RESUMO
OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis, Actinobacillus seminis, Histophilus somni in fresh ram semen samples. DESIGN: The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis, and the forward primer published for the detection of H somni; an alternative reverse primer for H somni was designed in this study. PROCEDURE: Culture and PCR of 295 fresh semen samples were carried out. RESULTS: The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. CONCLUSION: This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis, H somni and A seminis.
Assuntos
Actinobacillus seminis/isolamento & purificação , Brucella ovis/isolamento & purificação , Pasteurellaceae/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Sêmen/microbiologia , Animais , Contagem de Colônia Microbiana/veterinária , DNA Bacteriano/análise , Masculino , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , OvinosAssuntos
Doenças dos Bovinos/epidemiologia , DNA Bacteriano/análise , Mycobacterium avium subsp. paratuberculosis/classificação , Paratuberculose/epidemiologia , Animais , Austrália/epidemiologia , Técnicas de Tipagem Bacteriana/veterinária , Bovinos , Doenças dos Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Especificidade da EspécieRESUMO
An antigen extracted from Dichelobacter nodosus with potassium thiocyanate (KSCN) is currently used in enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of ovine footrot, but the test lacks specificity in mature sheep. Other antigens were therefore evaluated for use in this test. Structural components of the cell envelope of D. nodosus including outer membrane, cytoplasmic membrane, lipopolysaccharide and pilus and extracellular proteases were purified from cultured D. nodosus while recombinant membrane proteins, protease and pilus antigens were also evaluated. Many antigenic components of D. nodosus participated in reactions in ELISA that were not specific for infection with D. nodosus and apart from pilus, none of the antigens resulted in improved specificity of the ELISA. Using a positive-negative cut-off to yield sensitivity of 70%, ELISA using pili from cultured D. nodosus serogroup A had a specificity of 98.3% compared with 89.7% for the ELISA with KSCN-extract as antigen (P < 0.001). Recombinant pili morphogenetically expressed in Pseudomonas aeruginosa were unsuitable for use in ELISA due to copurification of Pseudomonas antigens to which apparently healthy sheep directed antibodies. The application of ELISA with D. nodosus pilus as antigen in footrot control programs is discussed.