RESUMO
An indirect enzyme-linked immunosorbent assay (IELISA), a competitive ELISA (CELISA), and a fluorescence polarization assay (FPA) for the presumptive serological diagnosis of swine brucellosis were evaluated using two populations of swine sera: sera from brucellosis-free Canadian herds and sera from Argentina selected based on positive reactions in the buffered antigen plate agglutination test (BPAT) and the 2-mercaptoethanol (2-ME) test. In addition, sera from adult swine from which Brucella suis was isolated at least once for each farm of origin were evaluated. The IELISA, CELISA, and FPA specificity values were 99.9, 99.5, and 98. 3%, respectively, and the IELISA, CELISA, and FPA sensitivity values relative to the BPAT and the 2-ME test were 98.9, 96.6, and 93.8%, respectively. Actual sensitivity was assessed by using 37 sera from individual pigs from which B. suis was cultured, and the values obtained were as follows: BPAT, 86.5%; 2-ME test, 81.1%; IELISA, 86.5%; CELISA, 78.5%; and FPA, 80.0%.
Assuntos
Brucelose/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Doenças dos Suínos/diagnóstico , Animais , Argentina , Brucella/imunologia , Brucelose/diagnóstico , Brucelose/imunologia , Brucelose/microbiologia , Testes Sorológicos/métodos , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologiaAssuntos
Animais , Brucelose , Doenças dos Suínos/diagnóstico , Ensaio de Imunoadsorção Enzimática , Brucelose , SuínosRESUMO
A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.
Assuntos
Antígenos de Bactérias/genética , Vacinas Bacterianas/genética , Brucella abortus/genética , Vetores Genéticos , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Vacinas Bacterianas/imunologia , Sequência de Bases , Brucella abortus/imunologia , Brucella abortus/fisiologia , Brucelose/microbiologia , Clonagem Molecular , DNA Bacteriano , Feminino , Expressão Gênica , Genes Reporter , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Trypanosoma cruzi/genéticaRESUMO
An indirect enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Brucella ovis infection was developed. The assay uses a mouse monoclonal antibody to bovine IgG1 horseradish peroxidase (HRPO) conjugate that cross-reacts with immunoglobulin from sheep and a purified antigen from Brucella ovis. The ELISA data were read and analyzed according to a targeting procedure. The ELISA results were compared with a cold complement fixation test (CFT). Sera from 675 rams from three uninfected flocks were used to determine the ELISA cut-off value (O.D. 405 nm: 0.095) and the diagnostic specificity of the ELISA (100%) and the CFT (99.69% +/- 0.42). The ELISA cut-off value was corroborated by receiver operating characteristic (ROC) analysis. Six hundred and forty semen and serum samples from 419 rams from two naturally infected flocks were collected before and after mating-time during two consecutive years. All semen samples were cultured and Brucella ovis was isolated from 28 samples. Sera from the 28 rams with positive semen were used to determine the diagnostic sensitivity of the ELISA (96.43% +/- 6.8) and of the CFT (including suspected positive samples with titers of 1:5; 88.89% +/- 11.85). Considering the CFT suspicious and the anti-complementary reactions as positive resulted in a diagnostic sensitivity value of 89.28% +/- 11.46. Six hundred and ten serum samples from the 640 sera were used to determine relative sensitivity (excluding sera with 1:5) at: ELISA/CFT 97.26% +/- 3.74 and CFT/ELISA was 71.72% +/- 8.87. The percent agreement, beyond chance measured by the Kappa index was 79.7. Relative sensitivity ELISA/CFT (including 1:5 titers in the CFT as positive) was 94.9% +/- 4.83 and CFT/ELISA was 72.84% +/- 8.59. The Kappa index was 79.4.
Assuntos
Anticorpos Antibacterianos/análise , Brucella/imunologia , Brucelose/veterinária , Imunoglobulina G/análise , Doenças dos Ovinos , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais , Coleta de Amostras Sanguíneas , Brucelose/diagnóstico , Brucelose/imunologia , Bovinos , Testes de Fixação de Complemento , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Masculino , Camundongos , Reprodutibilidade dos Testes , Sêmen/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Testes Sorológicos/veterinária , OvinosRESUMO
A Brucella ovis surface protein antigen (P-II), obtained by gel filtration with Sepharose 4B of a hot saline extract was characterized. The analysis of P-II over gradient sodium dodecylsulfate electrophoresis yielded an 18.5 and a 20 kDa band. In a radioimmunoprecipitation assay using P-II labeled with 125I, the antigen reacted specifically only with sera from rams experimentally infected with a naturally occurring rough strain of B. ovis and did not react with sera from rams experimentally infected with other smooth Brucella strains (B. abortus and B. melitensis).
Assuntos
Antígenos de Bactérias/análise , Brucella/imunologia , Animais , Antígenos de Bactérias/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Ligação Competitiva , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Masculino , Ensaio de RadioimunoprecipitaçãoRESUMO
Brucella ovis rough lipopolysaccharide (R-LPS) was studied with respect to its heterogeneity, chain length, sugar composition and immunological activity. R-LPS was mildly hydrolysed and oligosaccharides were recovered in the upper phase after partition with chloroform-methanol. Gel-filtration of the upper phase in a column of Bio-Gel P-2 yielded oligosaccharides of 2, 4, 6 and 7 monosaccharide units, 2-keto-deoxy-octulosonic acid (KDO), and monosaccharides. Strong acid hydrolysis followed by paper chromatography showed that the hexa- and heptasaccharides are both composed of glucose, KDO and an unidentified sugar while tetrasaccharide is composed of glucose, mannose and glucosamine. These three oligosaccharides were able to inhibit the LPS-antibody reaction in a solid phase radioimmunoassay, suggesting the oligosaccharides bear antigenic determinants of LPS.