Billy L. Deyoe

Effects of challenge dose on the clinical and immune responses of cattle vaccinated with reduced doses of Brucella abortus strain 19

Fifty-seven pregnant beef heifers that were unvaccinated or previously vaccinated with Brucella abortus S19, at a dose of either 10(9) or 10(10) colony-forming units (CFU), were challenge-exposed intraconjunctivally with virulent B. abortus S2308 at a dose of 9.4 X 10(6) CFU (Experiment 1) or 5.2 X 10(7) CFU (Experiment 2). In Experiment 1, S19 afforded significant protection (P less than 0.01) against challenge exposure in that 8 of 9 unvaccinated heifers, 1 of 11 vaccinated with 10(9) CFU, and 3 of 10 vaccinated with 10(10) CFU aborted or delivered weak, non-viable calves. In Experiment 2, vaccination did not afford significant protection (P greater than 0.05) in that 9 of 9 unvaccinated heifers, 8 of 10 vaccinated with 10(9) CFU, and 8 of 8 vaccinated with 10(10) CFU aborted. Serologic responses to B. abortus were determined by three standard tests, as well as a quantitative fluorometric immunoassay (FIAX) and an enzyme-linked immunosorbent assay. In Experiment 1, the early serologic response, 0-8 weeks after challenge, appeared greater for controls than for vaccinates, but in Experiment 2, the early response, 0-6 weeks after challenge exposure, appeared greater for vaccinates than for controls. The lymphocyte blast transformation assay, using heat-killed B. abortus as an antigen, was performed sequentially after challenge exposure. In general, mean responses were significantly higher (P less than 0.05) for vaccinated than for non-vaccinated heifers. For individual heifers, an association could not be established between the lymphocyte blast transformation assay and the clinical response to challenge exposure.

Conservation of Antigenicity in a 31-kDa Brucella Protein

A 31-kilodalton (kDa) protein extracted from Brucella abortus was previously cloned into Escherichia coli and expressed at high levels. The E. coli-derived protein can be purified by a simple 2-step procedure entailing detergent extraction followed by ion-exchange chromatography. Subsequent analyses show that the E. coli-derived protein is identical to the Brucella-derived protein in molecular weight and isoelectric point. A partial amino acid sequence of the N-terminus of the protein of E. coli origin matches the predicted sequence, based on DNA sequence data. Using specific antiserum raised against the E. coli-derived protein, 34 strains of Brucella, representing all 6 recognized species, were examined for expression of the 31-kDa protein by Western blotting. This protein was detectable in all, but one Brucella species (B. ovis), including all 8 biovars of B. abortus tested. This degree of conservation supports further study of the 31-kDa protein for potential exploitation as a vaccine or diagnostic component.

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 Freunds 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 fluorometric 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 X 10(7) 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 greater than 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 less than 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 greater than 0.20). There was a significantly lower (P less than 0.05) LBT response to heat-killed B. abortus in those heifers that aborted compared to those that did not.

Immunogenicity of Brucella abortus salt-extractable proteins

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.

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.

Differentiation by western blotting of immune responses of cattle vaccinated with Brucella abortus strain 19 or infected experimentally or naturally with virulent Brucella abortus

Brucella abortus strain 19 salt-extractable proteins fractionated by differential ammonium sulfate precipitation were used in a western blotting method to detect bovine immunoglobulin G antibodies to B. abortus. Sera from infected cattle and from cattle vaccinated with strain 19 and subsequently exposed to virulent B. abortus bound to a common group of antigens ranging in molecular weights from 31,000 to 45,000 daltons. Immunoglobulin G antibodies in sera from the latter group in addition also bound to antigens with molecular weights of 66,000 to 71,000 daltons. Some sera from cattle vaccinated when sexually mature reacted similar to those from infected cattle, while immunoglobulin G antibodies in sera from Brucella-free cattle and vaccinated calves did not bind to either group of antigens. In general, fractionation of the proteins by ammonium sulfate precipitation offered no advantage for detecting differences between groups of sera. Ammonium sulfate fraction 0 to 35% reacted with a larger number of sera from a naturally infected group than fraction 0 to 70%. Both fractions reacted equally well with sera from the other groups of cattle, while fractions 35 to 70% and 70 to 100% reacted poorly in this technique. The attractive feature of the blot is that sera from calfhood-vaccinated cattle did not react.

Immunogenic properties of ribosomes isolated from Brucella abortus

Abstract Ribosomes from Brucella abortus strain 19 were isolated by differential centrifugation. The 70S ribosomes were purified by pelleting through 0.5 M ammonium chloride and 30% sucrose. The resulting ribosomes were designated washed ribosomes and consisted of 64–65% RNA and 35–36% protein with sedimentation coefficients of 50S and 30S. When the washed ribosomes were tested by immunodiffusion against immune bovine sera, no precipitins were detectable. The immunogenic responses of the washed ribosomes were determined in guinea pigs, lemmings, and calves. Inoculation dosages as low as 1.0 microgram in guinea pigs and 0.1 microgram in lemmings protected against challenge with virulent B. abortus . Results of similar studies in a limited number of calves indicated that a dose of 1 mg may be protective. Brucella ribosomes did not appear to elicit marked undesirable side effects when used as immunizing agents.

Properties of a bactericidal extract fromBrucella abortus

A water-soluble bactericidal fraction was obtained by extractingBrucella abortus with aqueous methanol. Bactericidal activity was directed against the Gram-positive organism tested (Bacillus subtilis, Streptococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis), but not against the Gram-negative organisms tested (Escherichia coli and aPseudomonas sp.). Mild base hydrolysis, but not mild acid hydrolysis, potentiated the bactericidal activity of the extract. Vigorous acid hydrolysis destroyed the activity. Periodate oxidation and hydrolysis with glycosidases did not affect the activity, while hydrolysis with pronase decreased it by 15–20%. Bioactivity was retained by Sephadex gels, silica gel, and a cation exchange resin, but not by an anion exchange resin. The fact that the activity was precipitable with Fe3+, bound to a cation exchanger, and was extractable with ethanol and methanol suggests that the bioactive substance contains in addition to carbohydrate and lipid a cationic moiety, possibly a peptide.