Korean Lepr Bull.  2023 Jun;56(1):4.

Improved protocols for the purification and maintenance of Mycobacterium lepraestrains in athymic nude mice

• Kim JR
• Lee MS
• Kim JP

Abstract

Background
Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae), an obligate intracellular organism residing within host macrophages and Schwann cells. Currently, there is no artificial medium capable of culturing M. leprae, so it is mainly grown by inoculating animals such as nude mice and armadillos. In a laboratory environment, it is convenient to maintain and cultureM. lepraeusing athymic nude mice.
Objective
The maintenance and growth ofM. lepraestrains in nude mice is important for studying leprosy. In this study, we developed an efficient purification method to collect M. lepraestrain grown in nude mice.
Methods
Purification ofM. lepraestrain from nude mouse footpads was used of gentleMACS™ C Tube and DT-20 Dispersing tube without a tissue homogenizer. The isolated tissues were dissociated with gentleMACS™ C Tubes and gentleMACS Dissociators. In order to increase the bacterial recovery rate, bacilli from the remaining tissues (dermis, tendons, nerves and bones of the foot) were purified using a DT-20 Dispersing tube and IKA Tube Disperser. After the tissue dissociation, cells were separated by trypsin treatment and the contaminated bacteria were removed by sodium hydroxide (NaOH). The isolated bacteria were stained with methylene blue solution in the usual Ziehl-Neelsen method and the chromosomal DNA extracted by cracking of cell wall with bead beating was analyzed by DNA amplification by polymerase chain reaction (PCR).
Results
Using gentleMACS™ C Tube and DT-20 Dispersing tube instead of a tissue homogenizer, M. lepraestrain was efficiently obtained from tissues. Trypsin was suitable for tissue dissociation, resulting in an effective cell suspension and did not affect the survival of M. lepraestrain. The procedures showed high viability and high recovery rate and allowed simultaneous purification of different bacillary strains from each nude mice without contamination by NaOH treatment.
Conclusion
It is a useful method for high viability rate and high recovery rate of M. lepraestrain harvested from nude mouse. The procedures were possible method for extracting several strains at the same time.

Keyword

Leprosy; Mycobacterium leprae; Maintenance; Athymic nude mice