Silver staining or immunogold-silver staining is sometimes useful as an adjunct technique. Darkfield microscopy can also be used to detect Leptospira; however, this technique is non-specific and not very sensitive.
Culture is definitive, but the availability of this test is limited. Leptospira spp. can be isolated on a variety of media, but they are fastidious and grow slowly on primary isolation. Special transport media may be required for shipment to the laboratory. Depending on the serovar, culture can take up to 13 to 26 weeks. Identification to the species, serogroup and/or serovar level is done by reference laboratories, using genetic and immunological techniques.
Serology is often used to diagnose leptospirosis. Caution must be used in interpreting serological test results, as subclinical infections are common; the vaccination status of the animal must be considered; antibodies may not be present early in the illness; and antibody titers can become low or undetectable in some chronically infected animals. Paired acute and convalescent samples are preferred from most animals, and rising antibody titers are usually seen in acute cases. Single samples with high titers increase the suspicion of leptospirosis, although they are not definitive. However, a single positive sample from an aborted fetus is diagnostic. Herd tests are often used in ruminants. The most commonly used serological tests in animals are the microscopic agglutination test (MAT) and enzyme-linked immunosorbent assays (ELISAs). The MAT evaluates antibody responses to a selection of Leptospira serovars (often 5-7 in veterinary assays). This test is serogroup but not serovar specific, although it may suggest a likely serovar. False negatives are possible if the infecting serogroup was not included in the MAT panel. ELISAs, including a bovine milk ELISA, are available for some species of animals.