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1.
The outer membrane-peptidoglycan complex (OM-PG) from rough strains of Brucella abortus was tested for its ability to induce lymphocyte responsiveness in cattle. Six groups of heifers were immunized with varying doses and administration schedules of rough OM-PG and assayed for responsiveness of their lymphocytes in proliferation assays in vitro. All OM-PG preparations were emulsified in a commercial adjuvant for administration. Two other groups of heifers were immunized with strain 19 vaccine or adjuvant alone. Three groups of heifers received two inoculations of OM-PG antigens from a naturally-occurring rough strain at a 57-day interval. The doses of OM-PG given in these three groups were 400 micrograms, 1200 micrograms, and 4000 micrograms at each inoculation. The frequency of cows that responded in lymphocyte proliferation assays increased with the dose of OM-PG given. Two groups received single inoculations of OM-PG, either 2400 micrograms or 8000 micrograms. Although there were responsive cows in these immunization groups, their frequency was lower than in the groups receiving the same total dose in two inoculations. A sixth group of cows was inoculated with OM-PG from a rough transposon mutant of B. abortus, and the frequency of responsive cows in this immunization group was comparable to that of responsive cows immunized with the same dose of OM-PG from the spontaneous rough mutant. In comparisons of cows inoculated with strain 19 to those inoculated with OM-PG preparations, differences were observed in the relative responsiveness of their lymphocytes to whole cells and OM-PG in the in vitro lymphocyte proliferation assays. These differences suggested that lymphocytes stimulated by strain 19 vaccination have different specificities than those stimulated by immunization with OM-PG of rough mutant strains of B. abortus.  相似文献   

2.
It was shown in this study that complement-resistant Brucella abortus used were unable to activate complement in the absence of specific antibody. Complement-resistant isolates possessed O-antigen, but complement-sensitive organisms used are O-antigen deficient. Since B. abortus LPS does not activate the alternative pathway of complement, we concluded that activation of bovine complement must be due to some other mechanism. In this study, it was shown that bovine C1 binds to the outer membrane proteins of B. abortus. Isolated outer membrane proteins of both smooth (O-antigen positive) and rough (O-antigen negative) B. abortus used bind to C1q. However, only rough isolates were killed by complement. All of the O-antigen positive B. abortus isolates were complement-resistant. We propose that O-antigen shields outer membrane proteins and blocks C1q binding.  相似文献   

3.
Brucella abortus resists the microbicidal mechanisms of macrophages, and the expression of its heat shock proteins (HSPs) such as GroEL, GroES and HtrA may play a role in this resistance. Bacterial HSPs can be very immunogenic, inducing protective immunity in various types of bacterial infections. However, the significance of immune responses directed against B. abortus HSPs in the protection against brucellosis is currently unresolved. To elucidate the role of these proteins in protection against Brucella challenge, individual, divalent or trivalent baculovirus (BV) recombinants of B. abortus GroEL, GroES and/or HtrA were injected into BALB/c mice either as protein-expressing whole cells or as purified proteins. The preparations were given to mice in combination with Freund's or Ribi adjuvant, respectively. In addition, some mice were primed with a vaccinia virus-GroEL recombinant, followed by inoculation with purified GroEL-Ribi adjuvant combination. Antibodies were observed against B. abortus GroEL and HtrA, but not against GroES. Cellular immune response was demonstrated by observing significant IFN-gamma release by lymphocytes of mice immunized with the purified HtrA-Ribi adjuvant combination. However, none of the mice inoculated with individual, divalent or trivalent HSP-expressing cells combined with complete Freund's adjuvant or inoculated with purified B. abortus HSPs combined with Ribi adjuvant, were protected against challenge with B. abortus virulent strain 2308. Priming with vaccinia virus-GroEL recombinant and boosting with GroEL-Ribi combination did not induce protective immunity. Based on the results obtained, we suggest that although humoral and cell-mediated immune responses are induced, but protective immune response is not induced by B. abortus HSPs.  相似文献   

4.
Brucella abortus vaccines composed of native cell envelopes or outer membrane proteins of smooth strain 2308 were compared with a vaccine (PG) composed of the insoluble residue of strain 2308 cell envelopes which had been extracted with hot sodium dodecyl sulfate. Vaccines were given by injection in an oil base adjuvant containing trehalose dimycolate and muramyl dipeptide or without adjuvant. Mice vaccinated with 30 micrograms native cell envelopes or PG and challenged 4 weeks later with virulent B. abortus strain 2308 displayed equivalent levels of protective immunity at 1 and 4 weeks post-infection. Heifers were vaccinated with 5 mg of antigens in adjuvant; PG was also administered without adjuvant. Humoral and cell mediated immune (CMI) responses were tested at monthly intervals. PG without adjuvant induced negligible immune responses. Native cell envelope antigens induced significantly higher titers of whole cell agglutinins over a 3-month period than did PG, although revaccination with PG in adjuvant enhanced the production of agglutinins and both vaccines induced antibodies to the O polysaccharide. Lymphocyte blastogenesis responses and delayed hypersensitivity reactions to porin and group 3 proteins were stimulated by both native and PG vaccines, and the magnitude of the responses did not differ significantly between the treatment groups. These vaccines were therefore comparable in their capacity to induce protective immunity in mice and CMI responses in cattle, whereas antibody responses induced by PG in cattle were generally lower. These findings provide a basis for evaluation of nonliving B. abortus vaccines in cattle.  相似文献   

5.
Outer membrane-peptidoglycan complex from Brucella abortus was separated from cytoplasmic membrane and cytosol by either sucrose density gradient fractionation or differential (rate) centrifugation of surface labeled cells disrupted by sonication without the use of detergents. The outer membrane-peptidoglycan complex had a buoyant density of 1.22 gm/ml and contained 67 labeled SDS-soluble proteins when examined by SDS-PAGE. Included were four major bands exhibiting molecular masses of 88k, 40k, 35.7k and 26k daltons corresponding to previously described group 1, 2 and 3 outer membrane proteins. Lysozyme treatment of outer membrane-peptidoglycan complex increased its buoyant density to 1.25 gm/ml and released eight additional peptidoglycan-linked proteins.  相似文献   

6.
In an attempt to establish if cross protection can be induced by different strains of Haemophilus parasuis, three groups of 12 gnotobiotic pigs were immunized each with an aluminum hydroxide adsorbed whole cell bacterin of one of three H. parasuis strains. Two weeks later, four pigs within each vaccinated group were challenged with aerosols of live cultures of each of the three test strains and observed for response. Two virulent strains V1 and V2 protected all the vaccinated pigs, while all nonvaccinated controls succumbed to Glasser's disease when challenged with these strains. Vaccination with strain LV (of low virulence) protected the pigs against challenge with strain V2, but not against strain V1. Strain LV did not cause disease in the immunized animals and only in one of ten nonimmunized pigs upon second challenge. The results suggest that strains may differ in antigenicity and that virulence and immunoprotection are positively related. Strains to be used in commercial vaccines should therefore be selected carefully. Antibodies detected in the sera of vaccinated pigs were to outer membrane proteins of the bacteria, but not to lipopolysaccharides or capsular polysaccharides. This would suggest that for gnotobiotic pigs outer membrane proteins are more immunogenic than lipopolysaccharide or capsular antigens. Further work is needed to determine if outer membrane proteins also contribute protective immunogens.  相似文献   

7.
Immunogenicity of Brucella abortus salt-extractable proteins   总被引：5，自引：0，他引：5  
The immunogenic properties of salt-extractable proteins and chromatographic fractions thereof from Brucella abortus were evaluated in lemmings (Dicrostonyx rubricatus). The efficacy of the Brucella proteins as immunogens was determined after challenge with virulent B. abortus strain 2308 and was based on protection against clinical signs and gross lesions of brucellosis, as well as on numbers of viable Brucella in the spleen. Vaccination of lemmings with as little as 0.1 microgram of salt-extractable proteins (CSP) suppressed splenic infection, resulting in reduced numbers of viable organisms per spleen of 5-6 logs compared to non-vaccinated controls. Protein fractions separated by column chromatography were generally effective in reducing splenic infection, and contained proteins with molecular weights of 30,000, 20,000 and 12,000. Vaccines containing chemically modified dodecanoyl-CSP offered no additional advantage over unmodified CSP vaccines.  相似文献   

8.
Outer membrane antigens which bind to non-agglutinating antibodies (NAAb) elicited by smooth (S19) and rough (S45/20) Brucella abortus strains, were extracted from S45/20 by stirring in cold 2.5% NaCl and then analyzed by SDS-PAGE, electroblotting and enzyme-linked antibody test. Eight bands were observed in the gel stained with Coomassie blue. Seven antigenic fractions were transferred to nitrocellulose by blotting. A 27-kd band was recognized by bovine anti-S45/20 non-agglutinating serum and not by purified NAAb against surface antigens. Bands 10 kd and 14.3 kd bound to bovine anti-S45/20 NAAb from calves immunized with either S19 or S45/20. A 12.0-kd band was recognized by the serum and NAAb from calves immunized with S45/20 but not by those injected with S19. There are thus antigenic fractions shared by S19 and S45/20 which bind in vitro to NAAb.  相似文献   

9.
Peripheral blood mononuclear cells (PBM) from four normal cows with no known exposure to vesicular stomatitis virus (VSV) were cultured with a New Jersey (NJ) serotype (Ogden) VSV that had been UV-irradiated and inactivated. PBM from these animals produced no detectable proliferative response when incubated with varying concentrations of VSV-NJ (Ogden) ranging from 10 ng to 10 μg protein/ml. Two of these cows were immunized with an experimental VSV-NJ vaccine and their PBM were tested at various intervals after immunization. PBM tested 14 days after the initial immunization produced readily detectable antigen-specific proliferative responses when cultured with UV-irradiated strains of VSV-NJ. Following a second immunization, lower concentrations of antigen were sufficient to stimulate the proliferative response and the magnitude of the proliferative response was increased. The responsiveness persisted for at least 6 months after these two immunizations. The specificity of the proliferative response was examined by comparing the responses stimulated by one VSV-Ind and four VSV-NJ serotype strains. The PBM from the immunized cows produced proliferative responses that were essentially specific for the VSV-NJ serotype antigens. In dose titrations, the VSV-NJ antigens were 300–1000-fold more effective than was the VSV-Ind antigen. Thus, persistent antigen-specific proliferative responsiveness that is serotype specific can be stimulated by immunizing cattle with an inactivated VSV vaccine.  相似文献   

10.
This study is to (1) investigate the prevalence of Chlamydophila abortus infection in cows and goats in Taiwan, and (2) compare the genetic properties of Taiwanese isolates with abortion strains from other sources. Approximately 71% of aborted cows and 58% of aborted does had IgG against C. abortus in their sera. The seroprevalence rate in cows may be overestimated, because a certain degree of cross-reactivity with C. pecorum cannot be ruled out. Only 22.7% (from aborted cows) and 33.3% (from aborted dogs) of vaginal swabs that tested positive by polymerase chain reaction led to successful isolation of C. abortus by inoculation into chicken embryos, equivalent to 7.1% and 7.9% of isolation rates, respectively. The major outer membrane protein gene of 15 Taiwanese abortion isolates was compared with that of various strains by restriction fragment length polymorphism (RFLP) and nucleotide sequencing. Restriction enzyme CfoI was able to distinguish Taiwanese ruminant isolates, which have identical RFLP patterns, from C. felis (feline) and C. psittaci (avian) strains. Taiwanese isolates had 98.8-100% homology with known ruminant abortion strains and were phylogenetically closest to bovine LW508 strain.  相似文献   

11.
The aim of this study was to evaluate the effect of 3 Brucella ovis subcellular protein fractions: Outer membrane (OMP), inner membrane (IMP), and cytoplasm (CP), on cellular immune response by in vitro production of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma. Each fraction was inoculated 3 times into Balb/c mice, primary cultures of mice spleen cells were done, and these were then stimulated with the fractions. Culture supernatants were collected at 24, 48, 72, 96, and 120 h postinoculation. Cytokine concentration was measured by Duoset-enzyme-linked immunosorbent assay (ELISA). The OMP fraction induced highest cellular immune response of 1000 pg/mL of IL-2 at 24 h, which decreased to < 100 pg/mL by 96 h. The IL-2 response for the IMP fraction was low at 24 h, but exceeded that of the OMP fraction at 72, 96, and 120 h. The CP showed a poor IL response. Regarding the IFN-gamma production, OMP and IMP induced a high response at 120 h. These results open the possibility for the use of B. ovis outer and inner membrane proteins as a subcellular vaccine.  相似文献   

12.
Soluble fractions of Hypoderma lineatum third instar fat body, haemocytes and haemolymph were formulated with Quil A and used to immunize four groups of calves while a fifth group remained untreated. Calves received two subcutaneous injections of the soluble fractions, or adjuvant only delivered two weeks apart. Two weeks after the last injection the calves were exposed to 50 newly hatched larvae of H. lineatum which were placed on the skin and allowed to penetrate. Survival of larval stages was monitored by weekly palpation and collection of emergent third instars. Antibody responses to the immunogens were evaluated by immunoblots and following infestation antibody responses to first instar antigens were evaluated by an ELISA. Non-immunized calves and calves injected with adjuvant were all palpation positive for cattle grubs. In groups immunized with fat body, haemocyte and haemolymph components 100%, 33% and 33% were palpation positive for grubs respectively. First instar mortality, as reflected in palpable grubs, was high in the groups receiving injections with tissue components (99.3%, 95.1%, 95.8%, 83.9 and 80.4% mortality for those groups receiving fat body, haemocyte, haemolymph, adjuvant or control respectively). Second and third instar mortality was also higher in the immunized groups (100.0%, 91.7%, 91.7% for fat body, haemocyte, and haemolymph respectively) in comparison to the adjuvant only (14.0%) and unvaccinated (33.3%) groups. No viable flies emerged from pupae originating from larvae emergent from any of the immunized groups. Calves receiving the tissue extracts developed antibodies to several protein components following the second immunization which were still present 13 weeks post-infestation. Several proteins appeared to be common among the three tissue extracts and were recognized by antibodies from the immunized calves. All groups of calves became positive for antibodies to first instar antigens, although in some immunized calves the antibodies were transient.  相似文献   

13.
Hybridomas producing antibodies to determinants associated with the lipopolysaccharide (LPS) of Brucella abortus and B melitensis were obtained by polyethylene glycol fusion of the SP2/0 myeloma cell line with B lymphocytes harvested from a Sprague-Dawley-derived rat previously immunized with whole B abortus strain 1119 organisms. Two clones, BRU38 and BRU28 , were selected for their ability to react with whole B abortus organisms and purified smooth-LPS ( f5p ). The BRU38 monoclonal antibodies were absorbed with live, rough strain 45/20 and smooth strains of B abortus and B melitensis organisms, whereas only smooth strains absorbed the antibody activity from BRU28 . Complete inhibition of the monoclonal's activity could be achieved with crude smooth-LPS, a purified f5p fraction, and a water-soluble acid degraded polysaccharide. Absorption of BRU38 and BRU28 with rough Brucella LPS, polysaccharide-B antigen, keto- deoxyoctanoic acid, or with several sugars and fatty acids known to be components of the Brucella LPS complex had no effect on the monoclonals. The data indicate that antigenic determinants are associated with the smooth LPS complex, probably with the O-side chain, and are expressed patchwise and in different quantities on several strains of B abortus and B melitensis. The B abortus rough strain 45/20 contains surface determinants which lead to the agglutination of smooth strain 1119 organisms. The potential use of monoclonals in competitive enzyme-linked immunosorbent assays for diagnostic purposes is discussed.  相似文献   

14.
Biological properties of RB51; a stable rough strain of Brucella abortus   总被引：27，自引：0，他引：27  
A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.  相似文献   

15.
目的研究布鲁氏菌外膜蛋白OMP25的克隆、测序和表达,并对其进行免疫特性检测。方法采用PCR技术对牛布鲁氏菌外膜蛋白OMP25基因进行了扩增和克隆测序,并成功构建了原核表达质粒pGEX-OMP25。将其转入E.coliBL21,经IPTG诱导表达,用电洗脱纯化后获得了高纯度的目的蛋白。纯化后的蛋白与弗氏佐剂乳化后免疫家兔得到了较高效价的抗血清。结论通过ELISA、Western-blotting分析及特异性检测试验,证明目的蛋白不仅具有抗原性且有良好的免疫反性。  相似文献   

16.
A lysate of erythrocytes infected with Babesia divergens was subjected to isoelectric focusing within a pH range of 3.5-9.5. The focused proteins were split into 2 fractions depending on pH (1) those from 3.5-6.5 and (2) those from 6.5-9.5. The acidic fraction was used as an immunogen for groups of 5 adult cows using 2 different adjuvants, saponin and a mixture of muramyldipeptide (MDP) and incomplete Freund's adjuvant (IFA). Two other groups of 5 cows were simultaneously treated with the alkaline fraction and a sample of the complete lysate with the MDP/IFA adjuvant. The treated cows plus a control group were infected with 2.5 X 10(8) erythrocytes infected with a homologous strain of B. divergens. When compared with the controls all pre-treated groups were protected to some degree. There were significant differences between the treated groups with the group which received MDP/IFA combined with the acidic fraction showing most resistance to the infection. The experiment also showed that the MDP/IFA adjuvant produced more protection than the saponin.  相似文献   

17.
Serologic responses to the newly introduced rough Brucella abortus vaccine strain RB51 have been determined in a dot-blot format using gamma-irradiated RB51 cells as the antigen. Because gamma-irradiated cells are not easily prepared and the signal from cells was not always reliable, an alternative antigen was sought. Detergent extracts of B. abortus RB51 were prepared using zwittergent 3-14, Triton X-100, and sodium dodecyl sulfate (SDS) and examined in a dot-blot format. Zwittergent 3-14 extracts and gamma-irradiated RB51 cells gave the same titers. Unlike gamma-irradiated RB51 cells, zwittergent 3-14 extracts produced signals consistently, and the signals were easily interpreted. Triton X-100 extracts interfered with signal development, and SDS extracts resulted in a high background signal. Western blot analyses revealed several outer membrane proteins in the zwittergent 3-14 extract. The major antigens in the extract had apparent molecular weights of <20,000.  相似文献   

18.
Pregnant goats were inoculated intravenously or in uterine arteries with Brucella abortus, and tissues from the uterus and placenta were examined by electron microscopy. Identification of B. abortus in placentae was with antibody-coated colloidal gold. B. abortus was first seen in phagosomes of erythrophagocytic trophoblasts and in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Subsequently, trophoblast necrosis and ulceration of chorioallantoic membranes were present. Coincidently, B. abortus was present in the lumen of placental capillaries. In late stages of infection, placental vasculitis was present, and placentomal trophoblasts were separated from maternal syncytial epithelium. In lesions with vasculitis, large numbers of B. abortus were in connective tissue of chorionic villi. Within the placentome, trophoblasts that lined chorionic villi contained no intracellular bacteria and were separated from B. abortus by intact basement membranes. These results suggest that bacteremic B. abortus is endocytosed by erythrophagocytic trophoblasts and that B. abortus replicates in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Replication of brucellae in trophoblastic rough endoplasmic reticulum is unique; we believe that B. abortus may utilize endoplasmic reticulum for synthesis and glycosylation of bacterial membrane proteins or that B. abortus catabolizes trophoblast secretory proteins.  相似文献   

19.
Brucellosis is a highly infectious disease which is diagnosed using serological and microbiological methods. The objective of this study was to assess the viability of using conventional and real-time PCR assays as potential diagnostic tools for the detection of Brucella abortus in naturally infected cows. PCR assays that amplify various regions of the Brucella genome, IS711 genetic element, 31kDa outer membrane protein and 16S rRNA, were optimised using nine known Brucella strains. Real-time PCR was used to examine the detection efficiency of the IS711 assay which was estimated at 10 gene copies. Milk, blood and lymph tissue samples were collected from naturally infected animals. B. abortus was not detected in blood samples collected from naturally infected cows by conventional or real-time PCR, but was detected in a proportion of the culture-positive milk (44%) and lymph tissue (66% - retropharyngeal, 75% - supramammary) samples by the same methods. There was no difference between PCR and bacteriological detection methods. It is unlikely that conventional or real-time PCR will supersede current diagnostic methods for detection of B. abortus in clinical samples.  相似文献   

20.
The iron-regulated outer membrane proteins (IROMPs) of Pasteurella multocida A:3 strain 232 (Pm232), a bovine isolate, were investigated as potential immunogens in cattle. We addressed the ability of P. multocida IROMP-enriched fractions to induce antibody responses in cattle by different vaccination strategies and the protective efficacy of these antibodies using a P. multocida-induced pneumonia challenge model. Vaccination of cattle with outer membrane-enriched fractions derived from Pm232 grown on either iron-depleted (IROMPs) or iron-sufficient (OMPs) conditions induced significant antibody responses; however, the correlation with lung lesion scores was not significant (P = 0.01 and P < 0.07, respectively). SDS-PAGE, Western blots and densitometric analyses of Pm232 grown under iron-deficient conditions revealed five major IROMPs including an immunodominant 96 kDa protein band. Mass spectrometry analysis of the 96kDa protein band suggested homology with the heme acquisition system receptor (HasR) of avian P. multocida (strain Pm70) and was confirmed by DNA sequence analysis of the cloned Pm232 hasR gene. Further analyses indicated that Pm232 HasR is a surface-exposed OMP and conserved among most P. multocida isolates investigated. In addition, cattle vaccinated with live Pm232 or IROMPs had significantly higher antibody responses to the 96 kDa protein band and the correlation with lung lesion scores approached significance (P = 0.056). These results indicate that antibody responses in cattle are induced by P. multocida IROMPs, and that the 96 kDa HasR protein is an immunodominant IROMP.  相似文献   

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