A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses |
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| Authors: | Richtzenhain Leonardo José Cortez Adriana Heinemann Marcos Bryan Soares Rodrigo Martins Sakamoto Sidnei Miyoshi Vasconcellos Silvio Arruda Higa Zenaide Maria Morais Scarcelli Eliana Genovez Margareth Elide |
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| Institution: | 1. Department of Preventive Veterinary Medicine and Animal Health, University of São Paulo, São Paulo 05508-900, SP, Brazil;2. Biologic Institute, São Paulo, SP, Brazil;1. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, People’s Republic of China;2. College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin Province 130118, People’s Republic of China;3. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, Jiangxi Province 330045, People’s Republic of China;4. Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu Province 225009, People’s Republic of China;1. Department of Animal Breeding Genetics and Biostatistics, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, 680651, Kerala, India;2. Centre for Advanced Studies in Animal Genetics and Breeding, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, 680651, Kerala, India;1. Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, U.P., India;2. Goat Genetics and Breeding Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, U.P., India;3. Goat Health Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, U.P., India |
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| 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. |
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