RESUMO
Brucellosis remains as a major infectious disease of domestic animals and is considered a re-emerging zoonosis in several countries. B. abortus infections in bulls are related to reproductive tract infections, although infected animals show transient serological titers or nonreactor status. Thus, diagnosis of bovine brucellosis based exclusively on serological tests probably underestimates B. abortus infections in bulls. In this scenario, three hundred thirty-five serum samples from reproductively mature bovine bulls were subjected simultaneously to standard serodiagnosis using the rose Bengal test (RBT), 2-mercaptoethanol (2-ME), complement fixation (CFT), and fluorescence polarization assay (FPA). Furthermore, conventional semen plasma agglutination (SPA) and modified 2-ME, FC and, FPA were carried out in all bulls replaing serum by seminal plasma. Semen from all bulls was also analyzed for sperm viability, microbiological culture in Farrell media, and polymerase chain reaction (PCR). Only eight (2.38%) semen samples were considered improper for reproduction services (necrospermia and azoospermia), although none of these animals was positive in any of the diagnosis methods used. Five bulls (1.49%) were simultaneously positive in conventional RBT, 2-ME, SPA, modified 2-ME, microbiological culture in Farrell media, and in PCR for B. abortus strain 19. Two (1.67%) bulls were positive in PCR for B. abortus field strains and negative in all other tests, although semen was considered viable to reproduction service. The identification of B. abortus B19 strain in serum and semen of bulls occurred probably due to improper vaccination of males or infection by B19 strain shedding by vaccinated females that could to contaminated environment of farms. In addition, detection of B. abortus field strains only using PCR in bulls without sperm viability abnormalities indicate the need for including molecular methods to improve diagnosis of the disease in bovine bulls.
Assuntos
Brucella abortus , Brucelose Bovina/sangue , Brucelose Bovina/microbiologia , Sêmen/microbiologia , Animais , Bovinos , MasculinoRESUMO
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.
Assuntos
Brucella abortus/patogenicidade , Brucelose Bovina/sangue , Brucelose Bovina/imunologia , Doenças dos Bovinos/sangue , Doenças dos Bovinos/imunologia , Estresse Oxidativo , Adenosina , Animais , Anticorpos Antibacterianos/sangue , Biomarcadores/sangue , Brasil/epidemiologia , Brucelose Bovina/diagnóstico , Brucelose Bovina/epidemiologia , Catalase/sangue , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/epidemiologia , Feminino , Inflamação , Gravidez , Estudos Soroepidemiológicos , Testes Sorológicos , Superóxido Dismutase/sangueRESUMO
The aim of this research was to study if seropositivity for brucellosis in vaccinated cows against this disease hampers reproductive performance and milk production in high-yielding Holstein cows. For this purpose 1,026 healthy cows and 372 cows seropositive for brucellosis were enrolled in this study. Cows positive to card test and subsequently to the rivanol test were further subjected to the radial immunodiffusion (RID) test. It was found that only 11% of the presumably infected cows by brucellosis screening tests were really infected with this disease. The reproductive performance of the group of cows with 11% Brucella-infected animals was not impaired; overall pregnancy rate did not differ between seropositive and healthy cows (30.9 vs. 29.6%). The abortion rates were similar between seropositive cows (5.3%) and seronegative animals (6.9%). Cows in the herd with 11% Brucella-infected animals produced significantly more milk than unaffected cows over a 305-day lactation (10,684 ± 1,720 vs. 10,345 ± 1,736; mean ± SD; P < 0.05). It was concluded that in dairy herds vaccinated against brucellosis with both 19 and RB51 strains, supplemental tests such as RID need to be conducted on all reactors in order to maintain diagnostic accuracy. These results also indicate that 11% animal prevalence of brucellosis did not exert a detrimental effect on 305-day milk yield and reproductive performance in high milk-yielding Holstein cows.
Assuntos
Vacinas Bacterianas/imunologia , Brucelose Bovina/prevenção & controle , Aborto Animal/microbiologia , Aborto Animal/prevenção & controle , Animais , Brucelose Bovina/sangue , Brucelose Bovina/complicações , Bovinos , Feminino , Lactação , Leite , Gravidez , Taxa de Gravidez , ReproduçãoRESUMO
Between 2007 and 2009, a cross-sectional survey was carried out in Maranhão State, Brazil to estimate the seroprevalence of and risk factors for bovine brucellosis. In total, 749 herds and 6779 cows greater than two years of age were blood sampled. At the time of sampling a questionnaire to collect details on possible risk factors for bovine brucellosis was administered to the participating herd manager. A logistic regression model was developed to quantify the association between herd demographic and management characteristics and the herd-level brucellosis status. Spatial analyses were carried out to identify areas of the state where the presence of brucellosis was unaccounted-for by the explanatory variables in the logistic regression model. The estimated herd-level prevalence of brucellosis in Maranhão was 11.4% (95% CI 9.2-14) and the individual animal-level prevalence was 2.5% (95% CI 1.7-3.6). Herds with more than 54 cows older than two years of age, herds that used rented pasture to feed cattle, and the presence of wetlands on the home farm increased the risk of a herd being brucellosis positive. Infected farms were identified throughout the state, particularly in the central region and on the northwestern border. Spatial analyses of the Pearson residuals from the logistic regression model identified an area in the center of the state where brucellosis risk was not well explained by the predictors included in the final logistic regression model. Targeted investigations should be carried out in this area to determine more precisely the reasons for the unexplained disease excess. This process might uncover previously unrecognized risk factors for brucellosis in Maranhão.
Assuntos
Brucella/isolamento & purificação , Brucelose Bovina/epidemiologia , Brucelose Bovina/microbiologia , Animais , Brasil/epidemiologia , Brucelose Bovina/sangue , Bovinos , Estudos Transversais , Feminino , Mapeamento Geográfico , Modelos Logísticos , Mercaptoetanol , Modelos Biológicos , Prevalência , Fatores de Risco , Rosa Bengala , Estudos Soroepidemiológicos , Inquéritos e QuestionáriosRESUMO
Brucella abortus is a Gram-negative intracellular bacterium that causes infectious abortion in food-producing animals and chronic infection in humans. This study aimed to characterize a B. abortus S19 antigen preparation obtained by Triton X-114 (TX-114) extraction through immunoproteomics to differentiate infected from vaccinated cattle. Three groups of bovine sera were studied: GI, 30 naturally infected cows; GII, 30 S19-vaccinated heifers; and GIII, 30 nonvaccinated seronegative cows. One-dimensional (1D) and two-dimensional electrophoretic profiles of TX-114 hydrophilic phase antigen revealed a broad spectrum of polypeptides (10-79 kDa). 1D immunoblot showed widespread seroreactivity profile in GI compared with restricted profile in GII. Three antigenic components (10, 12, 17 kDa) were recognized exclusively by GI sera, representing potential markers of infection and excluding vaccinal response. The proteomic characterization revealed 56 protein spots, 27 of which were antigenic spots showing differential seroreactivity profile between GI and GII, especially polypeptides <20 kDa that were recognized exclusively by GI. MS/MS analysis identified five B. abortus S19 proteins (Invasion protein B, Sod, Dps, Ndk, and Bfr), which were related with antigenicity in naturally infected cattle. In conclusion, immunoproteomics of this new antigen preparation enabled the characterization of proteins that could be used as tools to develop sensitive and specific immunoassays for serodiagnosis of bovine brucellosis, with emphasis on differentiation between S19 vaccinated and infected cattle.
Assuntos
Brucella abortus/imunologia , Brucelose Bovina/sangue , Brucelose Bovina/imunologia , Proteoma/imunologia , Proteômica/métodos , Animais , Vacina contra Brucelose/imunologia , Brucelose Bovina/prevenção & controle , Bovinos , Humanos , Octoxinol , Polietilenoglicóis , Proteoma/análise , Testes SorológicosRESUMO
De outubro a dezembro de 2007, 845 amostras de sangue foram coletadas de 836 vacas (98,9%) e nove touros (1,1%), em um matadouro com inspeção federal no Estado do Tocantins. Cento e quarenta e duas amostras [(16,8%) IC 95% 14,3-19,4] tiveram reação positiva ao teste do antígeno acidificado e tamponado de Brucella abortus. Um touro foi positivo ao teste. Quinhentos e sessenta e um bovinos (66,3%) procederam do Estado do Pará e 284 (33,7%) do Estado do Tocantins. A prevalência de soropositividade foi de 17,2% [IC (95%) 13,6 - 19,9] e de 16,6% [IC (95%) 13,0 - 22,2] para o gado do Pará e Tocantins, respectivamente. Estas prevalências não são significativamente diferentes (p > 0,05). Nenhuma das vacas tinha registro de vacinação contra brucelose. Os exames "ante e post-mortem" não detectaram qualquer sinal ou lesão sugestiva à brucelose, como descargas vaginais, orquites, artrites ou bursites [(0,00%) IC 95% 0,00-0,43] em todos os bovinos da amostra. Esta situação, associada às altas prevalências da doença e à falta de diagnóstico da brucelose nos rebanhos, aumenta o número de bovinos infectados abatidos sob condições sanitárias inadequadas, o risco de contaminação dos trabalhadores e a colocação no mercado, sem restrição, de carne de animais infectados.
From October to December 2007, 845 blood samples were collected from 836 cows (98.9%) and nine bulls (1.1%), in a slaughterhouse with federal inspection in the state of Tocantins. One hundred and forty-two samples [(16.8%) 95% CI 14.3-19.4] had positive reaction to the plate agglutination test carried out with acidified and buffered antigen of Brucella abortus. One bull was positive to the test. Five hundred and sixty one bovines came from the State of Pará (66.3%) and 284 (33.7%) came from the State of Tocantins. The prevalence of serum positivity was 17.2% [CI (95%) 13.6 19.9] and 16.6% [CI (95%) 13.0 22.2] for cattle came from Pará and Tocantins, respectively, with no significant difference between these frequencies (p > 0.05). None of the cows had record of vaccination against brucellosis. "Ante and post-mortem" examinations could not find any sign or lesion attributable to brucellosis, such as vaginal discharges, joint inflammation or bursitis [(0.00%) 95% CI 0.00-0.43] in all bovines sampled. This situation, coupled with the high prevalence of the disease and the lack of brucellosis diagnostic at herds, increases the number of infected cattle slaughtered under inadequate sanitary conditions, the risk of contamination of workers and placing on the market, without restriction, the meat from infected animals.
Assuntos
Animais , Bovinos , Ferimentos e Lesões/veterinária , Brucella abortus/isolamento & purificação , Brucelose Bovina/sangue , Brucelose Bovina/epidemiologia , Brasil , Saúde Pública Veterinária , Inspeção SanitáriaRESUMO
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.
Assuntos
Animais , Bovinos , Brucella , Brucelose Bovina/sangue , Argentina , Brucelose Bovina/diagnóstico , Polarização de Fluorescência/métodos , Raposas/microbiologiaRESUMO
OBJECTIVE: To estimate receiver-operating characteristic (ROC) curves for a competitive ELISA (c-ELISA) that is used in serodiagnosis of brucellosis in water buffalo and cattle, to determine the most appropriate positive cutoff value for the c-ELISA in confirmation of infection, and to evaluate species differences in c-ELISA function. SAMPLE POPULATION: Sera from 4 herds of cattle (n = 391) and 4 herds of water buffalo (381). PROCEDURE: Serum samples were evaluated for Brucella-specific antibodies by use of a c-ELISA. On the basis of previous serologic test results, iterative simulation modeling was used to classify animals as positive or negative for Brucella infection without the use of a gold standard. Accuracy of c-ELISA for diagnosis of infection was compared between cattle and water buffalo by comparison of areas under ROC curves. RESULTS: A positive cutoff value of 30% inhibition for c-ELISA yielded sensitivity and specificity estimates, respectively, of 83.9 and 92.6% for cattle and 91.4 and 95.4% for water buffalo. A positive cutoff value of 35% inhibition yielded sensitivity and specificity estimates, respectively, of 83.9 and 96.2% for cattle and 88.0 and 974% for water buffalo. Areas under ROC curves were 0.94 and 0.98 for cattle and water buffalo, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: ROC curves can be estimated by use of iterative simulation methods to determine optimal cutoff values for diagnostic tests with quantitative outcomes. A cutoff value of 35% inhibition for the c-ELISA was found to be most appropriate for confirmation of Brucella infection in cattle and water buffalo.
Assuntos
Brucella abortus/isolamento & purificação , Brucelose Bovina/microbiologia , Búfalos/microbiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Animais , Anticorpos Antibacterianos/sangue , Brucelose Bovina/sangue , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Cadeias de Markov , Modelos Estatísticos , Método de Monte Carlo , Curva ROC , Sensibilidade e Especificidade , Trinidad e TobagoRESUMO
Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.
Assuntos
Aborto Animal/microbiologia , Brucella abortus/isolamento & purificação , Brucelose Bovina/diagnóstico , Leptospira interrogans/isolamento & purificação , Leptospirose/veterinária , Reação em Cadeia da Polimerase/veterinária , Animais , Brucella abortus/genética , Brucelose Bovina/sangue , Brucelose Bovina/microbiologia , Bovinos , Cricetinae , DNA Bacteriano/química , DNA Bacteriano/genética , Feminino , Leptospira interrogans/genética , Leptospirose/sangue , Leptospirose/diagnóstico , Leptospirose/microbiologia , Reação em Cadeia da Polimerase/métodos , Gravidez , Sensibilidade e EspecificidadeRESUMO
One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.