A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as betweeen vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.

Lipopolysaccharide (LPS) fractions were obtained from smooth cultures of Brucella abortus strains 2308 and S-19 by butanol extraction procedures. The LPS from the initial butanol extraction contained 10 to 15% protein and was reduced to less than 1% protein by treatment with proteinase K. The LPS fractions were identified and characterized on the basis of the chemical analysis, sodium dodecyl sulfate gel electrophoresis, cesium chloride gradients, electron microscopy, and gel immunodiffusion. Results indicated that the butanol procedure is a reliable method in the extraction of LPS from Brucella abortus cells. Proteinase K-treated LPS containing less than 1% protein from strain 2308 was used to vaccinate BALB/cByJ mice. Immune and protective criteria for vaccinated and nonvaccinated mice were increased immunoglobulin (IgG and IgM) titers in sera of prechallenge-exposed mice, reduced colony-forming units/spleen, and splenomegaly in post-challenge-exposed mice. Results indicated that proteinase K-treated LPS was immunuogenic as well as protective for mice.

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.

To determine their capacity to host Brucella abortus, face flies were examined 1 to 120 hours after feeding on broth containing bacteria and bovine erythrocytes. Brucella abortus was cultured in large numbers from whole flies for 12 hours after feeding, but not after 72 hours. Histologic analysis showed that brucellae were rapidly taken into the midgut, sequestered from erythrocytes, transiently stored, and shed in the feces; there was no evidence of bacterial replication within epithelial cells. Immunoperoxidase and immunogold techniques revealed that most brucellae in the gut were confined to the lumen by the peritrophic membrane, that brucellae were degraded in secondary lysosomes of midgut epithelial cells, and that intact brucellae passed into connective tissues surrounding the midgut. Bacterial excretion without midgut replication is consistent with transient, but not long-term, insect transmission in nature.

Serum and vaginal Brucella-specific immunoglobulin isotypes (IgGl, IgG2, IgM, and IgA), obtained from 62 crossbred beef heifers vaccinated with Brucella abortus salt-extractable proteins and subsquently 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 > 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.

Immunogenic or pathogenic factors of recombinant proteins (rBCSP20, rBCSP-31, and rBCSP45 of Brucella abortus strain 19) for mice were compared with factors of a proteinase K-treated lipopolysaccharide extracted from B abortus strain 2308. Mice were vaccinated with 4 products, using different inoculation schedules and were challenge exposed with a virulent culture of B abortus strain 2308. Blood samples were collected 2 weeks after vaccination and at necrospy and sera were obtained. Spleens were cultured for B abortus at necropsy (3 to 4 weeks after challenge exposure). Mice given proteinase K-treated lipopolysaccharide alone or in conjunction with rBCSP20 or rBCSP45 proteins were protected, but mice given rBCSP31 on the same day as challenge exposure were not. Vaccination with recombinant proteins alone neither provide protection nor significantly (P greater than 0.05) increase the pathogenic effect of the challenge-exposure culture. Seemingly, rBCSP31 might be a virulence factor of B abortus.

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.

The restriction endonuclease digestion DNA patterns from Brucella abortus strains 19 and 2308 were examined with 11 restriction enzymes (AvaI, BamHI, BglII, BstEII, DdeI, EcoRI, HindIII, KpnI, PstI, XbaI, and SalI)). The DNA electrophoretic banding patterns between the strains were highly similar, using this restriction enzyme analysis. Differences were not discernable between B abortus strains 19 and 2308 in any of the restriction banding patterns examined. Methylation at CCGG or GATC sites was not detectable on the basis of digestion with isoschizomers (HpaII and MspI, and DpnI, Sau3AI and MboI). Homology between B abortus strains 19 and 2308 was assessed, using solution-hybridization techniques followed by S1 nuclease assays. Results of these reassociation experiments indicated 98.6 to 99.3% homology between B abortus strains 19 and 2308 with 13.5 to 18.6% homology between B abortus (strains 19 and 2308) and the E coli HB101 control. We concluded that any DNA differences between the 2 B abortus strains are small and will require analysis at the DNA sequence level.

A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as betweeen vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.

A method has been developed for the detection of Brucella abortus in complex tissue homogenates. The technique uses tissue homogenization in the presence of sucrose and Triton X-100 and subsequent filtration through a 5-micrometer pore size filter to remove mammalian nuclei and cellular debris. The DNA from the bacteria is then extracted, dot blotted onto nitrocellulose, and hybridized with a biotinylated probe of B abortus strain 19 DNA. In the present study, BALB/C mice were inoculated intraperitoneally with either 10(9) or 10(11) B abortus strain 2308S organisms. After 6 days, the mice were euthanatized by cervical dislocation and the livers were removed, weighed, and the appearance of each was noted. The tissues were homogenized, and a viable cell count was performed to determine the number of bacteria in each organ. The DNA was extracted, blotted onto nitrocellulose, and hybridized with the Brucella probe. The biotin label was detected by use of a commercially available streptavidin/alkaline phosphatase system. In control experiments, the technique detected 10(5) organisms in a mixture of bacteria and 1 g of rat liver. The technique also detected 10(7) B abortus organisms/g of tissue from experimentally inoculated mice. The probe was specific for Brucella and had no affinity for contaminating bovine or bacterial DNA. ?