Serological diagnosis of brucellosis in goats: comparison of techniques

Goat production is an important item in the agricultural system of Argentina and brucellosis caused by Brucella melitensis is considered the main cause reproductive problems in this species. Although definitive diagnosis for this disease is made by isolation of the etiologic agent, but in practice the health status of a herd was evaluated by serological techniques indirect testing. To evaluate and compare the performance of different serological tests for diagnosis of brucellosis in goats, serum samples were analyzed. The following tests were performed: buffered plate antigen (BPA), rose bengal (RB RB 3% and 8%), fluorescence polarization (FPA) and the test tube and 2-mercaptoethanol (SAT/2ME). The validity of each test was determined in a relative manner by combination of indirect ELISA (IELISA) and complement fixation (CF). BPA was the most sensitive test, followed by SAT/2ME, 3% RB, FPA and, finally, RB 8%. FPA test, when used in series with BPA, significantly improved overall specificity. Based on these results it was proposed for brucellosis diagnosis in goats, BPA as a screening test, FPA as complementary test, and FC and IELISA as confirmatory tests. SAT/2ME is recommended only when it is not possible to perform other tests, due to the toxic effects of SAT/2ME reagents.Fil: Cisterna, C. Comisión de Investigaciones Científicas (CIC). Buenos Aires, ArgentinaFil: Conde, S. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología. Buenos Aires, ArgentinaFil: Hollender, D. CONICET. ArgentinaFil: Martino, P.E. Comisión de Investigaciones Científicas (CIC). Buenos Aires, ArgentinaFil: Samartino L. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología. Buenos Aires, ArgentinaLa producción caprina representa un rubro importante dentro del sistema agropecuario de la República Argentina y la brucelosis causada por Brucella melitensis es considerada el motivo principal de los problemas reproductivos en esta especie. El diagnóstico de certeza se realiza aislando el agente etiológico, aunque en la práctica se evalúa el estado sanitario de un hato mediante pruebas serológicas. El objetivo de este trabajo fue evaluar y comparar el desempeño de las distintas pruebas serológicas tamíz y complementarias para el diagnóstico de la brucelosis caprina. Para ello, se analizaron muestras de suero de animales sin vacunación, realizando antígeno buferado en placa (BPA), rosa de bengala (RB 3 % y RB 8 %), polarización fluorescente (FPA) y pruebas de tubo y 2 mercaptoetanol (SAT/2ME). La validez de cada prueba se determinó por combinación de ELISA Indirecto (IELISA) y Fijación de Complemento (FC). BPA resultó ser más sensible, seguido de SAT/2ME, RB 3%, FPA y por último RB 8%. Al realizar FPA en serie con BPA mejoró significativamente la especificidad global. Se propone BPA como prueba tamíz, FPA como complementaria y FC e IELISA como confirmatorias. Se recomienda SAT/2ME sólo en los casos en que no sea posible la realización de las otras pruebas debido a los efectos tóxicos de los reactivos empleados para su realización

Isolation of Leptospira sp. from an equine abortion

The differences observed between females and males were statistically significant (p 1⁄4 0, 001). It was detected a virus infection of equine Viral Arteritis in 53% of the samples obtained in feral donkeys, which could indicate some degree of viral circulation in these animal populations. It suggests later studies that they allow to characterize these viruses of donkeys, to see his phylogeny. Chile is free of EVA in horses, the disease has not been detected in domestic horses and it is of compulsory notification. The detection of antibodies anti-vEVA inwild species was communicated to the World Organization for Animal Health (OIE)

Diagnóstico serológico de brucelosis en caprinos: comparación de técnicas

La producción caprina representa un rubro importante dentro del sistema agropecuario de la República Argentina y la brucelosis causada por Brucella melitensis es considerada el motivo principal de los problemas reproductivos en esta especie. El diagnóstico de certeza se realiza aislando el agente etiológico, aunque en la práctica se evalúa el estado sanitario de un hato mediante pruebas serológicas. El objetivo de este trabajo fue evaluar y comparar el desempeño de las distintas pruebas serológicas tamíz y complementarias para el diagnóstico de la brucelosis caprina. Para ello, se analizaron muestras de suero de animales sin vacunación, realizando antígeno buferado en placa (BPA), rosa de bengala (RB 3 % y RB 8 %), polarización fluorescente (FPA) y pruebas de tubo y 2 mercaptoetanol (SAT/2ME). La validez de cada prueba se determinó por combinación de ELISA Indirecto (IELISA) y Fijación de Complemento (FC). BPA resultó ser más sensible, seguido de SAT/2ME, RB 3%, FPA y por último RB 8%. Al realizar FPA en serie con BPA mejoró significativamente la especificidad global. Se propone BPA como prueba tamíz, FPA como complementaria y FC e IELISA como confirmatorias. Se recomienda SAT/2ME sólo en los casos en que no sea posible la realización de las otras pruebas debido a los efectos tóxicos de los reactivos empleados para su realización.Goat production is an important item in the agricultural system of Argentina and brucellosis caused by Brucella melitensis is considered the main cause reproductive problems in this species. Although definitive diagnosis for this disease is made by isolation of the etiologic agent, but in practice the health status of a herd was evaluated by serological techniques indirect testing. To evaluate and compare the performance of different serological tests for diagnosis of brucellosis in goats, serum samples were analyzed. The following tests were performed: buffered plate antigen (BPA), rose bengal (RB RB 3% and 8%), fluorescence polarization (FPA) and the test tube and 2-mercaptoethanol (SAT/2ME). The validity of each test was determined in a relative manner by combination of indirect ELISA (IELISA) and complement fixation (CF). BPA was the most sensitive test, followed by SAT/2ME, 3% RB, FPA and, finally, RB 8%. FPA test, when used in series with BPA, significantly improved overall specificity. Based on these results it was proposed for brucellosis diagnosis in goats, BPA as a screening test, FPA as complementary test, and FC and IELISA as confirmatory tests. SAT/2ME is recommended only when it is not possible to perform other tests, due to the toxic effects of SAT/2ME reagents

Potential role of fibroblast-like synoviocytes in joint damage induced by Brucella abortus infection through production and induction of matrix metalloproteinases

Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated. Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion. The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate. These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during brucellar arthritis through the production of MMPs and proinflammatory mediators.Fil: Scian, Romina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Barrionuevo, Paula. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: de Simone, Emilio Adrian. Universidad de Buenos Aires. Facultad de Cs.veterinarias. Catedra de Fisiologia Animal; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Vanzulli, Silvia I.. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Fossati, Carlos Alberto. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Baldi, Pablo Cesar. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Delpino, María Victoria. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

An Oral Vaccine Based on U-Omp19 Induces Protection against B. abortus Mucosal Challenge by Inducing an Adaptive IL-17 Immune Response in Mice

As Brucella infections occur mainly through mucosal surfaces, the development of mucosal administered vaccines could be radical for the control of brucellosis. In this work we evaluated the potential of Brucella abortus 19 kDa outer membrane protein (U-Omp19) as an edible subunit vaccine against brucellosis. We investigated the protective immune response elicited against oral B. abortus infection after vaccination of mice with leaves from transgenic plants expressing U-Omp19; or with plant-made or E. coli-made purified U-Omp19. All tested U-Omp19 formulations induced protection against Brucella when orally administered without the need of adjuvants. U-Omp19 also induced protection against a systemic challenge when parenterally administered. This built-in adjuvant ability of U-Omp19 was independent of TLR4 and could be explained at least in part by its capability to activate dendritic cells in vivo. While unadjuvanted U-Omp19 intraperitoneally administered induced a specific Th1 response, following U-Omp19 oral delivery a mixed specific Th1-Th17 response was induced. Depletion of CD4+ T cells in mice orally vaccinated with U-Omp19 resulted in a loss of the elicited protection, indicating that this cell type mediates immune protection. The role of IL-17 against Brucella infection has never been explored. In this study, we determined that if IL-17A was neutralized in vivo during the challenge period, the mucosal U-Omp19 vaccine did not confer mucosal protection. On the contrary, IL-17A neutralization during the infection did not influence at all the subsistence and growth of this bacterium in PBS-immunized mice. All together, our results indicate that an oral unadjuvanted vaccine based on U-Omp19 induces protection against a mucosal challenge with Brucella abortus by inducing an adaptive IL-17 immune response. They also indicate different and important new aspects i) IL-17 does not contribute to reduce the bacterial burden in non vaccinated mice and ii) IL-17 plays a central role in vaccine mediated anti-Brucella mucosal immunity

Interaction of bovine chorioallantoic membrane explants with three strains of Brucella abortus.

Chorioallantoic membrane (CAM) explants were used to determine the in vitro growth and cytotoxic potential of 3 strains of Brucella abortus. Bovine CAM explants were inoculated with 2 x 10(7) colony-forming units of the pathogenic strain 2308, attenuated strain 19, or the rough strain RB51 of B abortus. After inoculation, the explants were harvested and examined at 2 or 4 hours, 12 or 14 hours, and 24 or 26 hours of incubation. Bacterial growth associated with each explant was determined by counting colony-forming units. The degree of cellular damage in each explant associated either with bacterial growth or bacterial toxins was evaluated by morphometric analysis after trypan blue staining. Significant differences were not detected in the numbers of bacteria of any strain of B abortus in the CAM explants at comparable time intervals. The rate of growth of the bacteria in CAM explants was higher between 2 and 12 hours after inoculation than between 12 and 24 hours after inoculation. Cytotoxic effects associated with strain 2308 were significantly (P less than 0.05) greater than that caused by other strains. Cytotoxic effects associated with strain 19 and rough strain RB51 were similar, and both were significantly (P less than 0.05) greater than the phosphate buffer solution control. Chorioallantoic membrane explants inoculated with a filtrate of heat-killed strain 2308 induced minimal cellular damage, compared with that caused by the viable bacteria. These results indicated that the number of B abortus in trophoblasts was independent of the degree of cellular damage

Methodology for the assessment of brucellosis management practices and its vaccination campaign: example in two Argentine districts.

BACKGROUND: In Argentina, vaccination with Brucella abortus Strain 19 vaccine is mandatory. The objective of the study was to develop and test a method for evaluating, in an innovative way, some farmers' and veterinarians' management practices in relation to brucellosis and to assess the vaccination campaign and coverage. The work took place in Brandsen and Navarro districts. Four questionnaires were designed (for officials from Local Sanitary Entities, vaccinators, vet practitioners and farmers). Responses were coded as "ideal" (0) and "not ideal" (1). To assess the relative weight of each question ("item"), experts ranked the items according to their impact on management practices and vaccination. A weighted score was then calculated. A higher weighted score was assigned to the worse practices. Farmers obtaining a global weighted score above the third quartile were classified as "inappropriately managed farms", to be compared per type of production system and district. To assess the immunization coverage, female calves were sampled 30 to 50 days post vaccination; they were expected to react positively to serological diagnostic tests (DT+). RESULTS: There were significantly more inappropriately managed farms and higher global scores among beef farmers and in Brandsen. Eighty three percent (83%) of female calves were DT+, significantly under the ideal immunization coverage (95%). Only 48% of farms were considered well vaccinated. DT+ results were positively associated with the Brandsen district (OR = 25.94 [4.60-1146.21] and with the farms having more than 200 cow heads ((OR = 78.34 [4.09-1500.00]). On the contrary, DT+ were less associated with vaccinators being veterinary practitioners (OR = 0.07 [0.006-0.78]). Farmers are well advised by their veterinary practitioners but they should improve some management practices. CONCLUSIONS: The vaccination campaign is globally well implemented, but the immunization coverage and some vaccinators' practices should be improved. This study leads to a better understanding of the most common used management and control practices regarding brucellosis, which affect its epidemiology. Any vaccination campaign should be periodically assessed to highlight possible fails. The described methodology can be extrapolated to other countries and different contexts