Accidental exposure to Brucella abortus vaccines and occupational brucellosis among veterinarians in Minas Gerais state, Brazil.

Brucellosis is an important occupational disease, mainly among veterinarians, because of their frequent contact with sick animals, contaminated secretions and live attenuated anti-Brucella vaccines. This study aimed to determine the prevalence of accidental exposure to S19 and RB51 vaccine strains and occupational brucellosis among veterinarians registered to administer vaccinations in Minas Gerais, Brazil, as well as to identify the risk factors associated with accidental exposure to anti-Brucella abortus vaccines. Data were collected through an online questionnaire. Three hundred and twenty-nine veterinarians were included in the analyses using stratified random sampling. A multivariate logistic regression analysis was used to evaluate the predictors of accidental exposure to S19 and RB51 strains. Nearly one third of the veterinarians registered to administer bovine brucellosis vaccination in Minas Gerais, 32.83% (108/329) (95% confidence interval [CI]: 27.78-38.19%), reported having been accidentally exposed to S19 or RB51 vaccine strains. The exposure factors associated with this outcome included a score of personnel protective equipment (PPE) use during work (odds ratio [OR], 0.94; 95% CI: 0.89-0.98) and a score of knowledge about brucellosis symptoms, classified as poor (base category), intermediate (OR, 0.26; 95% CI: 0.07-0.87) or good (OR, 0.22; 95% CI: 0.07-0.62). In addition, 4.56% (15/329) (95% CI: 2.57-7.41%) of veterinarians reported that they had brucellosis, of which 46.67% (7/15) considered that the disease was due to accidental exposure to anti-B. abortus live attenuated vaccine. The prevalence of accidental exposure to B. abortus vaccine strains among veterinarians from Minas Gerais enrolled in the control of bovine brucellosis was high. The reduced knowledge about human brucellosis symptoms and lack of appropriate PPE use were risk factors from unintentional contact with S19 and RB51 vaccine strains.

Interaction of Brucella abortus with Osteoclasts: a Step toward Understanding Osteoarticular Brucellosis and Vaccine Safety.

Osteoarticular disease is a frequent complication of human brucellosis. Vaccination remains a critical component of brucellosis control, but there are currently no vaccines for use in humans, and no in vitro models for assessing the safety of candidate vaccines in reference to the development of bone lesions currently exist. While the effect of Brucella infection on osteoblasts has been extensively evaluated, little is known about the consequences of osteoclast infection. Murine bone marrow-derived macrophages were derived into mature osteoclasts and infected with B. abortus 2308, the vaccine strain S19, and attenuated mutants S19vjbR and B. abortusΔvirB2 While B. abortus 2308 and S19 replicated inside mature osteoclasts, the attenuated mutants were progressively killed, behavior that mimics infection kinetics in macrophages. Interestingly, B. abortus 2308 impaired the growth of osteoclasts without reducing resorptive activity, while osteoclasts infected with B. abortus S19 and S19vjbR were significantly larger and exhibited enhanced resorption. None of the Brucella strains induced apoptosis or stimulated nitric oxide or lactose dehydrogenase production in mature osteoclasts. Finally, infection of macrophages or osteoclast precursors with B. abortus 2308 resulted in generation of smaller osteoclasts with decreased resorptive activity. Overall, Brucella exhibits similar growth characteristics in mature osteoclasts compared to the primary target cell, the macrophage, but is able to impair the maturation and alter the resorptive capacity of these cells. These results suggest that osteoclasts play an important role in osteoarticular brucellosis and could serve as a useful in vitro model for both analyzing host-pathogen interactions and assessing vaccine safety.

Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

Extended safety and efficacy studies of the attenuated Brucella vaccine candidates 16 M(Delta)vjbR and S19(Delta)vjbR in the immunocompromised IRF-1-/- mouse model.

The global distribution of brucellosis and high incidence in certain areas of the world warrant the development of a safer and efficacious vaccine. For the past 10 years, we have focused our attention on the development of a safer, but still highly protective, live attenuated vaccine for human and animal use. We have demonstrated the safety and protective efficacy of the vaccine candidates 16 MΔvjbR and S19ΔvjbR against homologous and heterologous challenge in multiple immunocompetent animal models, including mice and deer. In the present study, we conducted a series of experiments to determine the safety of the vaccine candidates in interferon regulatory factor-1-knockout (IRF-1(-/-)) mice. IRF-1(-/-) mice infected with either wild-type Brucella melitensis 16 M or the vaccine strain Brucella abortus S19 succumb to the disease within the first 3 weeks of infection, which is characterized by a marked granulomatous and neutrophilic inflammatory response that principally targets the spleen and liver. In contrast, IRF-1(-/-) mice inoculated with either the 16 MΔvjbR or S19ΔvjbR vaccine do not show any clinical or major pathological changes associated with vaccination. Additionally, when 16 MΔvjbR- or S19ΔvjbR-vaccinated mice are challenged with wild-type Brucella melitensis 16M, the degree of colonization in multiple organs, along with associated pathological changes, is significantly reduced. These findings not only demonstrate the safety and protective efficacy of the vjbR mutant in an immunocompromised mouse model but also suggest the participation of lesser-known mechanisms in protective immunity against brucellosis.

Immunopathological responses and kinetics of Brucella melitensis Rev 1 infection after subcutaneous or conjunctival vaccination in rams.

The innocuousness of the Brucella melitensis Rev 1 live attenuated vaccine strain has never been fully assessed in rams. The immunopathological responses and the kinetics and distribution of the infection induced by this strain were determined after subcutaneous or conjunctival vaccination in both young (3-4 months old) and adult (12 months old) rams. At regular intervals after vaccination the animals were bled for serological studies, and slaughtered for both pathological and bacteriological examinations. The serological response after conjunctival inoculation was of lower intensity and duration than that induced subcutaneously, being the differences more evident in young rams. No genital lesions were produced and genital organs and accessory sexual glands were never found infected, being Rev 1 infection restricted to lymph nodes and spleen. Immunostained Rev 1 bacteria were located intracellularly in plasmablasts, dendritic follicular cells and macrophages in the target lymph nodes, in which cellular hyperplasia was the dominant pathological response. Subcutaneous vaccination induced a generalized infection by 2 weeks after vaccination, being then restricted to the prescapular target lymph node. Infection after conjunctival vaccination was less generalized, being restricted essentially to the cranial lymph nodes. Rev 1 infection was fully cleared by 3 months after vaccination in all animals. These results confirm the innocuousness of B. melitensis Rev 1 vaccine in rams.

Residual virulence and immunogenicity of CGV26 and CGV2631 B. melitensis Rev. 1 deletion mutant strains in sheep after subcutaneous or conjunctival vaccination.

The CGV26 and CGV2631 strains are novel engineered Brucella melitensis Rev.1 mutant strains deleted for the bp26 gene or for both bp26 and omp31 genes, respectively, coding for proteins of diagnostic significance. The residual virulence and immunogenicity of both mutants were compared to the parental Rev.1 strain in sheep after subcutaneous or conjunctival vaccination. The deletion of the bp26 gene or both bp26 and omp31 genes had no significant effect on the intracellular survival of the Rev.1 strain in ovine macrophage cultures. The kinetics of infection induced by both mutants in sheep was similar to the Rev.1 strain, and inoculation by the subcutaneous route produced wider and more generalized infections than the conjunctival route. All strains were cleared from lymph nodes and organs within 3 months after inoculation. The CGV26 and CGV2631 mutants induced both specific systemic antibody response and lymphoproliferation in sheep. The kinetics of the responses induced by the mutants was quite similar to that of the parental Rev.1 strain, except for the intensity of the lymphoproliferative response, which was attenuated for the CGV2631 mutant. In conclusion, the residual virulence of both CGV26 and CGV2631 mutants in sheep was similar to that of the parental Rev.1 vaccine strain. These mutants induced also significant specific antibody and cell-mediated immunity in sheep and are suitable to be evaluated as potential vaccine candidates against B. melitensis and B. ovis infections in sheep.

Adverse events in humans associated with accidental exposure to the livestock brucellosis vaccine RB51.

Brucella abortus strain RB51 vaccine, is an attenuated live bacterial vaccine that was licensed conditionally by the Center for Veterinary Biologics, Veterinary Services, Animal and Plant Health Inspection Service, USDA, on 23 February 1996, for vaccination of cattle in the United States. Accidental human inoculations can occur during vaccination of cattle, and previous live Brucella vaccines designed for cattle have been known to cause brucellosis in humans. The Centers for Disease Control and Prevention (CDC) established passive surveillance for accidental inoculation with the RB51 vaccine in the United States to determine if this veterinary vaccine is associated with human disease, to describe the circumstances of accidental inoculation, to evaluate the potential efficacy of post-exposure chemoprophylaxis, and to develop recommendations for post-exposure management following exposure to RB51. Reports were received from 26 individuals. Accidental exposure to RB51 occurred by needle stick injury in 21 people (81%), conjunctival spray exposure in four (15%), and spray exposure of an open wound in one (4%) individual. At least one systemic symptom was reported in 19 (73%) people, including three (12%) who reported persistent local reactions with systemic involvement. One case required surgery, and B. abortus strain RB51 was isolated from the wound of that individual. Seven cases reported no adverse event associated with accidental exposure. Nine cases reported previous exposure to Brucella vaccines, including one case who also reported a previous diagnosis of brucellosis following exposure to S19 vaccine. Accidental needle stick injuries and conjunctival or open wound exposures of humans with the RB51 vaccine are associated with both local and systemic adverse events in the United States that are consistent with brucellosis; however, it remains undetermined if strain RB51 vaccine can cause systemic brucellosis in humans. Early culture attempts on those exposed and developing disease in the future and serologic diagnostic assays for anti-RB-51 antibodies are needed to define if these adverse events are due to RB51 and to define appropriate prophylaxis regimens.

Bacterial survival, lymph node pathology, and serological responses of bison (Bison bison) vaccinated with Brucella abortus strain RB51 or strain 19.

From August 1993 to June 1994, 3 month-old bison (Bison bison) were vaccinated with Brucella abortus strain RB51 (SRB51, n = 6), strain 19 (S19, n = 3), or with saline (n = 1) and serologic responses and persistence of vaccine strains within lymph nodes were monitored. Bison vaccinated with S19 had granulomatous lymphadenitis and greater peak numbers of B. abortus than those vaccinated with SRB51. Bison vaccinated with RB51 had similar histological lesions and B. abortus were still present in lymph nodes at 16 weeks. Although antibodies against RB51 were produced, standard tube agglutination test responses of RB51-vaccinates remained negative. The histological lesions of B. abortus infections in bison were similar to those observed in cattle, but bison did not clear SRB51 as rapidly as cattle.

Effect of brucellosis vaccination and dehorning on transmission of bovine leukemia virus in heifers on a California dairy.

Brucellosis vaccination and dehorning were examined for an association with bovine leukemia virus (BLV) infection in heifers on a California dairy between April 1984 and June 1987. Between December 1985 and June 1986, weaned heifers were dehorned using the gouge method at the time of brucellosis vaccination. Using logistic regression, the estimated probability for a nondehorned heifer to seroconvert within three months after brucellosis vaccination (0.08) was significantly less than that for heifers dehorned after a noninfected heifer (0.46) or than that for heifers dehorned after an infected heifer (0.85) (p = 0.039 and p less than 0.001, respectively). To evaluate risk of transmission by brucellosis vaccination, which was usually done within one month postweaning, cumulative proportions of heifers remaining uninfected were computed among heifers that did not seroconvert three months after dehorning. Because results of a Cox model analysis indicated that groups of heifers were 6.6 times more at risk of becoming infected if placed in pens holding gouge-dehorned heifers (where prevalence varied between 50 and 70%) (p less than 0.001) than other groups placed in pens without gouge-dehorned heifers (where prevalence varied between 10 and 30%), cumulative proportions of heifers remaining uninfected were computed for each type of group. The cumulative proportion of heifers remaining uninfected from weaning to first calving was 0.60 for the high prevalence group and 0.96 for the low prevalence group. No change in slope of cumulative proportions was observed before and after one month postweaning, suggesting that brucellosis vaccination was not an effective means of transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthropathy associated with Brucella abortus strain 19 vaccination in cattle. I. Examination of field cases.

Thirty cows presenting with lameness and persistent serological reactions to Brucella abortus had chronic granulomatous arthropathy of the femorotibial and occasionally other joints. Attempts to culture Brucella or other pathogens gave negative results but organisms of Brucella morphology were seen in fluorescent antibody-stained cryostat sections of synovial tissue. The synovial fluids contained high titres of antibodies to B. abortus and Yersinia enterocolitica O:9 and had elevated total protein and immunoglobulin concentrations showing an oligoclonal electrophoretic profile. Immune complexes and rheumatoid factor were detected in some of the fluids.

Reactivation of a residual Brucella abortus 19 vaccine infection in mice by a virulent challenge or by injection of brucellin or of Brucella lipopolysaccharide.

Facultative intracellular bacteria localize preferentially in reticulo endothelial system organs where they can either multiply or be destroyed, concomitantly or successively. Immunity may thus be estimated by counts of viable (surviving) bacteria at convenient time(s) after a standard challenge. When mice vaccinated with the living attenuated Brucella abortus strain 19 were intravenously challenged with the virulent B abortus strain 544, some mice exhibited unexpected high spleen counts. The vaccinal strain surviving at low level in some mice 30 days after a subcutaneous vaccination was reactivated by the virulent challenge as evidenced by a fast but temporary count increase. Reactivation was stronger in outbred OF 1 mice than in outbred CD-1 mice. Reactivation did not alter the normal course of the virulent infection as shown by comparison between mice already cured of the vaccinal infection or not, at time of challenge. Because reactivation was also induced by intravenous injection of either the brucellin allergen or the brucella lipopolysaccharide antigen, hypersensitivity reactions occurring inside the vaccinal granuloma foci may expel the surviving bacteria into the surrounding extracellular environment where a secondary growth may develop.

Histopathology of experimental brucellosis in rams following vaccination with Brucella ovis.

Lesions induced by inoculation of Brucella ovis into the epididymis were compared in rams previously vaccinated with B. ovis bacterin and unvaccinated rams. Inoculation of killed B. ovis did not produce significant lesions in either group whereas prior vaccination exacerbated epididymal lesions following inoculation of live B. ovis. Increased numbers of neutrophils, macrophages and lymphocytes were present in the interstitium and neutrophilic infiltration of the epididymal duct epithelium and intraepithelial cyst formation was more prominent. The inflammatory response surrounding extravasated spermatozoa was more severe in vaccinated rams but it was not determined if the response was directed at spermatozoa or intermixed brucellae, or both.