title: Development of a multiplex real-time PCR for contagious agalactia diagnosis in small ruminants.
abstract: Contagious agalactia is an important disease worldwide that affects small ruminants. Clinical manifestations vary from mastitis, pneumonia, arthritis and keratoconjunctivitis to septicemia. Four mycoplasmal etiological agents have been identified: Mycoplasma (M.) agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. The current procedure for direct diagnosis, recommended by the World Organization for Animal Health, involves the isolation of one or several causative agents from clinical specimens and further time-consuming identification steps. The present study reports the development of a new multiplex real-time PCR (including an internal positive control) that detects all four pathogens simultaneously and distinguishes M. agalactiae from the others. First, intra- and inter-species polymorphisms of the two target house-keeping genes, namely polC and fusA, were analyzed to design primers and probes adapted to the diversity of currently circulating strains. The specificity and the sensitivity of the assay were then challenged and the limit of detection was found to be as low as 6 to 12 copies of the target genes. The assay requires further assessment on clinical specimens but its performances (notably low intra- and inter-assay variability) are already very promising for use in large-scale diagnosis and prophylactic surveys of contagious agalactia.
authors.affiliation: Université de Lyon, VetAgro Sup, UMR Mycoplasmoses des Ruminants Anses VetAgro Sup, F-69280, Marcy l'Etoile, France. claire.becker@vetagro-sup.fr
authors.forename: Claire A M
authors.initials: CA
authors.lastname: Becker
authors.forename: Fabien
authors.initials: F
authors.lastname: Ramos
authors.forename: Eric
authors.initials: E
authors.lastname: Sellal
authors.forename: Sandrine
authors.initials: S
authors.lastname: Moine
authors.forename: François
authors.initials: F
authors.lastname: Poumarat
authors.forename: Florence
authors.initials: F
authors.lastname: Tardy
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D015511:Goat Diseases / Q000175:diagnosis* / Q000382:microbiology; D006041:Goats; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009174:Mycoplasma / Q000145:classification / Q000302:isolation & purification*; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D020653:Peptide Elongation Factor G / Q000235:genetics; D011110:Polymorphism, Genetic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000382:microbiology; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D020653:Peptide Elongation Factor G; C446929:PolC protein, bacteria; D004259:DNA-Directed DNA Polymerase