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PubMed2002

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.

Animals↗
PubMed1996

Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19.

The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins [BCSP]) of Brucella abortus 19 and later challenge exposed with B. abortus 2308. BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate. Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC). Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines. Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria. Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic. The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier. However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice. MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D. Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only. The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B. abortus infections.

Animals↗
PubMed1967

Antigenic relationship of Brucella ovis and Brucella melitensis.

Immune sera were prepared in rabbits by the injection of living and acetone-killed cells of Brucella ovis and smooth and rough B. melitensis. The use of whole-cell antigens in agglutination and agglutinin-absorption tests revealed little relationship between B. ovis and smooth B. melitensis, although there was extensive cross-agglutination between B. ovis and rough B. melitensis. The use of water-soluble antigens prepared from ultrasonically treated cells of the three strains revealed extensive cross-reactions in indirect hemagglutination, agar gel precipitation, and immunoelectrophoresis tests, as well as in allergic skin tests in rabbits. The most definitive results were obtained with the immunoelectrophoresis technique. B. ovis antigen produced at least 11 lines with its homologous serum. All were removed by absorption of the serum with rough B. melitensis antigen. All but three were removed by absorption with smooth B. melitensis antigen. Smooth B. melitensis antigen produced 11 lines with its homologous serum, and all but 3 were removed by absorption with B. ovis antigen. Rough B. melitensis produced nine lines with its homologous serum, and eight were removed by B. ovis antigen. The extensive cross-reactions between soluble antigens of B. ovis and B. melitensis are added evidence that B. ovis belongs in the genus Brucella.

Animals↗
PubMed1981

Cellular fatty acids of Brucella canis and Brucella suis.

The cellular fatty acid composition of Brucella canis and Brucella suis was determined by gas-liquid chromatography. The presence of relatively large amounts of a 19-carbon cyclopropane fatty acid in B. suis was a major distinguishing feature between these organisms. The gas-liquid chromatography test for cellular fatty acids provides an additional criterion for the distinction of antigenically rough strains of B. suis which cannot be differentiated from B. canis by conventional procedures.

Animals↗
PubMed1995

Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51.

Because the brucellosis eradication program uses slaughter and quarantine as control measures, it would benefit from faster methods of bacterial identification. Distinguishing vaccine strains from strains that cause infections among vaccinated herds in the field is essential. To accomplish this, our PCR-based, species-specific assay (B. J. Bricker and S. M. Halling, J. Clin. Microbiol. 32:2660-2666, 1994) was updated to identify Brucella abortus vaccine strains S19 and RB51. Three new oligonucleotide primers were added to the five-primer multiplex Brucella AMOS PCR assay. Identification is based on the number and sizes of six products amplified by PCR.

Animals↗
PubMed2002

Brucella spondylitis with paravertebral abscess due to Brucella melitensis infection: a case report.

This report describes the case of a 45-year-old woman with a 5-month history of fever, generalized malaise, myalgia, lower back pain and difficulty in walking. Serodiagnosis for brucella, carried out at the onset of symptoms 5 months previously, was negative. When the patient was admitted to our hospital there was contracture of the paraspinal muscles but no peripheral nerve damage. Laboratory tests showed positive agglutination for Brucella and an increase in the rate of dilution from 1/160 to 1/640 over 2 weeks. Radiographs and a computed tomography scan of the spine revealed bone erosion in the posterior borders of the L4-L5 vertebral end plates and a soft tissue mass surrounding the interposed disc and protruding into the spinal canal. Magnetic resonance imaging confirmed the presence of a paraspinal abscess around the affected disc and tissue edema. Culture tests of the blood and abscess tissue, taken by biopsy, were negative. Rifampicin treatment (600 mg daily), combined with a bust cast to immobilize the spine, led to clinical healing without the need for surgery. Because onset symptoms are nonspecific and insidious, in nonrisk subjects a diagnosis of brucellosis may sometimes be suspected only if there are local symptoms. The phenomenon of the absence of positivity in patients with a high antibody titer should also be considered Cases such as that described herein demonstrate the need for culture tests and serodiagnosis, even in nonrisk patients with persistent fever and arthralgia, to prevent the later complications of brucellosis.

Abscess↗
PubMed1989

Macrophage function in mammary glands of Brucella abortus-infected cows and cows that resisted infection after inoculation of Brucella abortus.

Nonvaccinated pregnant cows were segregated retrospectively into 2 groups following inoculation with Brucella abortus strain 2308. One group resisted infection (resistant cows) and the other group developed active infections (susceptible cows) and subsequently aborted. Mammary gland macrophages collected from the 2 groups of cows were compared, using in vitro functional assays. In a chemiluminescence assay, mammary gland macrophages from resistant cows produced significantly (P = 0.014) higher oxidative burst activity than did macrophages from susceptible cows. Macrophages from resistant cows had significantly (P = 0.038) greater bacteriostatic activity against B abortus than did macrophages from susceptible cows. Differences in lysosomal enzymatic activity or Fc receptor expression were not observed for macrophages from the 2 groups of cows. Differences in macrophage function may be one factor responsible for natural resistance to Brucella infection in cattle.

Abortion, Veterinary↗
PubMed1981

The bovine immune response to Brucella abortus. III. Preparation of antisera against a Brucella component precipitated by sera of some infected cattle.

Two methods are described for the partial purification of a high molecular weight, heat-resistant component (CO1) of sonicates of smooth and rough Brucella abortus which is precipitated by sera of some infected cattle. Method 1, a combination of gel filtration chromatography and polyacrylamide gel electrophoresis, was used to prepare CO1 from sonicates of a smooth field strain of B. abortus. Method 2, a combination of gel filtration chromatography and heat treatment, was used to obtain CO1, from sonicates of rough B. abortus strain 45/20. Rabbit antisera produced against CO1 prepared by either method contained only CO1 precipitins but were negative in standard agglutination and complement fixation tests conducted with whole cell antigens. Evidence is presented that CO1 is identical to Brucella antigen A2, and it is proposed that in future the designation A2 be employed.

Agglutination Tests↗
PubMed1982

Efficacy of Brucella melitensis Rev. 1 vaccine against Brucella ovis infection in rams.

The efficacy of Brucella melitensis strain Rev. 1 vaccine against a Brucella ovis experimental infection was judged on 30 rams, 15 of which had received 8.5 X 10(8) Rev. 1 at the age of 4 months. All were challenged 9 months later by conjunctival route with 5 X 10(8) B. ovis. The 30 rams were killed three months after challenge and were autopsied. Genital and other organs and lymph-nodes were searched for B. ovis. Only a few organs and lymph-nodes of three vaccinated rams shed B. ovis, contrary to controls which were all heavily infected. The distribution of infection indicated that the most frequently infected organs are the vesicular glands which, more than epididymes, seem to be the target organs of the infection. These results show that immunization with strain Rev. 1 is very effective against B. ovis infection. It is recommended to vaccinate rams of four to six months in an area where B. ovis or B. melitensis infection, or both, are prevalent.

Animals↗
PubMed1975

Serologic studies of Brucella suis and Brucella canis.

Brucella suis strain 3b and Brucella canis strain RM-6-66 were grown on tryptose agar, and aqueous ether extracts were prepared from the cells. The aqueous phase was clarified by centrifugation for a total of 10 hours at 144,700 times g and fractionated with gels and with diethylaminoethyl (DEAE) cellulose. Two fractions from B suis and 1 fraction from B canis were reactive in the indirect hemagglutination test. The sensitizing ability was destroyed by pronase, but was unaffected by ribonuclease or deoxyribonuclease. Apparently, protein or one of its subunits has an important role in antigenic specificity relating to indirect hemagglutination activity. Results in immunoelectrophoretic analysis indicated at least 22 antigens were in the ultracentrifugal supernate (144,700 times g) of B suis and 31 antigens were in a similar supernate of B canis. Results in immunodiffusion and agglutination absorption studies indicated a close relationship existed between B suis and B canis.

Animals↗
PubMedDate not supplied

Resistance of Brucella abortus infected mice to intravenous or intraperitoneal Brucella reinfection.

The development of acquired resistance to Brucella abortus was studied in mice infected intravenously with small numbers of live CO2-dependent B. abortus 544 used as vaccinal strain. CO2-independent B. abortus was used as challenge strain. The influence of several parameters in the development of acquired resistance was examined: vaccination--challenge interval, challenge route, dose of challenge, challenge-sacrifice interval. The behavior of the challenge strain in normal mice depended on the size of the inoculum. After an intravenous inoculum of about 1 X 10(6) bacteria, the level of infection in the liver or the spleen followed three phases: a phase of increase of the viable counts, a phase of decrease, and a phase of plateau. The decreasing phase was more pronounced in liver than in spleen. After an intraperitoneal injection of the same dose of bacteria, an infection developed following approximately the same pattern, except during the first day of infection. One month after vaccination by an inoculum of about 1 X 10(4) viable B. abortus, an accelerated appearance of the decreasing phase prevented almost completely the multiplication of an intravenous challenge of about 1 X 10(6) B. abortus in the spleens. In the livers the same phenomenon took place after a small increase of the bacterial population. After an intraperitoneal challenge, the numbers of Brucella isolated from the spleens and livers of vaccinated mice first fell sharply to practically nothing around the sixth day, and then rose to reach a phase of slow increase or of plateau until the twenty-fourth day. In our experimental conditions, the vaccine population remained undisturbed by the reinfecting inoculum.

Animals↗
PubMed1992

Control of brucellosis in Kuwait by vaccination of cattle, sheep and goats with Brucella abortus strain 19 or Brucella melitensis strain Rev. 1.

In Kuwait, approximately 12,000 dairy cows were vaccinated with a reduced dose of 3 x 10(9) Brucella abortus strain 19 and approximately 350,000 sexually mature sheep and goats with a reduced dose of 10(7) B. melitensis strain Rev. 1. Using the criteria of prevaccinal and postvaccinal incidences of antibodies, abortions, and human cases of brucellosis, the programme was very successful. Widespread vaccination of adult animals is the most effective method of controlling brucellosis among cattle, sheep and goats in many countries.

Animals↗
PubMed2003

Statistical procedures for calculating the residual virulence of Brucella abortus strain 19 (S19) and Brucella melitensis strain Rev 1 vaccines in mice: theoretical basis and practical applications.

Regular control of the biological quality of live Brucella abortus strain 19 (S19) and B. melitensis strain Rev 1 vaccines is essential for the successful management of ruminant brucellosis in affected countries. The reference procedures recommended by the OIE (World organisation for animal health) and the European Pharmacopoeia include the determination of residual virulence, expressed as the recovery time 50 (RT50), of the tested (problem) vaccine in a reference mouse model compared with the RT50 of the corresponding reference strains in the same assay. The underlying statistical procedure applied is based on a parallel line assay and a classical probit model. In practice, the currently recommended procedure for calculating the RT50 is based on a graphical method which has never been described in detail. This paper provides a full description of this graphical method with the aim of making the technique comprehensible and accessible to all interested biologists. The procedure is somewhat cumbersome and very few laboratories apply the OIE and European Pharmacopoeia recommendations on a regular basis. Moreover, since this reference graphical method shows some statistical inconsistencies, a dedicated internet interface has been developed to perform RT50 calculations and is now available free of charge on the web (www.afssa.fr/interne/rev2.html).

Animals↗
PubMed1985

Restriction endonuclease analysis of Brucella ovis and other Brucella species.

Brucella ovis DNA was analysed by using 11 different restriction endonucleases. The most clearly resolved DNA fragment patterns were obtained after digestion with the enzyme Hind III. When DNA preparations from 35 strains of B. ovis were digested with this enzyme, the fragment patterns appeared to be identical. The patterns obtained after Hind III digestion of DNA from one strain each of B. abortus, B. canis and B. melitensis were more similar to each other than to the B. ovis pattern.

Animals↗