Facing the Human and Animal Brucellosis Conundrums: The Forgotten Lessons.

Brucellosis is a major zoonotic disease caused by Brucella species. Historically, the disease received over fifty names until it was recognized as a single entity, illustrating its protean manifestations and intricacies, traits that generated conundrums that have remained or re-emerged since they were first described. Here, we examine confusions concerning the clinical picture, serological diagnosis, and incidence of human brucellosis. We also discuss knowledge gaps and prevalent confusions about animal brucellosis, including brucellosis control strategies, the so-called confirmatory tests, and assumptions about the primary-binding assays and DNA detection methods. We describe how doubtfully characterized vaccines have failed to control brucellosis and emphasize how the requisites of controlled safety and protection experiments are generally overlooked. Finally, we briefly discuss the experience demonstrating that S19 remains the best cattle vaccine, while RB51 fails to validate its claimed properties (protection, differentiating infected and vaccinated animals (DIVA), and safety), offering a strong argument against its current widespread use. These conundrums show that knowledge dealing with brucellosis is lost, and previous experience is overlooked or misinterpreted, as illustrated in a significant number of misguided meta-analyses. In a global context of intensifying livestock breeding, such recurrent oversights threaten to increase the impact of brucellosis.

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.

Effect of vaccination on the apparent prevalence of bovine brucellosis in the state of Tocantins, Brazil / Efeito da vacinação na prevalência aparente da brucelose bovina no estado de Tocantins, Brasil

A cross-sectional study on the epidemiological situation of bovine brucellosis was carried out in the state of Tocantins to evaluate the effectiveness of its vaccination program. The state was divided into five regions, and a predetermined number of farms was randomly selected in each one. Females aged 24 months or older were randomly selected in each farm and diagnosed with brucellosis by serial serology (AAT and 2-ME). A total of 6,846 animals from 756 farms were examined. The prevalence of seropositive herds in the state was 6.42% [CI95%: 4.76-8.62], and the prevalence of seropositive animals was 2.21% [CI95%: 1.05-4.01]. The prevalence of seropositive herds was homogeneously distributed among regions. The 2002/2003 study estimated the prevalence of seropositive herds in the state to be 21.22% [CI95%: 19.33-23.11]. In conclusion, the vaccination program implemented in Tocantins, reaching vaccination coverage above 70% as of 2010, significantly reduced the prevalence of seropositive herds. Thus, continuing the vaccination program in the state is recommended, preferably increasing the quality of the processes involved, from commercialization to inoculation in animals, since immunization remains the most effective means to reduce the prevalence of brucellosis. In addition, animal replacement remains a major risk factor for bovine brucellosis in Tocantins since 20022003; therefore, the state must implement a strong health education program explaining to farmers the importance of testing animals for brucellosis before introducing them into their herds.(AU)

Foi realizado um estudo seccional sobre a situação epidemiológica da brucelose bovina no estado de Tocantins com o objetivo de avaliar a eficácia do programa de vacinação implementado. O estado foi dividido em cinco regiões e em cada uma delas foi aleatoriamente amostrado um número pré-estabelecido de propriedades. Dentro de cada propriedade, fêmeas com idade igual ou superior a 24 meses foram aleatoriamente selecionadas e submetidas à sorologia em série para o diagnóstico da brucelose (AAT e 2-Mercaptoetanol). Ao todo foram examinados 6.846 animais oriundos de 756 propriedades. A prevalência aparente de focos no estado foi de 6,42% [IC95%: 4,76-8,62] e a de animais 2,21% [IC95%: 1,05-4,01]. A prevalência aparente de focos apresentou-se homogeneamente distribuída entre as cinco regiões. Como o estudo realizado em 2002/2003 estimou a prevalência aparente de focos no estado em 21,22% [IC95%: 19,33-23,11], conclui-se que o programa de vacinação implementado pelo Tocantins, atingindo coberturas vacinais acima de 70% a partir de 2010, reduziu a prevalência de maneira importante. Assim, recomendase que o estado continue seu programa de vacinação, dando grande ênfase para a qualidade dos procedimentos, desde a comercialização do insumo até a inoculação nos animais, pois a imunização ainda é a maneira mais racional de se reduzir a prevalência da brucelose bovina no seu território. Adicionalmente, o estado deve implementar uma forte ação de educação sanitária para que os produtores passem a testar os animais para brucelose antes de introduzi-los nos seus rebanhos, pois verificou-se que a reposição de animais permanece associada à condição de foco da brucelose bovina desde 2002/2003.(AU)

Mathematical modeling of bovine brucellosis control using the RB51 vaccine / Modelagem matemática do controle da brucelose bovina com a utilização da vacina RB51

The Brazilian Ministry of Agriculture, Livestock and Food Supply established the National Program for Control and Eradication of Brucellosis and Tuberculosis (PNCEBT) in 2001. One of the main strategies adopted by the program is the mandatory vaccination of heifers between three and eight months of age with the B19 strain. In 2007, Brazil allowed the use of RB51 vaccine in bovine females over 8 months of age as an option for the producer, but kept the B19 strain as a mandatory vaccine. This decision is based on the assumption that combining the two vaccines allows to achieve significant vaccination coverage sooner and, consequently, accelerates the fall speed of prevalence. Thus, this study aimed to measure the impact of the combined use of these two vaccines in reducing the prevalence, using as a tool the mathematical modeling. It was concluded that vaccination by RB51, if adopted as a complement to vaccination by B19, mean decrease in the prevalence of bovine brucellosis in less time.(AU)

O Ministério da Agricultura Pecuária e Abastecimento brasileiro instituiu o Programa Nacional de Controle e Erradicação da Brucelose e da Tuberculose Animal em 2001. Uma das principais medidas adotadas pelo programa é a vacinação obrigatória de fêmeas entre três e oito meses de idade com a cepa B19. Em 2007, o Brasil permitiu a utilização da vacina RB51 em fêmeas bovinas com mais de 8 anos de idade como uma opção para o produtor, porém manteve como vacina obrigatória a B19 em bezerras de 3 a 8 meses de idade. Essa decisão parte do princípio de que combinando as duas vacinas abrevia-se o tempo para a obtenção de coberturas vacinais expressivas na população de fêmeas em idade de procriar e, consequentemente, acelera-se a velocidade de queda da prevalência. Assim, o presente estudo objetivou medir o impacto do uso combinado dessas duas vacinas na redução da prevalência, utilizando como instrumento a modelagem matemática. Concluiu-se que a vacinação pela RB51, se adotada de forma complementar à vacinação pela B19, significaria queda da prevalência da brucelose bovina em menor intervalo de tempo.(AU)

Mathematical modeling of bovine brucellosis control using the RB51 vaccine / Modelagem matemática do controle da brucelose bovina com a utilização da vacina RB51

The Brazilian Ministry of Agriculture, Livestock and Food Supply established the National Program for Control and Eradication of Brucellosis and Tuberculosis (PNCEBT) in 2001. One of the main strategies adopted by the program is the mandatory vaccination of heifers between three and eight months of age with the B19 strain. In 2007, Brazil allowed the use of RB51 vaccine in bovine females over 8 months of age as an option for the producer, but kept the B19 strain as a mandatory vaccine. This decision is based on the assumption that combining the two vaccines allows to achieve significant vaccination coverage sooner and, consequently, accelerates the fall speed of prevalence. Thus, this study aimed to measure the impact of the combined use of these two vaccines in reducing the prevalence, using as a tool the mathematical modeling. It was concluded that vaccination by RB51, if adopted as a complement to vaccination by B19, mean decrease in the prevalence of bovine brucellosis in less time.

O Ministério da Agricultura Pecuária e Abastecimento brasileiro instituiu o Programa Nacional de Controle e Erradicação da Brucelose e da Tuberculose Animal em 2001. Uma das principais medidas adotadas pelo programa é a vacinação obrigatória de fêmeas entre três e oito meses de idade com a cepa B19. Em 2007, o Brasil permitiu a utilização da vacina RB51 em fêmeas bovinas com mais de 8 anos de idade como uma opção para o produtor, porém manteve como vacina obrigatória a B19 em bezerras de 3 a 8 meses de idade. Essa decisão parte do princípio de que combinando as duas vacinas abrevia-se o tempo para a obtenção de coberturas vacinais expressivas na população de fêmeas em idade de procriar e, consequentemente, acelera-se a velocidade de queda da prevalência. Assim, o presente estudo objetivou medir o impacto do uso combinado dessas duas vacinas na redução da prevalência, utilizando como instrumento a modelagem matemática. Concluiu-se que a vacinação pela RB51, se adotada de forma complementar à vacinação pela B19, significaria queda da prevalência da brucelose bovina em menor intervalo de tempo.

Immune response triggered by Brucella abortus following infection or vaccination.

Brucella abortus live vaccines have been used successfully to control bovine brucellosis worldwide for decades. However, due to some limitations of these live vaccines, efforts are being made for the development of new safer and more effective vaccines that could also be used in other susceptible species. In this context, understanding the protective immune responses triggered by B. abortus is critical for the development of new vaccines. Such understandings will enhance our knowledge of the host/pathogen interactions and enable to develop methods to evaluate potential vaccines and innovative treatments for animals or humans. At present, almost all the knowledge regarding B. abortus specific immunological responses comes from studies in mice. Active participation of macrophages, dendritic cells, IFN-γ producing CD4(+) T-cells and cytotoxic CD8(+) T-cells are vital to overcome the infection. In this review, we discuss the characteristics of the immune responses triggered by vaccination versus infection by B. abortus, in different hosts.

Brucella abortus S19 and RB51 vaccine immunogenicity test: Evaluation of three mice (BALB/c, Swiss and CD-1) and two challenge strains (544 and 2308).

The aim of the present study was to evaluate the use of different mouse strains (BALB/c, Swiss and CD-1) and different challenge strains (Brucella abortus 544 and 2308) in the study of B. abortus vaccine (S19 and RB51) immunogenicity test in the murine model. No significant difference in B. abortus vaccine potency assay was found with the use of B. abortus 544 or B. abortus 2308 as challenge strain. Results of variance analysis showed an interaction between treatment and mouse strain; therefore these parameters could not be compared separately. When CD-1 groups were compared, those vaccinated showed significantly lower counts than non-vaccinated ones (P<0.05), independently of the vaccine received (S19 or RB51). Similar results were observed on BALB/c groups. However, in Swiss mouse groups, S19 was more protective than RB51 (P<0.05), which showed protection when compared to the non-vaccinated group (P<0.05). In summary, data from the present study showed that CD-1, BALB/c and Swiss mice strains, as well as both challenge strains, B. abortus strains 544 and 2308, can be used in immunogenicity tests of S19 and RB51 vaccines.

T lymphocytes subsets and cytokine pattern induced by vaccination against bovine brucellosis employing S19 calfhood vaccination and adult RB51 revaccination.

The aims of this study were to address the protective immune response induced by S19 vaccination (n=10) and RB51 revaccination, in pregnant (n=9) and non-pregnant (n=10) S19 calfhood-vaccinated cattle as follows: evaluate the in vitro CD4(+) and CD8(+) T-lymphocytes specific proliferation, and in vitro expression of IFN-γ by CD4(+) and CD8(+) T-cells and IL-4 by CD4(+), CD8(+) and CD21(+) lymphocytes subset. Upon in vitro stimulation with γ-irradiated Brucella abortus 2308, blood mononuclear cells from S19 vaccinated and RB51 revaccinated cows exhibited significantly higher proliferation of CD4(+) and CD8(+) T-lymphocytes and CD4(+)IFN-γ(+) T-cells compared to non-vaccinated animals. RB51 revaccination, regardless of the pregnancy status, did not enhance the proliferation of CD4(+) or CD8(+) T-cells nor IFN-γ or IL-4 production. Data from the present study suggest that cattle's cellular immune response induced after brucellosis vaccination and revaccination is due to CD4(+) and CD8(+) T-lymphocytes, being CD4(+) T-cells the main source of IFN-γ.

Estudo das vacinas contra brucelose bovina: revisão / Study of vaccines against bovine brucellosis: review

A brucelose bovina é uma das zoonoses mais difundidas no mundo e provoca prejuízos econômicos decorrentes de problemas reprodutivos. Diversos investimentos e esforços têm sido feitos para preveni-la em várias partes do mundo. Em destaque no cenário da pesquisa mundial sobre brucelose, está a busca por uma vacina que seja inócua aos humanos e animais, garanta uma boa imunidade e, principalmente não interfira nos testes de diagnóstico. Desde a criação da primeira vacina contra brucelose, diversas outras foram desenvolvidas, no entanto, poucas foram aceitas e usadas em larga escala. As perspectivas para o futuro das vacinas contra brucelose, demostram ser tão variadas quanto o seu passado. Nesse contexto, o objetivo desse trabalho é fazer uma reflexão sobre as vacinas contra brucelose considerando as principais vacinas desenvolvidas no passado, as utilizadas na atualidade e as perspectivas para a vacinologia contra brucelose.

Bovine brucellosis is one of the most widespread zoonoses in the world and it causes economical losses due to reproductive problems. Several investiments and efforts have been done to prevent bovine brucellosis in many parts of the world. The search for a vaccine which is harmless to both human and animals, ensure good immunity and , mainly, does not interfere in diagnostic tests is featured on the scene of worldwide research on brucellosis. Since the creation of the first vaccine against brucellosis, several others have been created. However, few have been accepted and widely used. The perspectives for the future of vaccines against brucellosis can be seen as varied as they were in the past. In this context, the aim of this study is to reflect on brucellosis vaccines considering the main vaccines developed in the past, the ones in use today and the perspectives for the future of vaccinology againt brucellosis.

Avaliação genética das vacinas contra a brucelose bovina comercializadas no Brasil / Genetic evaluation of vaccine against bovine brucellosis marketed in Brazil

A prevenção contra infecções causadas por Brucella abortus em bovinos é realizada por meio da administração das amostras vacinais B19 e RB51. Existem relatos de que estas vacinas podem causar aborto em fêmeas vacinadas. Portanto, toda a ocorrência de aborto em animais vacinados merece um estudo aprofundado sobre a causa. No Brasil, não há registro sobre a origem das amostras B19 e RB51 utilizadas na produção das vacinas comerciais. Assim, um estudo para verificar possíveis mutações em relação às amostras referência USDA B19 e USDA RB51 de B. abortus se faz necessário, devido às amostras vacinais poderem reverter a sua virulência. Objetivou-se com este estudo caracterizar genotipicamente as amostras vacinais B19 e RB51 comercializadas no Brasil. A metodologia utilizada foi a genotipagem de genes marcadores destas amostras vacinais, por meio da amplificação pela reação em cadeia da polimerase. Os resultados obtidos permitiram a identificação do genótipo das vacinas comerciais B19 e RB51 disponíveis e utilizadas em bovinos no Brasil. A ausência de mutações nas vacinas testadas corrobora com a qualidade genética das mesmas, quanto à estabilidade dos genes analisados.(AU)

Vaccine strains B19 and RB51 are administered to cattle for prevention against infection by Brucella abortus. However, there are reports that these vaccines can cause miscarriages. Thus, every miscarriage among vaccinated animals should be thoroughly studied to determine the cause. In Brazil, there are no records on the origin of B19 and RB51 samples used in the preparation of commercial vaccines. Therefore, a study is needed to determine possible mutations in relation to the USDA reference samples of B. abortus due to the fact that vaccine samples could revert to the virulence of the disease. The aim of the present study was to perform a genotype analysis of vaccine strains B19 and RB51 used in Brazil. The methodology was based on the genotyping of marker genes of these vaccine strains by amplification using polymerase chain reaction. The results allowed the identification of the genotype of the B19 and RB51 commercial vaccine available for use on cattle in Brazil. The absence of mutations in the samples tested confirmed the genetic quality of the vaccines and stability of genes analyzed.(AU)

Avaliação genética das vacinas contra a brucelose bovina comercializadas no Brasil / Genetic evaluation of vaccine against bovine brucellosis marketed in Brazil

A prevenção contra infecções causadas por Brucella abortus em bovinos é realizada por meio da administração das amostras vacinais B19 e RB51. Existem relatos de que estas vacinas podem causar aborto em fêmeas vacinadas. Portanto, toda a ocorrência de aborto em animais vacinados merece um estudo aprofundado sobre a causa. No Brasil, não há registro sobre a origem das amostras B19 e RB51 utilizadas na produção das vacinas comerciais. Assim, um estudo para verificar possíveis mutações em relação às amostras referência USDA B19 e USDA RB51 de B. abortus se faz necessário, devido às amostras vacinais poderem reverter a sua virulência. Objetivou-se com este estudo caracterizar genotipicamente as amostras vacinais B19 e RB51 comercializadas no Brasil. A metodologia utilizada foi a genotipagem de genes marcadores destas amostras vacinais, por meio da amplificação pela reação em cadeia da polimerase. Os resultados obtidos permitiram a identificação do genótipo das vacinas comerciais B19 e RB51 disponíveis e utilizadas em bovinos no Brasil. A ausência de mutações nas vacinas testadas corrobora com a qualidade genética das mesmas, quanto à estabilidade dos genes analisados.

Vaccine strains B19 and RB51 are administered to cattle for prevention against infection by Brucella abortus. However, there are reports that these vaccines can cause miscarriages. Thus, every miscarriage among vaccinated animals should be thoroughly studied to determine the cause. In Brazil, there are no records on the origin of B19 and RB51 samples used in the preparation of commercial vaccines. Therefore, a study is needed to determine possible mutations in relation to the USDA reference samples of B. abortus due to the fact that vaccine samples could revert to the virulence of the disease. The aim of the present study was to perform a genotype analysis of vaccine strains B19 and RB51 used in Brazil. The methodology was based on the genotyping of marker genes of these vaccine strains by amplification using polymerase chain reaction. The results allowed the identification of the genotype of the B19 and RB51 commercial vaccine available for use on cattle in Brazil. The absence of mutations in the samples tested confirmed the genetic quality of the vaccines and stability of genes analyzed.