title: Long-term survival and virulence of Mycobacterium leprae in amoebal cysts.
abstract: Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT.
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: William H
authors.initials: WH
authors.lastname: Wheat
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Casali
authors.affiliation: STERIS SA R&D, Fontenay-aux-Roses, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Thomas
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: John S
authors.initials: JS
authors.lastname: Spencer
authors.affiliation: Department of Health & Human Services, HRSA, HSB, National Hansen's Disease Programs, Laboratory Research Branch, Baton Rouge, Louisiana, United States of America; School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.
authors.forename: Ramanuj
authors.initials: R
authors.lastname: Lahiri
authors.affiliation: Department of Health & Human Services, HRSA, HSB, National Hansen's Disease Programs, Laboratory Research Branch, Baton Rouge, Louisiana, United States of America; School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.
authors.forename: Diana L
authors.initials: DL
authors.lastname: Williams
authors.affiliation: Department of Research and Development, STERIS Corporation, Mentor, Ohio, United States of America.
authors.forename: Gerald E
authors.initials: GE
authors.lastname: McDonnell
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Gonzalez-Juarrero
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Brennan
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Mary
authors.initials: M
authors.lastname: Jackson
mesh_terms: D000656:Amoeba / Q000382:microbiology*; D000818:Animals; D018920:Coculture Techniques; D006801:Humans; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008819:Mice, Nude; D009166:Mycobacterium leprae / Q000254:growth & development / Q000472:pathogenicity*; D010587:Phagocytosis; D014774:Virulence