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The early diagnosis of canine leptospirosis is still a challenge for clinicians that in most cases only have basic routine tests as diagnostic tools. This study aimed to isolate and characterize leptospires from the blood of dogs with clinical signs suggestive of leptospirosis and to evaluate the results of some laboratory tests in hamsters experimentally infected with these isolates. The blood of 22 animals was submitted to bacteriological culturing, and the isolates recovered were characterized by serogrouping, partial DNA sequencing (secY), and multiple locus variable-number tandem repeat analysis (MLVA). Golden Syrian hamsters were infected with isolated leptospires and submitted to serology using microscopic agglutination test (MAT), polymerase chain reaction (PCR), complete blood counts, and serum biochemistry. Three isolates (13.63%) were obtained, and all were assigned to the serogroup Icterohaemorrhagiae. The DNA sequencing results showed that all the isolates belonged to the species Leptospira interrogans, and the MLVA results evidenced that the profile of the strains was compatible with Copenhageni/Icterohaemorrhagiae serovars. Regarding laboratory tests performed on hamsters infected with these isolated strains, marked leukocytosis by neutrophilia and monocytosis was observed, associated with an accentuated increase in renal and hepatic indicators. In addition, a titer of 800 was detected by MAT for only the serogroup Icterohaemorrhagiae, and blood, liver, kidneys, and lung samples were PCR positive. The presence of these isolates suggests that the dogs were infected with strains carried by rodents, and significant changes were found in routine laboratory tests, which, when correlated, can help veterinarians with early diagnosis. [ABSTRACT FROM AUTHOR]