Isolation and propagation of leptospires at 37 °C directly from the mammalian host
Richard L Hornsby1, David P Alt1, Jarlath E Nally2
1Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA.
Abstract:
The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to months, and is limited to semi-solid media at 28-30 °C. Here we present an alternative media formulation, HAN, compared to commercially available 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 IC:20:001 in semi-solid media at 29 °C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29 °C. In addition, HAN supported primary isolation at 37 °C. Both T80/40/LH and HAN supported primary isolation of strain IC:20:001 in liquid media at 29 °C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37 °C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37 °C: 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.
Insights
A new HAN media formulation improves the primary isolation of pathogenic leptospires, bacteria causing leptospirosis. This faster method aids in diagnosing infections from host tissues at various temperatures.
Area of Science:
- Veterinary Microbiology
- Infectious Diseases
- Bacteriology
Background:
- Leptospirosis is caused by pathogenic Leptospira species, posing a risk to both animals and humans.
- Current primary isolation methods for Leptospira are time-consuming, requiring weeks to months in specialized media at specific temperatures.
- Efficient isolation is crucial for diagnosing and controlling leptospirosis outbreaks.
Purpose of the Study:
- To evaluate a novel HAN media formulation for the primary isolation of pathogenic Leptospira species.
- To compare the efficacy of HAN media against established EMJH and T80/40/LH media.
- To assess the performance of HAN media at different temperatures (29°C and 37°C) and in both liquid and semi-solid formats.
Main Methods:
- Developed and tested the HAN media formulation in both semi-solid and liquid compositions.
- Compared HAN media with commercially available EMJH and specialized T80/40/LH media.
- Used kidney tissue samples from hosts infected with diverse Leptospira species and serovars (L. interrogans and L. borgpetersenii).
- Incubated cultures at 29°C and 37°C to evaluate temperature tolerance.
Main Results:
- All tested media (HAN, EMJH, T80/40/LH) supported the isolation of L. interrogans serovar Copenhageni at 29°C in semi-solid media.
- HAN and T80/40/LH media supported the growth of L. borgpetersenii serovar Hardjo at 29°C.
- HAN media demonstrated successful primary isolation at 37°C for both species.
- HAN media supported liquid culture isolation for both strains at 29°C and 37°C, outperforming T80/40/LH for L. borgpetersenii.
Conclusions:
- The HAN media formulation is effective for the primary isolation of fastidious pathogenic Leptospira directly from host tissues.
- HAN media offers improved versatility by supporting growth at both 29°C and 37°C and in liquid or semi-solid formats.
- This defined media represents a significant advancement for optimizing Leptospira isolation and studying diverse species and serovars.
