| Summary: | <i>Leptospira</i> was investigated in kidneys (<i>n</i> = 305) from slaughtered livestock in the Gauteng Province abattoirs, South Africa, using a culture medium to isolate <i>Leptospira</i>, followed by the <i>LipL32</i> qPCR to detect <i>Leptospira</i> DNA. The <i>SecY</i> gene region was amplified, sequenced, and analyzed for <i>LipL32</i> qPCR-positive samples or <i>Leptospira</i> isolates. The overall frequency of isolation of <i>Leptospira</i> spp. was 3.9% (12/305), comprising 4.8% (9/186), 4.1% (3/74), and 0% (0/45) from cattle, pigs, and sheep, respectively (<i>p</i> > 0.05). However, with <i>LipL32</i> qPCR, the overall frequency of <i>Leptospira</i> DNA was 27.5%, consisting of 26.9%, 20.3%, and 42.2% for cattle, pigs, and sheep, respectively (<i>p</i> = 0.03). Based on 22 <i>SecY</i> sequences, the phylogenetic tree identified the <i>L. interrogans</i> cluster with serovar Icterohaemorrhagiae and the <i>L. borgpetersenii</i> cluster with serovar Hardjo bovis strain Lely 607. This study is the first molecular characterization of <i>Leptospira</i> spp. from livestock in South Africa. The reference laboratory uses an eight-serovar microscopic agglutination test panel for leptospirosis diagnosis, of which <i>L. borgpetersenii</i> serovar Hardjo bovis is not part. Our data show that pathogenic <i>L. interrogans</i> and <i>L. borgpetersenii</i> are circulating in the livestock population. Diagnostic use of molecular methods will eliminate or reduce the under-reporting of leptospirosis in livestock, particularly sheep, in South Africa.
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