Isolation and propagation of leptospires at 37C directly from the mammalian host

Authors: Hornsby, Richard; Alt, David; Nally, Jarlath

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2020
Publication Date: 6/15/2020
Citation: Hornsby, R.L., Alt, D.P., Nally, J.E. 2020. Isolation and propagation of leptospires at 37 degree C directly from the mammalian host. Scientific Reports. 10(9620). https://doi.org/10.1038/s41598-020-66526-4.
DOI: https://doi.org/10.1038/s41598-020-66526-4

Interpretive Summary: Leptospirosis is a global zoonotic disease. Pathogenic species of Leptospira are excreted in urine from asymptomatic carrier hosts which facilitates disease transmission to incidental hosts. The primary isolation of leptospires from reservoir hosts has always been a laborious, time consuming and inefficient task requiring weeks in semi-solid media at 28-30C. Yet it is an essential task that can provide a definitive diagnosis, an isolate for serotyping and epidemiological studies, a relevant serovar for inclusion in bacterin based vaccines to immunize animal species, and appropriate antigen for inclusion in diagnostic microscopic agglutination test (MAT) panels. Here we present a novel media formulation, HAN, for the isolation and propagation of fastidious pathogenic leptospires directly from host tissue, and demonstrate its improved effectiveness relative to EMJH and T80/40/LH, in both liquid and semi-solid media, at both 29 and 37C.

Technical Abstract: Leptospirosis is a global zoonotic disease affecting over one million people each year. The causative agent includes multiple serovars and species of pathogenic leptospires that colonize the renal tubules of reservoir hosts of infection, including domestic and wild animal species, from which they are excreted via urine into the environment. The primary isolation of leptospires from reservoir hosts has always been a laborious, time consuming and inefficient task requiring weeks in semi-solid media at 28-30C. Yet it is an essential task that can provide a definitive diagnosis, a strain for serotyping and epidemiological studies, a relevant serovar for inclusion in bacterin based vaccines to immunize animal species, and appropriate antigen for inclusion in diagnostic microscopic agglutination test (MAT) panels. Here we present an alternative media formulation, HAN, compared to EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and serovars of pathogenic leptospires directly from host kidney tissue. All three media types supported the isolation and propagation of L. interrogans serovar Copenhageni strain ICO2001 in semi-solid media at 29C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29C. In addition, HAN supported primary isolation at 37C. Both T80/40/LH and HAN supported primary isolation of strain ICO2001 in liquid media at 29C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37C: this formulation represents a more defined media for the continued optimization of growth factors required to support the primary isolation of the large and diverse range of species and serovars within the genus Leptospira circulating within domestic and wild animal populations.