Leptospirosis is a popularly zoonotic disease of the worldwide. The sero-prevalence of leptospirosis was reported in the domestic dogs in the Mekong Delta. Recently, the dogs suspecting leptospirosis with the clinical syndromes, such as jaundice, mucosal hemorrhage, oculo-nasal discharge, abdominal pain and renal dysfunction were recorded in the veterinary clinics in the investigating area. The objective of this study was to detect the Leptospira species causing leptospirosis in the dogs. One hundred and eleven (111) urine samples were collected from the suspected dogs to detect the presence of leptospira by a dark-field microscopy; leptospira from the positive samples was inoculated in the EMJH
medium, then Leptospira species were identified by PCR assay, and 16S rRNA gene sequence. The results of dark-field microscopic examination showed that leptospira was present in 63 out of 111 (56.75%) urine samples, however leptospira was cultured successfully on EMJH medium from only 3 out of 63 positive urine samples, accounting for 4.76%. The results of analysing leptospira gene sequences revealed two species, i.e. Leptospira interrogans and Leptospira fainei presenting in the urine of the investigated dogs, of which L. interrogans belonged to the mainly pathogenic group and L. fainei belonged to the associated pathogenic group. This is the first successful study on isolation and identification of pathogenic Leptospira from the urine of the dogs suspecting leptospirosis in Viet Nam.