M. leprae suspensions were purified by NaOH treatment as described [27]. (1∶1 to 5∶1), ingestion of live M. leprae did not exert any observable adverse effect on amoebae that divided normally over several days. The resulting mean fluorescence intensities (MFI) were acquired as one-color histograms and increases in MFI were plotted against time using GraphPad Prism software.
Duppre J.A.C. The acquisition of red fluorescence by the amoebae as a function of time at 32°C is shown in Fig. Amoebae were infected with viable M. leprae, or M. leprae isolated from armadillo tissues (presumed non-viable) that were first stained with the red fluorescent vital PKH26 membrane dye. Staining of long-term (6 months) encysted M. leprae/amoebae cocultures (maintained in encystment buffer at 32°C) with auramine/rhodamine for acid-fast organisms confirmed the presence of bacilli in A. polyphaga, A. castellanii and H. vermiformis cysts. H. N.C.
Results were gathered from gates of uniform size as determined from uninfected samples. Bwayo J.A.C. Both fluorescence and confocal microscopes were used to visualize extracted and internalized M. leprae by all amoebae spp. Future experimentation will likely reveal answers to these rather intriguing questions. A.-M. J.T. Future experimentation including testing for appearance of disease by transferring M. leprae from our other existing cocultures of A. lenticulata and H. vermiformis strains to nu/nu mouse FPs and determining whether mice challenged directly with M. leprae-infected amoebae (cysts or trophozoites) display any differences in progress to disease should resolve some of these issues. Wilson
In cases of nuclear staining of amoebae or mouse tissues, a DAPI filter (358 nm excitation/461emission) was utilized. Currently, only a few endemic countries remain where relatively high number of cases persists.
Altogether, these results suggest that the acid-fast bacilli observed in Figs. J.L. Oxborrow The results described in this current work do not demonstrate that M. leprae is capable of multiplying inside amoebae but are suggestive of a role for FLA providing sustenance to maintain viability of the bacilli; (C) whether the leprosy bacillus requires periodic excystment as is likely the case in the natural world in order to re-infect emergent trophozoites or human host cells; and, finally (D) whether the virulence of M. leprae is affected either positively or negatively by its passage through amoebae. Prior to flow cytometric analysis the aliquots were centrifuged 3X at 600 X g to pellet and remove any cell-free bacilli.
Furthermore, we show that acid-fast bacilli extracted from M. leprae/amoebae cocultures with A. lenticulata, A. castellanii, A. polyphaga, H. vermiformis str. Mice were injected a total of 3 times every other week for one month. that provides viability to the amoebae be "hijacked" by the captured M. leprae to provide long-term viability to the bacillus? ATCC 50237, and H vermiformis str. 1.5×107 bacilli were used to infect 3×106 (MOI = 5) amoebae and, based on microscopic field counts, the estimated number of M. leprae harvested from amoebic cysts and injected into FPs was between 105–106 per injection. Dockrell 9B; panel i) and in 4 out of 5 of those from each category challenged with bacilli extracted from 35 day cocultures of A. castellanii or A. polyphaga (Fig. Viable M. leprae was obtained from the National Hanson's Disease Programs, Baton Rouge, LA. Fig.
Cunanan A. 9A, panel 2; compare photo insets). The dead and live bacteria were assessed by direct observation of fluorescent red (PI+) and green (Syto9+) bacilli respectively under a fluorescence microscope using appropriate single bandpass filter sets [FITC filter (480 nm excitation/500 nm emission for Syto9); TRITC filter (488 excitation/653 excitation for PI)]. P.C. (, Blackwell Arrows indicate acid-fast+ staining peripherally in amoebic cysts. 7 (panels A–J) compares uninfected vs. M. leprae-infected emergent trophozoites for all five of the amoebae genera/strain cocultures and shows that auramine/rhodamine+ bacilli (arrows) were present within the cytoplasm of all emergent trophozoites. Athymic FoxN1nu/FoxN1nu (designated as “nu/nu” throughout this manuscript) mice, five in each group, were challenged in the plantar surface of the left hind foot with M. leprae bacilli extracted from A. castellanii or A. polyphaga cysts as described [27]. Recently, it was shown that M. leprae could be taken up by FLA, survive and remain viable intracellularly in these protozoa for a period of at least 72 hr [42]. These data provide a proof of concept that M. leprae can be phagocytized and lysosomally occupy common environmental FLA trophozoites, survive encystment while remaining viable and are fully capable of infectivity under suboptimal conditions endured by the amoebic cyst.
per unit time.
Extracellular bacilli were removed by centrifugation at 600xg and washing the amoebae pellet in HBSS (Hank's Balanced Salt solution) 3 times.
L. Y. Virtually all of the bacilli extracted from long-term cocultures at 32°C were propidium iodide negative (PI-) and Syto9+ indicative of viable organisms. C.H. Bacilli extracted from amoebae cyst cultures were washed in normal saline (0.90% NaCl w/v) and incubated for 15 min at RT with a final concentration of 1.67 mM Syto9 and 18.3 mM propidium iodide (PI). This procedure was performed monthly for 6 months. Plata Auramine/rhodamine was used to visualize acid-fast bacilli (such as mycobacteria) using fluorescence microscopy. 10). In addition, detection of RLEP in archeological samples is performed using the more sensitive TaqMan PCR methodology. [35]. In the current study, we demonstrate that M. leprae can survive and remain virulent for at least 35 days within amoebal cysts from both A. castellanii and A. polyphaga as determined by their ability to transfer infection to recipient nu/nu mouse FPs. 7D) indicative of being arranged in such a manner within the cysts. Kwaso R.R. Significant amounts of red-staining bacilli were observed in biopsies derived from the positive control samples (Panel B, i) as well as both amoebae cocultures (Panel B; iii and iv). (, Eichenwald It is a rod-shaped, Gram-positive organism that is acid-fast when stained by the Ziehl–Nielsen or the better Fite methods. (, Klatser E. Interestingly, intact auramine/rhodamine stained bacilli in long-term cocultures were observed to be somewhat distorted (more rounded as opposed to rod-shaped) as has been observed in other mycobacteria-amoebae systems [10], [11], [16]. Aliquots were taken at the time of infection and each hr after 2 hr of incubation and analyzed by flow cytometry.
This confirms that the acid-fast bacilli observed in FP lesions produced by challenge with bacilli extracted from amoebae are most likely M. leprae and that the bacilli remain viable and capable of transmitting FP pathology for up to 35 days in cocultures of two different Acanthamoeba spp.
J.A.C. https://doi.org/10.1371/journal.pntd.0003405.g003. T. Malema Tekle Haimanot That is to say, the process of the selection of environmental microorganisms for resistance to digestion by predatory FLA behaving as feral macrophages might be a driving force in the evolution of pathogenic environmental bacteria. L.M.
5 and 7). Thuc Because the causative organism, Mycobacterium leprae cannot be cultured in vitro, it is virtually impossible to assess exposure, and the onset of infection and disease. (, Schurr R.R.P. (, Abel Irgens As a consequence, the chain of infection, considered as the relationships between M. leprae, transmission and human host, is poorly understood. During the 6.5 months post-challenge, measurable swelling of the left FP was consistently evident in the positive control animals. Yes Other means such as transmission through insects [40] [41] has been considered but there has not been any substantial evidence supporting that claim. (, Walsh
Gros Collectively, these data confirm that M. leprae can indeed survive for extended periods of time in encysted FLA cultures and is capable of growth in nu/nu mice FP. Shaw It will be fascinating to determine whether FLA in general provide an environmental sanctuary possibly facilitating virulence and contributing to microbial survival in harsh conditions along with aiding transmission to susceptible hosts. To assess growth and counting efficiency of M. leprae in FPs real-time TaqMan PCR assays were performed. Qimaging and Slidebook software (Intelligent Imaging Innovations, Inc., Denver, CO) were used for image acquisition and analysis on a Macintosh G5 dual processor computer (Apple Computer, Cupertino, CA).
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