title: A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses.
abstract: Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.
authors.affiliation: Department of Preventive Veterinary Medicine and Animal Health, University of São Paulo, 05508-900, Brazil. leonardo@usp.br
authors.forename: Leonardo José
authors.initials: LJ
authors.lastname: Richtzenhain
authors.forename: Adriana
authors.initials: A
authors.lastname: Cortez
authors.forename: Marcos Bryan
authors.initials: MB
authors.lastname: Heinemann
authors.forename: Rodrigo Martins
authors.initials: RM
authors.lastname: Soares
authors.forename: Sidnei Miyoshi
authors.initials: SM
authors.lastname: Sakamoto
authors.forename: Silvio Arruda
authors.initials: SA
authors.lastname: Vasconcellos
authors.forename: Zenaide Maria Morais
authors.initials: ZM
authors.lastname: Higa
authors.forename: Eliana
authors.initials: E
authors.lastname: Scarcelli
authors.forename: Margareth Elide
authors.initials: ME
authors.lastname: Genovez
mesh_terms: D000034:Abortion, Veterinary / Q000382:microbiology*; D000818:Animals; D002003:Brucella abortus / Q000235:genetics / Q000302:isolation & purification*; D002007:Brucellosis, Bovine / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D002417:Cattle; D006224:Cricetinae; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005260:Female; D007921:Leptospira interrogans / Q000235:genetics / Q000302:isolation & purification*; D007922:Leptospirosis / Q000097:blood / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011247:Pregnancy; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial