Vaccination of cattle with chemically modified and unmodified salt-extractable proteins from Brucella abortus

Beef heifers were vaccinated on Day 0 with either salt-extractable protein (CSP) or chemically modified CSP (dCSP) from Brucella abortus Strain 19 in Freund's complete adjuvant (FCA). Six weeks later, vaccination was repeated, and heifers received either the homologous or heterologous vaccine. Another group of heifers received only FCA and saline. Vaccinations with CSP or dCSP stimulated marked antibody responses to B. abortus, as detected by standard serologic tests, an enzyme-linked immunosorbent assay, or a quantitative fluorametric immunoassay. Twelve percent of the heifers were seropositive by the CARD test 1 year after vaccination. Vaccination stimulated an increased cell-mediated immune response as measured by lymphocyte blast transformation (LBT) to B. abortus antigens. Fifty-six weeks after the initial vaccination, the heifers were challenged intraconjunctivally with 1.9 × 10⁷ colony-forming units of B. abortus strain 2308. Sixty to 83% of the heifers aborted in each group and 70–83% of the heifers were culture positive. There were no significant differences (P>0.05) among groups with respect to the number of abortions or the number of culture-positive heifers. Antibody responses increased rapidly within 4 weeks after challenge. Overall, antibody responses were greater for heifers that aborted than for those that did not abort. These differences were significant (P<0.05) only as measured by the fluorometric procedure. The LBT responses appeared to be higher for vaccinates than for the control group, but these differences were not significant (P>0.20). There was a significantly lower (P<0.05) LBT response to heat-killed B. abortus in those heifers that aborted compared to those that did not.     

Brucella abortus-specific immunoglobulin isotypes in serum and vaginal mucus from heifers vaccinated with Brucella abortus salt-extractable proteins and challenge exposed with virulent Brucella organisms

Serum and vaginal Brucella-specific immunoglobulin isotypes (IgG1, IgG2, IgM, and IgA), obtained from 62 crossbred beef heifers vaccinated with Brucella abortus salt-extractable proteins and subsequently challenge exposed with B abortus S2308, were studied. Brucella-specific IgG antibodies and Brucella-specific immunoglobulin isotypes were quantitated by a fluorometric immunoassay. Serum and vaginal immunoglobulin responses were evaluated as a method of distinguishing infected from noninfected heifers. Rivanol precipitation, complement-fixation, buffered-antigen brucellosis tests and an ELISA were performed on sera. For immunoglobulin isotypes, vaccinated heifers had mean antibody responses higher than baseline mean antibody responses for at least 31 weeks after vaccination. After challenge exposure, significant differences (P greater than 0.05) were not detected between mean antibody responses of vaccinated and nonvaccinated heifers. Vaginal Brucella-specific antibody responses did not correlate with protection from disease. Vaginal Brucella-specific IgM was detected only at the time of abortion. Vaginal IgA appeared specific for identification of virulent B abortus infection. All serotests appeared adequate in distinguishing baseline titers from titers of heifers that had aborted and were considered bacteriologic culture-positive. Results of serotests neither consistently distinguished vaccinates from challenge-exposed cattle nor distinguished heifers that were challenge exposed, had aborted, and were considered bacteriologic culture-positive adequately from heifers that were challenge-exposed, had not aborted, and were considered bacteriologic culture-negative. Brucella-specific IgA appeared to be the most effective in distinguishing vaccinated heifers from challenge- exposed heifers and heifers that were challenge exposed and had aborted, from heifers that were challenge exposed and had not aborted. Brucella-specific serum IgA was detected up to 13 weeks after abortion.     

Characterization of salt-extractable protein antigens from Brucella abortus by crossed immunoelectrophoresis and isoelectricfocusing

Salt-extractable protein antigens (CSP) from Brucella abortus strains 19 and 2308 (vaccine and virulent strains, respectively) were analysed by crossed immunoelectrophoresis (CIE) using rabbit antisera to protein antigens and by isoelectricfocusing (IEF) in polyacrylamide gels. The reference immunoelectrophoretic profiles developed for proteins from strain 19 and 2308 of B. abortus contained 20 and 25 immunoprecipitates, respectively. Serum from cows experimentally infected or hyperimmunized with live organisms produced up to 5 immunoprecipitates in CIE with the protein antigens. Absorption of rabbit sera with homologous B. abortus cells reduced, but did not eliminate all of the immunoprecipitates from rabbit sera, suggesting that the majority, but not all of the protein components, are exposed on the surface of the cell. In contrast, antibody to protein antigens in agglutinin-free absorbed serum from infected cattle could still be demonstrated by CIE, even though CIE with protein extracts from whole cells radioiodinated with the cell surface labeling reagent, diazoiodusulfanilic acid, indicated that these antigens may be at or near the surface of the cell. From CIE in heterologous systems we concluded that all proteins present in strain 19 preparations were partially or completely identical to those in strain 2308. The IEF studies paralleled the CIE studies and revealed that the protein profile from strain 2308 was more complex than the profile from strain 19. Major differences between the 2 strains were found in the pH region from 3.9 to 5.0, where strain 2308 exhibited 4 additional protein bands.     

Monophosphoryl lipid A-induced immune enhancement of Brucella abortus salt-extractable protein and lipopolysaccharide vaccines in BALB/c mice.

A study was conducted to determine the effect of monophosphoryl lipid A (MPL) and trehalose dimycolate (TDM) as adjuvants on the protective responses in BALB/c mice vaccinated with Brucella abortus salt-extractable protein (BCSP) or proteinase-K-treated B abortus lipopolysaccharide (PKLPS). Mice were vaccinated with different doses of BCSP or PKLPS given alone or in combination with MPL or TDM. Mice were challenge-exposed 4 weeks later with virulent B abortus strain 2308. Two weeks after challenge exposure, the number of B abortus colony-forming units (CFU) per spleen, spleen weights, and spleen cell interleukin 1 production were measured. Serum IgG and IgM concentrations specific for vaccinal immunogens were measured before and after challenge exposure with B abortus. Spleen weights and mean B abortus CFU per vaccine group were significantly lower in BCSP- and PKLPS-vaccinated mice, compared with those of nonvaccinated control mice. Monophosphoryl lipid A enhanced the suppression of splenic infection when given with the BCSP vaccine, but not when given with the PKLPS vaccine. Trehalose dimycolate had no effect on mean CFU when given with BCSP, but incorporation of TDM resulted in a significant increase in mean CFU when given with PKLPS. Spleen weights in BCSP- or PKLPS-vaccinated mice were not different when these vaccines were combined with MPL or TDM. Because of the wide variation in the results, we could not conclude that vaccination with BCSP or PKLPS alone, or in combination with MPL altered spleen cell interleukin-1 production in B abortus-infected mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogenicity of subcellular fractions of Brucella abortus: measurement by in vitro lymphocyte proliferative responses

Five groups of heifers were immunized with various subcellular fractions of Brucella abortus and tested for their responsiveness in lymphocyte proliferative responses in vitro. The five subcellular fractions used as immunogens were: (1) a mixture of recombinant outer membrane proteins fused to Escherichia coli beta-galactosidase, (2) a mixture of outer membrane proteins BaomI, BaomIIB1, and BaomIII1, (3) a mixture of outer membrane proteins 7.5 kDa and 8.8 kDa, (4) a complex of smooth lipopolysaccharide and proteins, and (5) a complex of outer membranes and peptidoglycan (OM-PG complex) from a rough strain. All immunogens were emulsified in adjuvant and administered twice at a 61-day interval. Two other groups of cows were included; one immunized with strain 19 and the other with adjuvant only. Strain 19 and the rough OM-PG complex induced responsiveness in lymphocyte proliferation assays in a high percentage of immunized cows. The smooth lipopolysaccharide-protein complex induced responsiveness in fewer cows. The lowest frequencies of responding cows were found in groups that received either recombinant proteins or purified protein mixtures. Based on these results, we concluded: (1) cellular immunity, as measured by in vitro lymphocyte proliferative responses, can be induced with subcellular fractions of B. abortus and (2) the more complex the immunogen, the greater the frequency of responding cows.     

Frequent exposure of mice to crude Brucella abortus proteins down-regulates immune response

Mice repeatedly immunized via the intraperitoneal route with a Brucella abortus antigen lost their ability to develop a strong in vitro lymphoproliferative response. This result correlates with a decreased tendency of the lymphoid population to produce interferon-gamma when stimulated in culture with the immunizing antigen. With respect to the humoral response, as the number of immunizations increased, the animals produced more specific immunoglobulin M and immunoglobulin G1 antibodies. It is postulated that the long-term exposure of an animal to Brucella antigen changes the nature of the immune response from a T-cell-mediated response to a humoral response favouring the establishment of the disease.     

Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.     

SDS-soluble and peptidoglycan-bound proteins in the outer membrane-peptidoglycan complex of Brucella abortus

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.     

Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis

To overcome the limitations of serological diagnosis, including false positive reactions caused by other pathogens, specific antigens for diagnosis of brucellosis other than LPS have been required. The present study was conducted to separate and identify immuno-dominant insoluble proteins of Brucella abortus against the antisera of cattle infected with B. abortus, or/and Yersinia enterocolitica, or the sera of non-infected cattle. After separating insoluble proteins of B. abortus by two dimensional electrophoresis (2-DE), their immuno-reactivity was determined by western blotting. A portion of the immunogenic spots against the positive antisera of B. abortus that have the potential for use as specific antigens were identified by MS/MS analysis. Overall, 18 immunogenic insoluble proteins of B. abortus 1119-3 showed immuno-reactivity against only the positive antisera of B. abortus, but failed to have immunogenicity toward both the positive sera of Y. enterocolitica and the negative sera of B. abortus. Identification of these proteins revealed the following: F0F1 ATP synthase subunit β, solute-binding family 5 protein, 28 kDa OMP, Leu/Ile/Val-binding family protein, Histidinol dehyddrogenase, Hypothetical protein, Twin-arginine translocation pathway signal sequence domain-containing protein, Dihydroorotase, Serine protease family protein, β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA, Short-chain dehydrogenase-/reductase carbonic anhydrase, Orinithine carbamoyltransferase, Leucyl aminopeptidase, Cold shock DNA-binding domain-containing protein, Cu/Zn superoxide dismutase, and Methionine aminopeptidase. The 18 immunogenic proteins separated in the present study can be considered candidate antigens to minimize cross reaction in the diagnosis of brucellosis and useful sources for Brucella vaccine development.     

Restriction endonuclease analysis of Brucella abortus

The restriction endonuclease profiles of bacterial DNA from Brucella abortus isolates were evaluated. It was not possible to distinguish between vaccine strain 19 and virulent (biotype 1 and biotype 2) strains of B abortus. Restriction endonuclease analysis is therefore not a suitable epidemiological tool in bovine brucellosis investigations. The genetic homogeneity of the Brucella genus was reinforced by these findings.     

Mice immune responses to Brucella abortus heat shock proteins. Use of baculovirus recombinant-expressing whole insect cells,purified Brucella abortus recombinant proteins,and a vaccinia virus recombinant as immunogens

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.     

Cloning and kinetics of expression of Brucella abortus heat shock proteins by baculovirus recombinants

In an effort to develop genetically engineered Brucella abortus (BA) vaccines, the genes encoding heat shock proteins (HSPs) GroEL, GroES, and HtrA were cloned and expressed in the BAC-TO-BAC Baculovirus System, and the kinetics of protein expression were analyzed using various insect cell lines in suspension cultures, different cell densities in suspension cultures, multiplicities of infection and recombinant virus replication times. Trichoplusia ni cells expressed only BA HtrA, but Spodoptera frugiperda (Sf9) cells expressed all three recombinant proteins. The best GroEL expression was achieved by infecting 2x10(6) Sf9 cells/ml with an MOI 10 of recombinant virus and harvesting the cells after 96h of virus replication. GroES and HtrA were best expressed when infecting 2x10(6) Sf9 cells/ml with an MOI 1 of recombinant viruses and harvesting the cells after 120h of virus replications. Under these conditions BA recombinant HSPs were expressed as follows: GroEL at 16% of the total cellular proteins (105microg/ml concentration); GroES 2% (15.25microg/ml); and HtrA 8% (84.48microg/ml). This is the first report of cloning and expression of BA genes in the baculovirus system.     

Experimental Brucella abortus infection in pigs

Sixteen pigs inoculated by the intra-conjunctival, intravenous or subcutaneous routes with Brucella abortus Strain 544 developed a short-lived infection usually accompanied by conjunctival and vaginal excretion of the organism for up to 99 days post-inoculation. Serological tests performed by the agglutination, complement fixation, Rose Bengal plate, antiglobulin, immunodiffusion or ELISA procedures with B. abortus antigens disclosed wide variations in the antibody responses of individual animals. In some cases the serological tests were negative even though the animal was shown to be excreting B. abortus. The intradermal test for delayed hypersensitivity to Brucella antigens gave more consistent results, especially when supported by histological evaluation of the skin reactions.     

Immune response of cattle to Brucella abortus outer membrane proteins measured by lymphocyte blastogenesis

Lymphocytes from cattle were tested in a blastogenesis test with outer membrane proteins isolated from smooth strain 2308 and rough strain 45/20 of Brucella abortus. The titration assay developed for measuring blastogenesis to microbial antigens (Baldwin, Antczak and Winter, this issue, pp. 319-333) was used to assess the response to both group 2 (porins) (Douglas et al., 1984) and group 3 proteins (Verstreate et al., 1982). Blastogenesis was evaluated for distinguishing cattle infected with virulent B. abortus strain 2308 from unimmunized cattle, cattle vaccinated with attenuated strain 19, or inoculated with Escherichia coli 0116:H31, known to cause serological cross-reactions with B. abortus (Nielsen et al., 1980). Strain 45/20 porin was the most effective for this purpose and data analyses utilizing the titration assay were better than those relying on a single point assay. When compared with BASA, an antigen preparation used in other studies (Kaneene et al., 1978a), responses to porin provided a more specific index of infection with B. abortus. Reactions to 45/20 porin occurred, however, in some heifers vaccinated as adults with strain 19 or inoculated with E. coli 0116:H31. Furthermore, nonpregnant heifers had negligible or only transient blastogenesis responses to the porin during the first 14 weeks after infection even though they developed strong 0 antibody responses. We do not recommend the blastogenesis test in its present form as a useful adjunct to serological tests, and could allow measurement of cell mediated immune responses relevant to protective immunity.     

Recent advances in Brucella abortus vaccines

Brucella abortus vaccines play a central role in bovine brucellosis control/eradication programs and have been successfully used worldwide for decades. Strain 19 and RB51 are the approved B. abortus vaccines strains most commonly used to protect cattle against infection and abortion. However, due to some drawbacks shown by these vaccines much effort has been undertaken for the development of new vaccines, safer and more effective, that could also be used in other susceptible species of animals. In this paper, we present a review of the main aspects of the vaccines that have been used in the brucellosis control over the years and the current research advances in the development of new B. abortus vaccines.     

牛布鲁氏菌膜蛋白的免疫抗原筛选

试验旨在通过免疫蛋白质组学筛选布鲁氏菌保护性抗原。利用双向电泳技术对试验条件下培养的布鲁氏菌强毒株544A膜蛋白进行分离,结合Western blotting技术分别用豚鼠、牛抗布鲁氏菌血清寻找发生免疫反应的蛋白质。16个免疫蛋白点经胶内酶切、质谱(LC-MS/MS)进行鉴定。利用生物信息学工具发现这些蛋白不全是膜蛋白,还存在胞质蛋白,其功能涉及生物合成和物质代谢等领域。成功建立了布鲁氏菌膜蛋白的免疫蛋白质组学研究方法,为寻找保护性抗原及为新型疫苗抗原候选提供新思路。