Development and evaluation of a species-specific PCR assay for the detection of Brucella ovis infection in rams

Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses worldwide. The goal of this study was to develop a PCR test targeting specific B. ovis genomic sequences. Specific primer pairs were designed targeting 12 of those ORFs. Samples of blood, serum, semen, urine, and preputial wash were collected from experimentally infected rams (n = 9) every other week up to 180 days post infection (dpi), when tissue samples were obtained. Blood, serum, semen, urine, and preputial wash samples were obtained, in weekly intervals for 1 month, from eight rams belonging to a B. ovis-free flock. Semen samples were also obtained from rams belonging to naturally infected flocks (n = 40). The limit of detection of this PCR protocol was 100, 10, and 1 CFU/mL for semen, urine and prepucial wash samples, respectively. Sensitivity and specificity values obtained with this PCR method were similar to that of bacteriology when evaluating biological samples. Agreement between PCR and bacteriology results was greater than 90%. These results clearly indicate that this speciesspecific PCR method is highly efficient for the diagnosis of B. ovis infection in semen, urine, preputial wash and tissue samples from infected rams.Estación Experimental Agropecuaria BarilocheFil: Xavier, Mariana N. Universidade Federal de Minas. Escola de Veterinária. Departamento de Clínica e Cirurgia Veterinária; BrasilFil: Silva, Teane M.A. Universidade Federal de Minas. Escola de Veterinária. Departamento de Clínica e Cirurgia Veterinária; BrasilFil: Costa, Erica A. Universidade Federal de Minas. Escola de Veterinária. Departamento de Clínica e Cirurgia Veterinária; BrasilFil: Paixao, Tatiane A. Universidade Federal de Minas. Escola de Veterinária. Departamento de Clínica e Cirurgia Veterinária; BrasilFil: Moustacas, Valeria S. Universidade Federal de Minas. Escola de Veterinária. Departamento de Clínica e Cirurgia Veterinária; BrasilFil: Carvalho Junior, Custodio A. Universidade Federal de Minas. Escola de Veterinária. Departamento de Clínica e Cirurgia Veterinária; BrasilFil: Sant’Anna, Felipe M. Universidade Federal de Minas. Escola de Veterinária. Departamento de Clínica e Cirurgia Veterinária; BrasilFil: Robles, Carlos Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. Grupo de Sanidad Animal; ArgentinaFil: Gouveia, Aurora M.G. Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Medicina Veterinaria Preventiva; BrasilFil: Lage, Andrey P. Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Medicina Veterinaria Preventiva; BrasilFil: Tsolis, Renee M. University of California. Department of Medical Microbiology and Immunology; Estados UnidosFil: Santos, Renato L. Universidade Federal de Minas. Escola de Veterinária. Departamento de Clínica e Cirurgia Veterinária; Brasi

ADAM23 Negatively Modulates alpha(v)beta(3) Integrin Activation during Metastasis

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation. [Cancer Res 2009;69(13):5546-52]FAPESP[04/09088-9