Optimized Protocols for <em>Mycobacterium leprae</em> Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Abstract

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1 nu ). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Autophagy Is an Innate Mechanism Associated with Leprosy Polarization

“…leprae Thai-53 isolate was maintained by serial passage in the footpads of athymic nude mice; and mycobacteria was harvested aseptically and analyzed for viability using a method described elsewhere [72]. Dead M .…”

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

“…To evaluate efficacy of the molecular markers, M. leprae’s purified DNA was used as a positive control, which was extracted after processing a footpad of infected nude mice, as previously described (Trombone et al ). In addition, to check DNA detection limit, a standard curve was prepared from the purified DNA.…”

Murine experimental leprosy: Evaluation of immune response by analysis of peritoneal lavage cells and footpad histopathology

“…The M. leprae suspension was obtained from the footpad of nude mice, previously inoculated with the Thai‐53 strain. After maceration of the footpads in Hank's saline solution (Sigma), the suspension was filtered through a 40‐μm sterile nylon membrane (BD Falcon) to remove tissue debris . The bacillary suspension was fixed on slides and evaluated using the Ziehl‐Neelsen staining.…”

“…The total number of bacilli was determined according to Laboratory Techniques for Leprosy . Viability of the M. leprae was determined by vital staining with fluorescence stain according to Trombone et al and it was estimated to be above 80%.…”