turkeys, marble spleen disease (MSD) in pheasants, and splenomegaly in chickens (SMC) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by protein immunoblotting with polyclonal antibodies to HE virus (HEV). The viral polypeptides II, III, IV, V, VI, and VII were detected on SDS-PAGE with the size range from 18 to 97 kDa in HEV. Viral polypeptides II, III, V, VI, and VII were detected in MSD virus and virus of SMC. Protein immunoblotting of viral proteins with anti-HEV serum revealed antigenic differences between the 3 viruses of avian adenovirus type-II examined. The differences were that the polypeptides II, III, IV, V, VI, and VII were identified in HEV and the polypeptides II, V, VI, and VII were identified in MSD virus and virus of SMC. The bands of penton base (polypeptide III) and fiber (polypeptide IV) were seen in HEV only by protein immunoblotting.  相似文献   

15.
Anaplasma marginale is the etiological agent of anaplasmosis, a tick-transmitted disease with an important economic impact that affects cattle throughout the world. Although, North American isolates of A. marginale and their antigens have been extensively studied, relatively little information is available on the antigenic composition of South American isolates. The characterization of diverse geographical isolates of A. marginale will result in a thorough antigenic profile and may lead to the identification of additional diagnostic and immunoprophylactic tools. Short-term cultures of a Venezuelan isolate (Ta) of A. marginale were maintained for up to 13 days in vitro. During that period, the A. marginale remained viable and were propagated in the bovine erythrocyte culture system. During the initial days of culture, cell division and reinvasion were evidenced by a significant rise in parasitemia up to a 50%. A. marginale antigens were identified by metabolic labeling with (35S) methionine, followed by fractionation and immunoprecipitation with homologous and heterologous bovine sera. This yielded a complete antigenic set for the Ta isolate of A. marginale, including soluble, secreted and corpuscular polypeptide antigens. Fifteen immunodominant polypeptides were recognized by the bovine sera in the soluble and corpuscular fractions with relative molecular weights of 200, 150, 100-110, 86, 60, 50, 47, 40, 37, 33, 31, 25, 23, 19 and 16kDa. Seven polypeptides were present in the exoantigen fraction. The 31 and 19kDa antigens were recognized by the ANAR76A1 and ANAF16C1 monoclonal antibodies, respectively which are specific for MSP-4 and MSP-5 from North American isolates of A. marginale. Metabolic labeling with (14C) glucosamine prior to immunoprecipitation with bovine sera allowed the identification of glycoprotein antigens of 200, 100-150, 60, 55, 50, 45-43, 37, 33, 31, 22, 19 and 16kDa in the soluble fraction.  相似文献   

16.
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.  相似文献   

17.
《Veterinary microbiology》1997,54(2):167-183
The outer membrane proteins (OMPs) of P. multocida serotypes A3 (7 isolates), A4 (2 isolates), A3,4 and A2 (one isolate each) obtained from pneumonic cattle (10 isolates) and from one pig isolate were investigated to identify potential immunogens. SDS-PAGE of P. multocida OM isolated by SDG centrifugation of spheroplasts revealed eight major OMPs. Outer membranes isolated by sarcosyl extraction or SDG had similar protein composition on Coomassie blue-stained SDS-PA gel and on immunoblots. Two major OMPs (Mrs of 35 and 46 kDa at 100°C) demonstrated heat modifiability with apparent Mrs of 30 and 34 kDa at 37°C, respectively. The N-terminal aa sequences of these heat modifiable proteins revealed homology with E. coli OmpA and Hib P1 proteins, respectively. Protease treatment of whole cells followed by western immunoblots using bovine convalescent sera identified several immunogenic, surface-exposed and conserved OMPs among the eleven P. multocida isolates examined. The whole organism SDS-PAGE profiles of the eleven P. multocida isolates differed such that six patterns were seen. These patterns could potentially be used as a typing system for P. multocida bovine isolates based on the molecular weights of whole cell proteins. The above observations have potentially important implications relative to the immunity to infection.  相似文献   

18.
Ewe placental and lamb intestinal isolates of Chlamydia psittaci recovered from flocks affected with ovine enzootic abortion were examined by inclusion morphology, indirect immunofluorescence (IIF) and immunoblot analysis. Chlamydiae recovered from the faeces of sheep from two flocks free of clinical disease were also examined. In cell culture ovine abortion (OA) and intestinal isolates were distinguishable by inclusion development and morphology. Similarly, in two-way IIF tests with one week mouse antisera isolates fell into two distinct groups: abortion or intestinal. Immunoblotting with convalescent sheep abortion antiserum identified 30 out of at least 40 silver staining polypeptides as antigenic both in OA and intestinal isolates. The serum produced a similar reaction pattern to the resolved proteins of each OA isolate, indicating a higher degree of antigenic conservation among these isolates. Considerable cross reactivity between the OA and intestinal isolates was identified, but the serum also showed apparent molecular weight differences between antigens of the two types in the 87-116 kDa, 38-44 kDa and 26-28 kDa regions. Furthermore, the immunoblotting analysis revealed heterogeneity among the intestinal isolates, particularly in antigens between 87-116 kDa and 38-44 kDa.  相似文献   

19.
Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS–PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.  相似文献   

20.
Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 31 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 31 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.  相似文献   

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