The defect in the metabolism of erythritol of the Brucella abortus B19 vaccine strain is unrelated with its attenuated virulence in mice.

The role of the defect in erythritol catabolism in the attenuated virulence of Brucella abortus B19 vaccine strain in mice was investigated by means of five different strains: (i) the erythritol sensitive B19 vaccine strain; (ii) a natural erythritol tolerant (NET) mutant obtained spontaneously from B19; (iii) an erythritol resistant derivative from B19 (FJS19) obtained by gene replacement of the deleted ery region; (iv) the erythritol resistant B. abortus 2308 reference virulent strain; and (v) an erythritol sensitive mutant (227 strain) obtained from strain 2308 by transposon insertion in the chromosomal ery region. Besides virulence for mice, erythritol oxidation as well as other phenotypic markers were tested in all the strains. The 2308 and FJS19 strains grew in the presence of erythritol and oxidized the sugar, whereas the B19 and 227 strains did not. The NET strain grew in presence of erythritol but was unable to oxidize it. The B19 vaccine strain and its two erythritol resistant derivatives, NET and FJS19, showed similar residual virulence and splenic time courses in mice. Moreover, the virulent strain 2308 and its erythritol sensitive derivative (227 strain) exhibited similar levels of splenic infection. Altogether, these results demonstrate that the genetic region implicated in erythritol catabolism is not related to the low virulence exhibited by B19 in mice.

Comparison of two selective media for the isolation of Brucella melitensis from naturally infected sheep and goats.

The efficacy of two selective media for the isolation of Brucella melitensis from naturally infected sheep and goats was compared. Two hundred and eighty sheep and 60 goats belonging to B melitensis-infected flocks were slaughtered and samples of milk (when available) and seven tissues were taken from each animal for bacteriological analyses. A modified Thayer-Martin's medium was more sensitive than Farrell's medium; by the combined use of both media 142 infected sheep and 40 infected goats were detected, and 486 of the samples from the sheep and 179 of the samples from the goats were found to be infected.

The Brucella abortus RB51 vaccine does not confer protection against Brucella ovis in rams.

The protective efficacy against Brucella ovis of the live vaccine Brucella abortus strain RB51 has been evaluated in rams using the attenuated B. melitensis strain Rev 1 as a reference vaccine. Sixteen Brucella-free rams, 6 months of age, were vaccinated subcutaneously with 4.18 x 10(10) c.f.u. RB51. Sixteen rams of the same condition and age were vaccinated subcutaneously the same day with 1.1 x 10(9) c.f.u. Rev 1. Fifteen similar rams were kept unvaccinated as controls. Six months after vaccination all rams were challenged with 3 x 10(9) c.f.u. B. ovis and slaughtered 8 weeks thereafter for bacteriological and pathological studies. The percentage of rams that were found infected was 68% (Rev 1), 100% (RB51) and 100% (controls). At necropsy, the percentage of organs found to be infected was significantly lower (p < 0.0005) in Rev 1-vaccinated (34%) than in RB51-vaccinated rams (74%) or controls (69%). In conclusion, the RB51 vaccine did not confer protective immunity against B. ovis in rams.

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.

Efficacy of different Rose Bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats.

Fifteen different Rose Bengal antigens showed large differences with respect to pH, cell concentration and agglutination with the international standard anti-Brucella abortus serum, demonstrating the lack of international standardisation. Their sensitivity and specificity, compared with that of the complement fixation test, were evaluated for the diagnosis of B melitensis infection in culture-positive sheep, brucella-free ewes, and sheep and goats belonging to field flocks under different epidemiological conditions. All the Rose Bengal antigens and the complement fixation test had 100 per cent specificity when testing brucella-free sheep or animals belonging to flocks in unvaccinated brucellosis-free areas, but there were large differences in sensitivity between the Rose Bengal antigens with sera from culture-positive sheep or from animals belonging to infected flocks. When using the most sensitive antigen, no difference was observed in Rose Bengal sensitivity between animals infected with either biovar 1 or biovar 3 of B melitensis. The relationship between the sensitivity of the Rose Bengal antigens and cell concentration was unclear, but their sensitivity was related to the standardisation of the antigens with the international standard serum. The complement fixation test was less sensitive than the Rose Bengal test when testing culture-positive sheep. When testing sera from animals belonging to infected flocks with antigens standardised according to European Union rules, no great differences were observed in the sensitivities of the two tests. However, great differences in sensitivity between the Rose Bengal antigens were observed with sera from animals belonging to flocks with low levels of prevalence.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective immunity to Brucella ovis in BALB/c mice following recovery from primary infection or immunization with subcellular vaccines.

Experiments were performed with BALB/c mice to elucidate the roles of humoral and cell-mediated immune responses in the acquisition of protective immunity to Brucella ovis and to compare infection immunity with immunity developed through vaccination with a hot saline extract (HS) of B. ovis. Mice convalescing from a primary infection with B. ovis displayed a high level of resistance to reinfection, as evidenced by splenic bacterial counts decreased over 10,000-fold from control groups at 2 weeks after challenge. Passive transfer assays revealed that protection was mediated by both T lymphocytes and antibodies but that antibodies had a substantially greater role on the basis of log units of protection that were transferred. Antibodies specific for HS proteins in sera from convalescent mice were predominantly of the immunoglobulin G 2a and 3 isotypes. Vaccination with HS conferred good protection against B. ovis, but protection was greatly enhanced by the incorporation of QS-21 or other adjuvants. Protection provided by the HS vaccine resulted largely from immune responses to its protein moieties. A critical evaluation of the protective efficacy of the rough lipopolysaccharide component of HS was precluded by its poor immunogenicity in BALB/c mice. HS-QS-21 afforded protection against challenge infection with B. ovis as good as that which developed after a primary infection and as good as or better than that provided by attenuated Brucella melitensis vaccine strain Rev 1. Passive transfer experiments confirmed that the magnitudes of both humoral and cell-mediated forms of protective immunity were equivalent in mice vaccinated with HS-QS-21 and those recovering from a primary infection. Protective immunity to B. ovis in mice therefore resembled that to Brucella abortus, except that the relative roles of humoral and cell-mediated immunity, rather than being equivalent, were shifted toward a greater role for antibodies.

Efficacy of Brucella suis strain 2 vaccine against Brucella ovis in rams.

The protective efficacy against Brucella ovis of live vaccine Brucella suis strain 2 (S2) and Brucella melitensis strain Rev 1 has been evaluated in rams. Fourteen 4-month-old Brucella-free Aragonesa rams were vaccinated conjunctivally with 2 x 10(9) c.f.u. S2. Sixteen rams of the same breed, condition and age were conjunctivally vaccinated the same day with 1.6 x 10(9) Rev 1. Thirteen rams were unvaccinated controls. Eight months after vaccination all rams were challenged with 6 x 10(9) c.f.u. B. ovis and slaughtered 2 months thereafter for bacteriological and pathological studies. The percentage of infection in the group vaccinated with Rev 1 (43.7%) was significantly lower (p < 0.05) than that of the S2-vaccinated animals (78.6%) and unvaccinated controls (84.6%). No significant differences were found when comparing the percentages of infection corresponding to S2-vaccinated and control groups. The degree of infection (percentage of necropsy samples infected) was significantly lower in Rev 1-vaccinated (13%) than in S2-vaccinated (36.9%) or control groups (47.4%) (p < 0.001). However, no significant differences were found when comparing S2-vaccinated and control groups.

Evaluation of whole cell and subcellular vaccines against Brucella ovis in rams.

Five antigen preparations from Brucella ovis strain REO 198 were incorporated with the pluronic polymer L-121 and muramyl dipeptide and tested as vaccines against B. ovis infection of rams. The antigenic preparations were: (1) a fraction enriched in outer membrane proteins and rough lipopolysaccharide (hot saline extract, HS); (2) the proteins from HS substantially free of lipopolysaccharide; (3) outer membrane blebs; (4) outer membrane-peptidoglycan complexes extracted with detergent; (5) killed whole cells. The experimental vaccines were compared with two standard vaccines, rough Brucella abortus 45/20 whole killed cells in an oil based adjuvant, and live Brucella melitensis Rev 1. Immunizations with non-living vaccines were performed on two occasions, 18 weeks apart. The rams were challenged with a virulent strain of B. ovis 31 weeks after the second vaccination and slaughtered 15 weeks thereafter. Rates of infection in groups vaccinated with Rev 1 (33%), and HS (40%) were significantly lower (P < 0.005 and P < 0.025, respectively) than that in the non-vaccinated control group (87%). Strain 45/20 was the only other vaccine that conferred a significant level of protection (50%) (P < 0.05). The organ distribution of the infection and the level of colonization of infected organs did not differ significantly between infected animals in the various vaccine groups and those in the unvaccinated control group. No statistically significant relationship was detected between the magnitude of the antibody responses to the HS extract, to outer membrane proteins, or to the rough lipopolysaccharide, and freedom from infection. The results indicate that the HS extract of B. ovis may represent a useful alternative to B. melitensis Rev 1 or B. abortus 45/20 as a vaccine against B. ovis.

Evaluation of vaccines and of antigen therapy in a mouse model for Brucella ovis.

A mouse model was developed to study Brucella ovis infection. The evolution of the number of B. ovis per spleen of mice inoculated intravenously, intraperitoneally or subcutaneously was found to be independent of the sex of the mice. The number of B. ovis increased in the spleen when increasing the challenge dose up to 1.7 x 10(7). At higher doses of challenge, the response remained constant. In this model it was observed that the inoculation of Brucella melitensis Rev 1 vaccine or subcellular B. ovis hot saline antigens during both the acute and chronic phases did not modify the time course of B. ovis infection. Finally, the model was found suitable to determine the efficacy of anti-B. ovis vaccines. B. melitensis Rev 1 (2.2 x 10(5) c.f.u.) and B. suis strain 2 (1.2 x 10(7) c.f.u.) live vaccines but not the inactivated B. melitensis H38 vaccine conferred protection against B. ovis.

An ELISA with Brucella lipopolysaccharide antigen for the diagnosis of B. melitensis infection in sheep and for the evaluation of serological responses following subcutaneous or conjunctival B. melitensis strain Rev 1 vaccination.

An indirect enzyme-linked immunosorbent assay (ELISA) with unpurified Brucella melitensis smooth lipopolysaccharide (S-LPS) as antigen was evaluated for the serological diagnosis of B. melitensis infection in sheep in comparison with the Rose Bengal (RB), complement fixation (CF), radial immunodiffusion (RID), microplate agglutination (MA) and rivanol agglutination (RIV) tests. Tests RB and CF detected as positive each of the 77 sera from B. melitensis-infected animals tested, the RID (74), MA (76) and the RIV (72) were less sensitive. However, all tests compared were negative when 77 sera from Brucella-free rams were tested. While subcutaneous Rev 1 vaccination induced high response levels in any of the tests, low level responses were obtained upon conjunctival vaccination, particularly in ELISA and RID tests.

Comparison of subcutaneous and conjunctival routes of Rev 1 vaccination for the prophylaxis of Brucella ovis infection in rams.

The serological response and protection conferred against Brucella ovis by the Rev 1 vaccine was evaluated in both adult (experiment 1) and young rams (experiment 2) vaccinated either subcutaneously or conjunctivally. In experiment 1 the Rev 1 vaccine protected 55.5 per cent and 100 per cent, respectively, of subcutaneously and conjunctivally vaccinated rams against three consecutive challenges that infected 100 per cent of unvaccinated controls. In experiment 2, Rev 1 protected 100 per cent of rams vaccinated subcutaneously and 70 per cent of those vaccinated conjunctivally against a challenge dose able to infect all the unvaccinated controls. The serological response after vaccination was significantly lower in rams vaccinated conjunctivally than in those vaccinated subcutaneously.

Comparison of three serological tests for Brucella ovis infection of rams using different antigenic extracts.

The sensitivity and specificity of the complement fixation, gel diffusion and ELISA tests for the diagnosis of Brucella ovis infection of rams have been compared using three different antigenic preparations. The antigens obtained by petroleum ether - chloroform - phenol, or cold saline extractions gave poorer diagnostic results than those obtained by hot saline extraction in all the tests. The best sensitivity was obtained with the ELISA (97.6 per cent) followed by the gel diffusion (96.4 per cent) and complement fixation tests (92.7 per cent). The gel diffusion test detected as positive the two rams negative in the ELISA, while the complement fixation test did not improve the sensitivity of the other tests. Under these conditions all the tests were 100 per cent specific when testing sera from rams free of B ovis.

Efficacy of long-acting oxytetracycline alone or in combination with streptomycin for treatment of Brucella ovis infection of rams.

Twenty-four rams inoculated with Brucella ovis by conjunctival and preputial routes were treated with a long-acting oxytetracycline alone or in combination with dihydrostreptomycin sulfate. The combined treatment eliminated Brucella ovis from 11 of 12 (91.6%) treated rams. Only 4 of 12 (33.3%) rams treated with oxytetracycline alone were bacteriologically negative. Neither treatment resolved clinical epididymitis in 2 rams affected before treatment. Many rams had pathologic lesions in the epididymis and ampullae, which limited the efficacy of antibiotic treatment.

Responses of ewes to B. melitensis Rev1 vaccine administered by subcutaneous or conjunctival routes at different stages of pregnancy.

An experiment to determine the innocuousness and the humoral immune response of B. melitensis Rev1 vaccine was carried out in pregnant ewes. Thirty-three ewes were vaccinated on day 55 of pregnancy: 9 ewes received 2 X 10(9) Rev1 of French origin by the subcutaneous route, 9 ewes were conjunctivally vaccinated with 5 X 10(8) of the same strain, 7 ewes were subcutaneously vaccinated with 10(9) Rev1 of Spanish origin and 8 ewes received 1.8 X 10(9) Spanish strain Rev1 by conjunctival route. Another group (8 animals) were subcutaneously vaccinated with a reduced dose (10(6) of the French strain between days 90 and 120 of pregnancy. Finally, 20 ewes were vaccinated on day 120 of pregnancy with the French strain of which 11 received 10(9) Rev1 by the subcutaneous route and 9 were conjunctivally vaccinated with the same dose. Rev1 strain was isolated from the vaginal discharge of a significantly higher number of ewes vaccinated subcutaneously than in conjunctivally vaccinated ewes (24/35 versus 9/26 respectively; P less than 0.001). No difference between the two vaccine strains was recorded in the number of ewes excreting Rev1. Ewes subcutaneously vaccinated on day 55 of pregnancy aborted more frequently than conjunctivally vaccinated ewes (11/16 versus 2/17, respectively), hence a significantly reduced (P less than 0.001) length of pregnancy. Although 10 of the 11 animals subcutaneously vaccinated on day 120 of pregnancy excreted Rev1 strain, only one aborted. There was no significant effect of the time of vaccination on both abortions and length of pregnancy in ewes conjunctivally vaccinated with the French strain.(ABSTRACT TRUNCATED AT 250 WORDS)