Culture Strategies for Isolation of Fastidious Leptospira Serovar Hardjo and Molecular Differentiation of Genotypes Hardjobovis and Hardjoprajitno

Abstract

The Leptospira serovar Hedjo belongs to the serogroup sejroe and this serovar is the most prevalent in bovine herds worldwide. The sejroe serogroup is the most frequently detected by serology in Brazilian cattle herds suggesting that it is due serovar Hardjo. In the molecular classification, this serovar has two genotypes: Hardjobovis and Hardjoprajitno. This serovar is as considered as fastidious pathogens, and their isolation is one of the bottlenecks in leptospirosis laboratories. In addition, its molecular characterization using genomic approaches is oftentimes not simple and time-consuming. This study describes a method for isolating the two genotypes of serovar Hardjo using culture medium formulations and suggests a get-at-able molecular characterization. Ten cows naturally infected which were seropositive were selected from small dairy farms, and their urine was collected for bacterial isolation. We evaluated three modifications of liquid Leptospira medium culture supplemented with sodium pyruvate, superoxide dismutase enzyme and fetal bovine serum, and the isolates were characterized by molecular techniques. After isolation and adaptation in standard culture medium, the strains were subcultured for 1 week in the three modified culture media for morphologic evaluation using electronic microscopy. Strains were molecularly identified by multilocus variable-number tandem-repeat analysis (MLVA), partial sequencing and phylogenic analyses of gene sec Y. Combining the liquid culture medium formulations allowed growth of the Leptospira serovar Hardjo in three tubes. Two isolates were identified as genotype Hardjobovis, and the other as genotype Hardjoprajitno. Morphologically, compared with control media, cells in the medium supplemented with the superoxide dismutase enzyme were more elongated and showed many cells in division. The cells in the medium supplemented with fetal bovine serum were fewer and lost their spirochete morphology. This indicated that the additional supplementation with fetal bovine serum assisted in the initial growth and maintenance of the viable leptospires and the superoxide dismutase enzyme allowed them to adapt to the medium. These culture strategies allowed for the isolation and convenient molecular characterization of two genotypes of serovar Hardjo, creating new insight into the seroepidemiology of leptospirosis and its specific genotypes. It also provides new information for the immunoprophylaxis of bovine leptospirosis.

Isolation of pathogenic Leptospira strains from naturally infected cattle in Uruguay reveals high serovar diversity, and uncovers a relevant risk for human leptospirosis

“…As a first approach to address this issue, the particularly fastidious L . borgpetersenii serovar Hardjo was chosen [ 30 ] to perform in vitro tests of viability kinetics in bovine urine. Indeed, a critical maximum time of exposure was defined at less than 2 h ( S4 Table ), above which subsequent isolation success rates decreased significantly.…”

Review of Diagnostic Procedures and Approaches to Infectious Causes of Reproductive Failures of Cattle in Australia and New Zealand

“…Although diagnosis can be made by histological observation of the organism in the heart, intestine, liver and kidney using silver staining ( 114 ), multiplex PCR or quantitative PCR (qPCR) can be performed for a rapid and comprehensive diagnostic test ( 116 , 117 , 118 ). Detection of Leptospira by isolation is not recommended, given the difficulty of culturing this organism ( 119 ).…”

Identification of Pathogenic Leptospira Species in the Urogenital Tract of Water Buffaloes (Bubalus bubalis) From the Amazon River Delta Region, Brazil

“…The positive uterine swab sample (MN723893), belonging to L. interrogans, showed homology to sequences characterized as belonging to serovar Hardjo strains, including an autochthonous sample (CP043041.1) isolated from the urine of dairy cattle in Paraná, in the southern region of Brazil (42). This finding may suggest that sexual transmission may be occurring among these animals.…”