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To identify potential zoonotic reservoirs of pathogenic leptospires in the Peruvian Amazon basin, wild mammals were trapped from July 1997 to December 1998 near the city of Iquitos. After extraction of nucleic acids from animal kidneys, DNA of pathogenic leptospires was identified by polymerase chain reaction (PCR) assays using one of two primer sets, one amplifying a region of the 23S rRNA gene, and the other amplifying a gene fragment specific for Leptospira spp (G1/G2 primers). Overall, 29% (40 of 136) of the mammals tested showed evidence of renal infection by Leptospira spp., including 20% (13 of 64) of the rodents, 39% (20 of 51) of the marsupials, and 35% (7 of 20) of the chiropterans (bats). Marsupials and chiropterans were implicated as more significant reservoir hosts of leptospires pathogenic to humans than previously recognized.