Levantamento sorológico de brucelose em bovinos monitorados no estado da Bahia / Serological survey of brucellosis in cattle monitored in the estate of Bahia

A brucelose é uma doença bacteriana de grande importância para a economia pecuária e para a saúde pública por se tratar de uma zoonose. É uma doença infecto-contagiosa que tem com agente etiológico bactérias do gênero Brucella. Em bovinos, as espécies do gênero é a Brucella abortus, que são cocobacilos gram negativo, intracelulares facultativos, imóveis e não esporulado. A infecção apresenta evolução crônica e acomete animais de todas as idades, sendo mais frequente em indivíduos sexualmente maduros. O objetivo desse trabalho é investigar, por meio da sorologia para brucelose bovina, utilizando a técnica do ELISA indireto, amostras de animais reagentes abatidos em frigoríficos inspecionados no estado da Bahia. Foram utilizados 666 animais, selecionados aleatoriamente no momento do abate. O sangue foi coletado com finalidade de obtenção de soro, todas as amostras foram submetidas à prova de triagem do Antígeno Acidificado Tamponado (AAT), prova do 2mercaptoetanol (2-ME) e ELISA Indireto. Das amostras reagentes no teste do AAT, obteve-se uma prevalência estimada em 1,2%. A prevalência no teste do ELISA foi de 13,21% (n=86). Esse resultado sugere a ocorrência de falsos negativos quando se utiliza a prova do antígeno acidificado tamponado.

Brucellosis is a bacterial disease of great importance to the livestock economy and to public health because it is a zoonosis. It is an infectious disease that has etiologic agent with bacteria of the genus Brucella. In cattle, the species of the genus Brucella is Brucella abortus that are gram negative, facultative intracellular, real estate and not sporulated. The infection presents chronic and affects animals of all ages, being more frequent in sexually mature individuals. This study aimed to investigate through serology for brucellosis, using the technique of indirect ELISA, samples from positive animals slaughtered in slaughterhouses inspected in the state of Bahia. A total of 666 animals were used, randomly selected at the time of slaughter. Blood was collected in order to obtain serum, all samples were subjected to a screening test Antigen Buffered Acidified (AAT), proof of 2-mercaptoethanol (2-ME) and Indirect ELISA. Of reagents in the test samples of AAT obtained an estimated prevalence of 1.2%. The prevalence in the ELISA test was 13.21% (n = 86). This result suggests the occurrence of false negatives when using the buffered acidified antigen test.

Inquérito sorológico de brucelose em machos e fêmeas bovinas em fazendas do Estado de Mato Grosso do Sul / Serological survey of brucellosis in males and females of cattle in the State of Mato Grosso do Sul

Objetivou-se avaliar a ocorrência de animais soro reagentes à brucelose bovina em fazendas localizadas no Estado de Mato Grosso do Sul, por meio de exame sorológico utilizando o Antígeno Acidificado Tamponado (AAT) e discutir as possíveis diferenças entre as soroprevalências de fêmeas e machos. Foram avaliados, a partir do teste de triagem com Antígeno Acidificado Tamponado (AAT), 724 bovinos da raça Nelore, sendo 274 machos e 450 fêmeas, provenientes de oito propriedades com histórico de problemas reprodutivos. O teste foi procedido conforme o protocolo determinado pelo Ministério da Agricultura, Pecuária e Abastecimento (MAPA). Os resultados demonstraram baixa soroprevalência da doença nos bovinos testados, sendo detectada prevalência para a doença de 1,10% nos machos e 2,88% nas fêmeas.Quando se considera o touro isoladamente nos rebanhos, pode-se perceber que a fertilidade é muito mais importante nos machos do que nas fêmeas individualmente, uma vez que os touros podem se acasalar com um número muito maior de fêmeas, seja na monta natural ou na inseminação artificial, demonstrando a importância do inquérito epidemiológico na população geral, principalmente nos machos. A maior frequência da doença foi encontrada nas fêmeas podendo estar relacionada à infecção por Brucella spp. no ambiente decorrente de parto ou aborto tornando as fêmeas transmissoras permanentes da doença.

The objective of this study was to evaluate the occurrence of seroreactive animals to bovine brucellosis in farms located in the State of Mato Grosso do Sul, by means of a serological examination using the Acidified Buffered Antigen (AAT) and to discuss the possible differences between the seroprevalence of females and males. A total of 724 Nellore cattle, 274 males and 450 females, from eight farms with a history of reproductive problems, were evaluated using the screening test with Acidified Buffered Antigen (AAT). The test was carried out according to the protocol determined by the Ministry of Agriculture, Livestock and Food Supply (MAPA).The results showed a low seroprevalence of the disease in the tested cattle, with a prevalence of 1.10% in males and 2.88% in females.When considered the bull alone in herds, it can be shown that fertility is much more important in males than in females individually, since bulls can mate with a much larger number of females, either in natural mating or in artificial insemination, demonstrating the importance of epidemiological survey in the general population, especially in males. The highest frequency of the disease was found in females and may be related to infection by Brucella spp. in the environment from childbirth or abortion making females permanent transmitters of the disease.

Comparative evaluation of fluorescence polarization assay and competitive ELISA for the diagnosis of bovine brucellosis vis-a-vis sero-monitoring.

Brucellosis is an important zoonosis that constitutes a serious public health hazard which is caused by a bacterium belonging to the genus Brucella. In the present study, two highly specific serological tests for brucellosis diagnosis, fluorescence polarization assay (FPA) and competitive ELISA (cELISA) were standardized in the laboratory, evaluated and compared with rose bengal plate test (RBPT), indirect ELISA (iELISA) and commercial cELISA kit. For test evaluation, 1386 serum samples [apparently healthy animals (n = 260), samples from Brucella infected farms (n = 701) and B. abortus S19 vaccinated animals (n = 425)] were analyzed to assess suitable diagnostic test in B. abortus S19 post vaccinated bovine population. In apparently healthy brucellosis free farms, RBPT, iELISA, in-house FPA and cELISA were found to be highly specific than commercial cELISA. Commercial cELISA kit was comparatively more sensitive than other serological tests in samples collected from infected farms. The FPA showed sensitivity nearly equal to RBPT and in-house cELISA showed greater sensitivity than RBPT in infected farms. In animals with persistent vaccinal antibodies, only in-house FPA and cELISA recorded higher specificity of 87.64 and 90.27%, respectively. The other tests, RBPT and iELISA displayed similar reactivity with vaccine antibodies to that of infection antibodies whereas commercial cELISA kit showed an intermediate specificity of 47.69%. With these findings, RBPT, iELISA and cELISA are suggested for screening infected herds, and in-house developed FPA and cELISA tests with a proven specificity can be used for confirmatory diagnosis of brucellosis in B. abortus S19 post vaccinated animal populations.

Triple Immunochromatographic System for Simultaneous Serodiagnosis of Bovine Brucellosis, Tuberculosis, and Leukemia.

An immunochromatographic test system has been developed for the simultaneous rapid multiplex serodiagnostics of bovine brucellosis, tuberculosis, and leukemia. The test system is based on the use of a conjugate of gold nanoparticles with the chimeric protein Cysteine-A/G and three analytical zones with immobilized pathogen antigens: Brucella abortus lipolysaccharide, recombinant proteins MPB64 and MPB83-MPB63 of Mycobacterium bovis, and recombinant protein p24 of the bovine leukemia virus. Prototypes of the test system were tested on 98 samples of sera from healthy and infected animals. The diagnostic sensitivity of the developed test system was 92% for brucellosis, 92% for tuberculosis, and 96% for leukemia. False positive test results were not observed.

Avaliação das técnicas de imunodifusão em gel de ágar, ensaio imunoenzimático indireto e reação em cadeia da polimerase no diagnóstico da brucelose ovina / Evaluation of technical, immunodiffusion agar gel, enzyme-linked immunosorbent assay and polymerase chain reaction in the diagnosis of ovine brucellosis

A brucelose na espécie ovina tem recebido destaque, uma vez que se trata de uma enfermidade que acomete o sistema reprodutivo dos animais, provocando sério comprometimento no setor produtivo. Dessa forma, objetivou-se a avaliação de três métodos para o diagnóstico da brucelose ovina: o ensaio imunoenzimático indireto (ELISAi), a técnica imunodifusão em gel de ágar (IDGA) e a reação em cadeia da polimerase (PCR). Para tanto, utilizaram-se 211 amostras de sangue de ovinos oriundos de propriedades de nove municípios da microrregião homogênea de Teresina, Piauí. As 211 amostras de sangue foram submetidas aos testes sorológicos e à PCR, visando detectar anticorpos anti-B. ovis e DNA de Brucella ovis, respectivamente. Foram obtidos resultados positivos nos testes sorológicos, sendo 36 (17,06%) positivos no teste IDGA e sete (3,31%) positivos no teste ELISAi, contudo não houve resultados positivos na técnica de PCR. Dos métodos de diagnóstico utilizados neste estudo, o teste IDGA foi o que apresentou melhor desempenho na detecção de animais reagentes, quando comparado ao teste ELISAi e à PCR em amostras de sangue, e o percentual de animais soropositivos sugere uma ampla distribuição de ovinos infectados por Brucella ovis na região em estudo, o que pode causar prejuízos aos produtores.(AU)

Brucellosis in sheep has received a major focus, since it is a disease that affects the reproductive system of animals, causing serious impairment in the productive sector. Thus, three methods for the diagnosis of ovine brucellosis were evaluated as goal, the indirect Linked Immunosorbent Assay (ELISAi) test, the Immunodiffusion Agar Gel (AGID) technique and the Polymerase Chain Reaction (PCR). Therefore, we used 211 sheep blood samples from properties of nine municipalities of the homogeneous micro-region of Teresina, Piaui. The 211 blood samples were subjected to serologic testing and PCR to detect anti-B. ovis antibodies, and Brucella ovis DNA, respectively. Positive results in serological tests were obtained, 36 (17%) positive in the AGID test and seven (3.3%) positive to the ELISAi test, however, there were no positive results in the PCR technique. Of the diagnostic methods used in this study, the AGID test was the one that presented the best performance in the detection of reactive animals, when compared to ELISAi and PCR in blood samples and, the percentage of seropositive animals suggests a wide distribution of Brucella ovis infected sheep in the study region and could cause loss to producers.(AU)

Occurrence of oxidative stress in dairy cows seropositives for Brucella abortus.

Bovine brucellosis is an important zoonotic disease caused by the bacterium Brucella abortus that leads to economic losses due to animal discard and commercial restrictions. Since positive animals for brucellosis are culled, little is known about the pathogenesis of this disease. Therefore, the aims of this study were to evaluate possible changes in the activity of deaminase adenosine (ADA) and the oxidative stress in cows seropositives for brucellosis (Experiment I), and to evaluate the seroprevalence of B. abortus in dairy cows from the Western state of Santa Catarina, Southern Brazil (Experiment II). The Experiment I evaluated 20 pregnant cows: ten seropositives for B. abortus and ten seronegatives that were used as controls. The ADA activity and markers of oxidative stress (TBARS, catalase (CAT) and superoxide dismutase (SOD)) were evaluated in these animals. A reduction in the activity of ADA and catalase enzymes in seropositive animals was observed (p < 0.001). Conversely, there was an increase in TBARS levels and superoxide dismutase activity in cows infected by B. abortus (p < 0.001). The presence of oxidative stress and a reduction of ADA might be related to the modulation of the inflammatory response. The experiment II was performed due to a high number of herds with restrictions imposed by cases of brucellosis in the state of Santa Catarina in the last two years, and thus, the seroprevalence for B. abortus was evaluated in 1242 serum samples of cows of 69 herds. The serodiagnosis was performed using two tests: buffered acidified antigen and 2-mercaptoethanol. However, none of the serum samples were positive for B. abortus. Although we did not find seropositive animals for brucellosis in our study, the disease still requires continued surveillance, due to its economic impact, and to the oxidative stress caused by it, which may have contributed to cases of abortion in three seropositive cows (Experiment I) in the final third of the gestation.

Detecção de Brucella abortus em tecidos bovinos utilizando ensaios de PCR e qPCR / Detection of Brucella abortus in bovine tissue using PCR and qPCR

Objetivou-se no presente estudo avaliar as técnicas reação em cadeia da polimerase (PCR) e PCR em Tempo Real (qPCR) para detectar Brucella abortus, a partir de tecidos bovinos com lesões sugestivas de brucelose. Para isto, 21 fragmentos de tecidos bovinos coletados em abatedouros de Mato Grosso do Sul foram processados e submetidos ao cultivo microbiológico e extração do DNA genômico para realização das reações de PCR e qPCR. No cultivo microbiológico, oito amostras apresentaram crescimento bacteriano e cinco foram confirmadas como B. abortus por PCR. Diretamente das amostras de tecido, DNA do gênero Brucella (oligonucleotídeos IS711) foi detectado em 13 (61,9 por cento) amostras de tecido e 17 (81 por cento) amostras de homogeneizado. Já com os oligonucleotídeos espécie-específicos BruAb2_0168F e BruAb2_0168R, 14 (66 por cento) amostras de tecido e 18 (85,7 por cento) amostras de homogeneizado foram amplificadas. Seis amostras positivas na PCR espécie-específica foram sequenciadas e o best hit na análise BLASTn foi B. abortus. Na qPCR, 21 (100 por cento) amostras de tecidos e 19 (90,5 por cento) amostras de homogeneizado foram positivas para B. abortus. Dez amostras de DNA de sangue bovino de rebanho certificado livre foram utilizadas como controle negativo nas análises de PCR e qPCR utilizando-se os oligonucleotídeos BruAb2_0168F e BruAb2_0168R. Na PCR nenhuma amostra amplificou, enquanto que na qPCR 2 (20 por cento) amplificaram. Conclui-se que as duas técnicas detectam a presença de B. abortus diretamente de tecidos e homogeneizados, porém a qPCR apresentou maior sensibilidade. Os resultados obtidos indicam que a qPCR pode representar uma alternativa rápida e precisa para a detecção de B. abortus diretamente de tecidos, e ser utilizada em programas de vigilância sanitária, por apresentar sensibilidade e especificidade satisfatórias.

The aim of the study was to evaluate the technical polymerase chain reaction (PCR) and Real-Time PCR (qPCR) to detect Brucella abortus from bovine tissues with suggestive lesions of brucellosis. For this, 21 fragments of bovine tissues collected at abattoirs of Mato Grosso do Sul were processed and subjected to microbiological culture and extraction of genomic DNA to perform the PCR reactions and qPCR. Eight samples of microbiological culture showed bacterial growth and five samples were confirmed as B. abortus by PCR. DNA of Brucella (IS711 primers) was detected in 13 (61.9 percent) directly from tissue samples and 17 (81 percent) from tissue homogenate samples. With the species-specific set of primers BruAb2_0168F and BruAb2_0168R, 14 (66 percent) tissue samples and 18 (85.7 percent) tissue homogenate samples were positive. Six positive samples in the species-specific PCR were sequenced and the best hit in the BLASTn analysis was B. abortus. By qPCR, 21 (100 percent) tissue samples and 19 (90.5 percent) tissue homogenate samples were positive for B. abortus. Ten samples of DNA from bovine blood from an accredited-free herd were used as negative control in PCR and qPCR analysis using the primers BruAb2_0168F and BruAb2_0168R, and no one amplified by PCR, whereas two samples were amplified by qPCR (20 percent). In conclusion, both techniques detect the presence of B. abortus directly from tissues and homogenized, but the qPCR showed high sensitivity. The results indicate that qPCR can represent an alternative tool for faster and more accurate detection of B. abortus directly from tissues, and use in health surveillance programs by presenting satisfactory sensitivity and specificity.

Confirmação de infecção por Brucella abortus em um rebanho bovino certificado livre em Minas Gerais: relato de caso / Brucella abortus infection in an officially brucellosis-free bovine herd in Minas Gerais state - Brazil: a case report

Relata-se a ocorrência de um surto de brucelose em um rebanho de aproximadamente 1000 animais, livre da doença há 18 anos, certificado pelo Ministério da Agricultura, Pecuária e Abastecimento desde 2006. Dois animais reagiram aos testes sorológicos de diagnóstico por ocasião dos procedimentos de recertificação em 2008. Após o sacrifício deles, Brucella abortus, biovariedade 1, amostra não vacinal, foi isolada e identificada por meio de provas bioquímicas e de biologia molecular (PCR AMOS). A origem do agente no rebanho é de difícil determinação. No entanto, a adoção de procedimentos preconizados pelo Programa Nacional de Controle e Erradicação da Brucelose permitiu evitar a disseminação da enfermidade. Ocorrências como essas, em que rebanhos livres foram infectados após anos sem a ocorrência de brucelose, nunca haviam sido relatadas no Brasil.

A Brucellosis outbreak is reported in a bovine herd free from the disease for over 18 years, officially free since 2006. One heifer and one cow tested positive in serological tests for the 2008 annual recertification in a herd of almost 1000 animals. Isolation and identification by biochemical tests and molecular biology (AMOS PCR) confirmed the infection by a Brucella abortus biovar 1 field strain. It wasn't possible to find the source of the infection. However, adoption of standard procedures prescribed by the Brazilian National Brucellosis and Tuberculosis Control and Eradication Program hindered the spread of the disease. This is the first report of a Brucella infection in an officially Brucellosis-free bovine herd in Brazil.

Intercorrência entre leucose enzoótica e brucelose em búfalos (Bubalus bubalis) em sistema de produção extensivo / Intercurrence of enzootic leucosis and brucellosis in buffaloes (Bubalus bubalis) in extensive production system

Objetivou-se com este estudo avaliar a intercorrência entre leucose enzoótica e brucelose em búfalos (Bubalus bubalis) em sistema de produção extensivo, pelo estabelecimento da prevalência de bubalinos reagentes às provas diagnósticas específicas. Foram analisados sorologicamente 232 animais, pela técnica de imunodifusão dupla em gel de agarose para diagnóstico de leucose enzoótica e o teste do antígeno acidificado tamponado, seguida pelos testes confirmatórios 2-mercaptoetanol e soroaglutinação lenta em tubos para diagnóstico de brucelose. As prevalências de búfalos que apresentaram positividade para leucose e brucelose foram 4,21% (10/232) e 5,18% (12/232), respectivamente. Os resultados obtidos nesta pesquisa permitiram concluir que as infecções estudadas encontram-se presentes na população avaliada, entretanto, com baixa prevalência e sem dependência de ocorrência entre ambas.

This study was carried out to evaluate the intercurrence of brucellosis and enzootic leucosis in buffaloes in extensive production system by establishing the prevalence of reagent buffaloes for specific diagnostic tests. We analyzed 232 animals serologically by the technique of double immunodiffusion in agarose gel (AGID) for diagnostic of leucosis and the tampon acidified antigen (TAA) test, followed by confirmatory tests of 2-mercaptoethanol (2-ME) and slow serum agglutination (SSA) in tubes for diagnostic of brucellosis. The prevalence of buffaloes that were positive for leucosis and brucellosis tests were 4.21% (10/232) and 5.18%(12/232) respectively. The results of this investigation indicated that the diseases occur in the population studied, however, with low prevalence and without depending on the occurrence of both.

Avaliação de testes sorológicos de triagem para diagnóstico da brucelose bubalina / Evaluation of screening serological tests for brucellosis diagnosis in buffaloes

The study compared the performance of three screening serological tests: buffered plate antigen (BPA), Rose-Bengal produced with 1119-3 Brucella abortus strain (RB1119-3), and Rose-Bengal produced with 99 Brucella abortus strain (RB99). Sera from 696 adult female animals were submitted to BPA, RB1119-3, RB99, 2-mercaptoethanol test (ME), and complement fixation test (FC). The gold standard was the combination of CF and ME. The Kappa values for BPA, RB99, and RB1119-3 were 0.82, 0.74, and 0.70, respectively. The relative sensitivity and specificity for the same tests were 0.98 and 0.96, 0.92 and 0.94, and 0.95 and 0.92, respectively. These results indicate that BPA is a better screening test than RB for buffalo, regardless of the B. abortus strain in RB.

Estudo comparativo entre os testes adotados pelo PNCEBT para o diagnóstico sorológico da brucelose em bovinos / Comparative study of serological tests for bovine brucellosis diagnosis adopted by the Brazilian Program for animal brucellosis and tuberculosis control and eradication

Avaliaram-se comparativamente as provas do antígeno acidificado tamponado (AAT), a combinação das provas de soroaglutinação lenta e 2-mercaptoetanol (2-ME) e a reação de fixação de complemento (RFC), provas preconizadas pelo Programa Nacional de Controle e Erradicação de Brucelose Tuberculose (PNCEBT). Para tanto, foram alisadas 1.061 amostras de soros bovinos. Os dados foram analisados pelo indicador kappa, adotando-se como ponto de corte o título 25 no 2-ME e 4 na RFC. Constatou-se sensibilidade relativa de 99,6%, 98,8% e 91,1%, respectivamente, para o AAT, a 2-ME e a RFC, e especificidade relativa de 83,9%, 96,2% e 100,0%. A comparação entre os testes adotados pelo programa apontou concordância boa entre o teste de triagem (AAT) e os testes confirmatórios (kappa: 2-ME = 0,80; e RFC = 0,73) e concordância ótima quando os testes confirmatórios foram comparados entre si (kappa = 0,86). No entanto foram encontrados soros com título elevado em um dos testes confirmatórios e resultado negativo no outro, o que reforça a ideia de que o diagnóstico sorológico da brucelose é mais confiável quando obtido por meio dos resultados de vários testes.

Serum samples from 1,061 bovine were analyzed by serological diagnostic techniques adopted by the Brazilian Program for Animal Brucellosis and Tuberculosis Control and Eradication in order to compare, as a screening test, the rose Bengal plate test (RBPT), and as confirmatory tests, the 2-mercaptoethanol plus standard tube agglutination test (2-ME), and the complement fixation test (CFT). Relative sensitivity of 99.6%, 98.8% and 91.1% were observed, respectively, for RBPT, 2-ME and CFT, and relative specificity of 83.9%, 96.2% and 100.0%. The agreement between tests was analyzed by the kappa statistic (2-ME cut-off = titer 25, and CFT cutoff = titer 4). RBPT showed a substantial agreement (kappa: 2-ME = 0.80; and RFC = 0.73) with the confirmatory tests, and excellent agreement between the confirmatory tests (kappa = 0.86) was observed. However sera with a negative result in one of the confirmatory tests and a high titer in the other were observed, reinforcing that the serological diagnosis of bovine brucellosis is more reliable when based on the results of several tests.

Alterações microscópicas em linfonodos de bovinos sorologicamente positivos para brucelose / Microscopic changes in cattle lymph odes serologically positive for brucellosis / Cambios microscópicos en ganglios linfáticos de bovinos serológicamente positivos para brucelosis

A Brucelose é uma doença infecto-contagiosa provocada por bactérias intracelulares facultativas do gênero Brucella spp., zoonose de distribuição mundial. As principais manifestações clínicas da doença provocam redução na produção dos animais, e suas implicações econômicas causam barreiras na comercialização. Objetivou-se pesquisar alterações microscópicas nos linfonodos de bovinos sorologicamente positivos para brucelose, relacionando à reação inflamatória ganglionar como achado indicativo para o diagnóstico histopatológico da doença. Foram colhidas amostras de linfonodos cervicais superiores em vinte bovinos fêmeas, 10 da raça Gir e 10 da raça Nelore, enviadas ao Laboratório de Patologia Animal da Universidade de Uberaba. Ao realizar o exame macroscópico dos linfonodos, verificou-se que todos se apresentavam reacionais, mesmo que discretamente. Na leitura das lâminas histopatológicas, diagnosticou-se em 100% deles linfadenite crônica, compatível com o quadro de brucelose. O impacto econômico e zoonótico da doença justificam a necessidade definitiva do estabelecimento de programas de controle e erradicação, sendo que o diagnóstico representa grande relevância neste processo, e o exame histopatológico perfaz importante auxílio na confirmação do diagnóstico sorológico.

Brucellosis is an infectious and contagious disease caused by intracellular bacteria of the genus Brucella spp., zoonosis of worldwide distribution. The main clinical manifestations of this disease cause reduction in the animals’ production, and their economic implications provoke barriers in trade. The aim of this study is to find microscopic changes in cattle lymph nodes from animals serologically positive for brucellosis, associating the inflammatory reaction nodes as indicative findings for the histopathologic diagnosis of the disease. Samples were collected from upper cervical lymph nodes in twenty cows, ten from Gir breed, and ten, Nellore, sent to Laboratory of Animal Pathology at the University of Uberaba. In macroscopic examination of the lymph nodes, it was found that all had been reactive even if discretely. In reading the histopathological slides, it was diagnosed in 100% of cases, the same chronic lymphadenitis, compatible with the framework of brucellosis. The economic impact and the zoonotic characteristic of the disease justify the definitive need to establish programs of control and eradication, as the diagnosis is very relevant in this process, histopathological examination turns an important aid to confirm serological diagnosis.

La brucelosis es una enfermedad infectocontagiosa causada por bacterias intracelulares facultativas del género Brucella spp, zoonosis de distribución mundial. Las principales manifestaciones clínicas de la enfermedad provocan reducción en la producción de los animales, y sus implicaciones económicas causan obstáculos en la comercialización. En esta investigación se buscó encontrar cambios microscópicos en los ganglios linfáticos de ganados sorológicamente positivos para brucelosis, listando la reacción inflamatoria de los ganglios linfáticos como indicativo para el diagnóstico histopatológico de la enfermedad. Las muestras fueron recolectadas en los ganglios linfáticos superiores del cuello de veinte vacas, diez de la raza Gir, y diez de la raza Nelore, y enviadas al Laboratorio de Patología Animal de la Universidad de Uberaba. Al realizar el examen macroscópico de los ganglios linfáticos, se comprobó que todos se presentaban reactivos, aunque discretamente. En la lectura de las diapositivas histopatológicas, se diagnosticó en 100% de ellos linfadenitis crónica, compatible con el cuadro de brucelosis. El impacto económico y zoonótico de la enfermedad justifican la necesidad definitiva de establecer programas de control y erradicación, siendo que el diagnóstico representa gran relevancia en este proceso, y el examen histopatológico se convierte en importante ayuda en la confirmación del diagnóstico serológico.

Monitoreo epidemiológico para Brucella abortus en fincas doble propósito del Municipio Machiques de Perijá, Venezuela: parte II validez y seguridad de las pruebas anillo de la leche y rosa de bengala / Epidemiologic surveillance system for Brucella abortus in dual purpose farms from Machiques de Perijá county, Venezuela: part II validity and accuracy of ring test and rose bengal test

Se evalúa la capacidad operativa de las pruebas diagnósticas: anillo de la leche (PAL), inmunoensayo enzimático indirecto (ELISA-i), rosa de bengala (RB) y ELISA competitivo (ELISA-c), utilizadas tras 5 años (2003-2007) de vigilancia epidemiológica para brucelosis bovina (Brucella abortus) en el municipio Machiques de Perijá, partiendo de la revalorización de un conjunto de muestras de leche y suero probados positivos y negativos. La garantía de diagnóstico previamente positivo fue alcanzada a través de evidencias epidemiológicas y positividad comprobada en suero sanguíneo al ensayo con antígeno buferado en placa y confirmación mediante fluorescencia polarizada (FP); sólo se consideraron negativos probados, los sueros y leches globales provenientes de animales negativos a FP que a lo largo del estudio no presentaron antecedentes serológicos alguno. Los resultados en leche muestran superioridad de ELISA-i versus PAL, en sensibilidad, 98,55 contra 56,52 por ciento; especificidad 99,41 y 98,82 por ciento; predicción positiva 98,58 y 95,12 por ciento; predicción negativa 99,49 y 84,84 por ciento; y cocientes de probabilidad. El nivel de concordancia entre los resultados y el verdadero estatus de las muestras fue también preferente para ELISA-i, 97,8 contra 55,3 por ciento. Las evidencias sobre sueros probados dejan en duda la contribución de RB en el diagnóstico, control y erradicación de la enfermedad en el área de estudio, al demostrarse valores medios de validez (169,92 versus 197,21) y seguridad (171,74 versus 197,02), además de bajos coeficientes de verosimilitud entre sus resultados. ELISA-c, en cambio, mantuvo excelente capacidad diagnóstica.

The performance of the following diagnostic tests: Milk Ring Test (MRT), Indirect Enzyme-linked Immunosorbent Assay (iELISA), Rose Bengal Test (RBT) and Competitive ELISA (cELISA) used for 5 years (2003-2007) in the epidemiological surveillance system for bovine brucellosis (Brucella abortus) in Machiques de Perijá County was evaluated. For this purpose, milk and sera samples previously assessed through the abovementioned tests were reassessed to define their true condition (true positive or true negative). Samples were considered as true positive through epidemiological findings and a positive result in Buffered Plate Antigen Test, which was confirmed through Fluorescence Polarization Assay (FPA); samples were considered as true negative only when they were taken from FPA-negative animals that did not seroconvert during the study. Results from tests in milk showed a higher performance of iELISA versus MRT, in sensibility: 98.55 and 56.52 percent; in specificity: 99.41 and 98.82 percent; in positive predictive value: 98.58 and 95.12 percent; in negative predictive value: 99.49 and 84.84 percent; and in coefficients of probability, respectively. The level of agreement between test and true status of samples was higher in iELISA: 97.8 versus 55.3 percent. Results from true positive and true negative samples warrant a reappreciation of the contribution of RBT in diagnostics, control and eradication of brucellosis in the studied area, taking into account its medium values of validity (169.92 versus 197.21) and accuracy (171.74 versus 197.02), as well as its low coefficients of probability. By contrast, cELISA showed an excellent diagnostic performance.

Brucelosis en el zorro gris pampeano (Pseudalopex gymnocercus) en la provincia de La Pampa (Argentina) / Brucelosis in the grey fox of the pampas (Pseudalopex gymnocercus) in La Pampa province (Argentina)

Brucella abortus (Br abortus) es una de las principales causas de abortos y pérdidas reproductivas en el ganado bovino. Los fetos abortados y sus fluidos fetales son la mayor fuente de infección. Es así como la enfermedad puede llegar a animales silvestres cuando ingieren esos tejidos. El objetivo de este trabajo fue determinar la prevalencia serológica de brucelosis en el zorro gris pampeano (Pseudalopex gymnocercus). Se tomaron muestras de sangre de 41 zorros de la región centro-este de la provincia de La Pampa, Argentina. Para determinar la presencia de anticuerpos específicos contra Br abortus se utilizaron las Pruebas de Polarización de la Fluorescencia (FPA), Seroaglutinación en Microplaca (SAP) y Seroaglutinación en Microplaca con 2-mercaptoetanol (2-ME). Sobre un total de 41 sueros procesados por FPA, en 7 (17,1%) se detectaron anticuerpos contra Br abortus. Sólo 34 sueros fueron analizados por las pruebas de SAP y 2-ME, encontrando 5 (14,7%) y 4 (11,8%) muestras positivas, respectivamente. Los resultados permiten inferir que el zorro gris pampeano es susceptible a infectarse con Br. abortus a una tasa de incidencia importante. Es necesario realizar futuros estudios para establecer el rol del zorro en la transmisión de la enfermedad y sus consecuencias en esta especie.

Brucellosis is produced by several species of Brucella. Brucella abortus causes abortion and reproductive loss in bovine cattle. In the epidemiology of brucellosis, aborted fetus and their fetal fluids are the main source of infection and dissemination. Although the biological cycle and the disease consequences to domestic cattle have been widely studied, it is not the case with wild fauna. The objective of this study was to determine the serologic prevalence to brucellosis in the grey fox of the pampas (Pseudalopex gymnocercus). To that purpose, 41 foxes were sampled in the centre-east area of La Pampa province (Argentina). Blood samples for serologic studies were collected. A test of agglutination in microplate (SAP), another SAP with the addition of 2-mercapto-etanol (2-ME) and a test of polarization of the fluorescence (FPA) were used for the diagnosis of antibodies against Brucella abortus. For this, 17.1%, 14.7% and 11.8% was the prevalence found by FPA, SAP and SAP with 2-ME. More studies will be necessary to know the role of foxes in disease transmission and maintenance as well as the consequence of the diseases in foxes.

Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals.

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.

The use of the milk ring test and rose bengal test in brucellosis control and eradication in Nigeria.

In this study, milk and blood samples collected simultaneously from 532 trade cows to be slaughtered at Bodija abattoir, Ibadan (southwestern, Nigeria) were examined for antibodies to Brucella using the milk ring test (MRT) and the rose bengal test (RBT). Overall, 18.61% of the milk samples were positive according to the MRT, while 9.77% of the serum samples were positive according to the RBT. The difference was highly significant (Chi-square value 16.33; P < 0.05); only 32 (6.02%) of the samples were positive for both tests. The Red Bororo breed of cattle and the White Fulani had the highest positive rates, namely 20.93% and 11.69% for the MRT and RBT respectively. No conclusion can be drawn about sensitivity because we do not know the true status of the animals tested. It is, however, obvious that although the MRT and RBT are 1st-line screening tests for brucellosis in cows in some countries, their lack of specificity is of concern. Therefore, the requirement for other confirmatory tests that are more specific should be considered for control and eradication of the disease, especially in Nigeria.

Avaliação do teste do anel em leite na vigilância epidemiológica da brucelose bovina em rebanhos e em laticínios / Evaluation of the ring test in an epidemiological surveillance of bovine brucellosis in herds and dairies

Analisaram-se 464 amostras individuais de leite e 54 amostras de leite de latões, oriundos de leite dos mesmos animais, por meio do teste do anel do leite (TAL) visando à sua aplicação no diagnóstico individual e de rebanhos da brucelose bovina. Foram também avaliadas 464 amostras de soro sangüíneo por meio de provas do antígeno tamponado acidificado (ATA), soroaglutinação lenta em tubos (SAL) e 2-mercaptoetanol (2-ME), todas para brucelose. Cento e vinte e três amostras (26,5 por cento) testadas pelo TAL apresentaram resultados positivos. Dessas, 30 resultaram positivas ao ATA, 28 ao ATA, à SAL e ao 2-ME e 18 à SAL. Das amostras positivas ao TAL, 95 pertenciam a animais sorologicamente negativos ao 2-ME, caracterizando 77,2 por cento (95/123) das reações falso-positivas; dos resultados negativos ao TAL, 4 pertenciam a animais sorologicamente positivos, caracterizando 1,2 por cento (4/341) de reações falso-negativas no TAL individual. Das 54 amostras de leite de latões analisadas pelo TAL, 17 foram consideradas positivas, das quais uma foi caracterizada como falso-positivo, pois todos os animais que a compunham foram negativos ao 2-ME. De 37 latões considerados negativos ao TAL, três continham leite de animais positivos ao 2-ME, caracterizando 8,1 por cento de falso-negativos. O TAL individual demonstrou elevado percentual de resultados falso-positivos, enquanto o TAL em amostras de leite obtidas em latões detectou 84,2 por cento de latões contaminados e 75 por cento de rebanhos infectados.

The ring test (RT) was analyzed regarding its application for the individual and herd bovine brucellosis diagnoses. Individual samples of milk from 464 cows and 54 composite samples of milk bucket from these animals were evaluated. The results were analyzed considering the serological results obtained by the rose bengal (RBT), tube agglutination (TAT) and 2-mercaptoethanol (2-ME) tests. From the 464 individual milk samples analyzed by the RT, 123 (26.5 percent) presented positive results. From those, 30 were positive only to the RBT, 28 by the RBT/TAT/2-ME and 18 by the TAT. From the 123 positive samples by the RT, 95 came from the 2-ME seronegative animals, characterizing 77.2 percent of false-positive reactions and 4/341 negative reactions came from the seropositive animals, characterizing 1.2 percent of false-negative reactions in the individual RT. From the 54 samples of compositive milk analyzed by the RT, 17 were positive. From these positive samples, one was considered false positive since all the animals that composed it were negative by the 2-ME. Three of the composite milk samples that were negative to RT(3/37) were constituted by milk from positive animals, characterizing 8.1 percent of false-negative results by the RT. The individual RT showed high percentage of false-positive results, while the RT in samples from bucket detected 84.2 percent of the contaminated milk and 75 percent of the infected herds.