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Lyme disease is an infectious multisystemic illness with dermatologic, neurologic, cardiac, and rheumatic manifestations. A total of 4,355 ticks was collected in Colombia, of which 2,805 were identified as Ixodes spp. The midgut contents of 2,600 specimens were fixed on microscope slides and examined by an indirect immunofluorescence assay (IFA) using monoclonal antibodies, anti-ospA H5332, and anti-flagellin 9724. All of the ticks examined by IFA tested negative.