TEMPERATURE INDUCED CHANGES IN THE FEEDING FORM OF THREE SPECIES OF ACANTHAMOEBA: POTENTIAL RELATIONSHIPS TO FRANCISELLA TULARENSIS, THE CAUSATIVE AGENT OF TULAREMIA James R. Palmieri, Ph.D., Department of Microbiology, Virginia College of Osteopathic Medicine and Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg VA, Katherine J. Barter, B.S., MT., CMMID, Virginia-Maryland Regional College of Veterinary Medicine and Muna Suliman, Ronald E. McNair Post baccalaureate Achievement Program, Center for Academic Enrichment and Excellence, Virginia Tech, Blacksburg, VA IntroductionConclusionDiscussionReferencesAbstract

1. Abd et al. 2003. Survival and Growth of Francisella tularensis in Acanthamoeba castellanii. Applied and Environmental Microbiology. Vol. 69 No. 1 p. 600-606.

2. Francine Marciano-Cabral and Guy Cabral. 2003. Acanthamoeba species. as Agents of Disease in Humans. Clinical Microbiology Reviews. Vol.16 No.2, p. 273307.Experimental DesignWe are currently studying the mechanics of survival of F. tularensis colonies and its symbiotic relationship within the trophozoite and cyst forms of three species of Acanthamoeba subjected to decreased temperature: A. astronyxis (nonpathogenic), A. castellanii (semi-pathogenic) and A. culbertsoni (highly pathogenic). In the environment, F. tularensis may be capable of surviving extended periods of time under adverse conditions; Acanthamoebae are able to remain protected in cyst stage for up to 24 years. When acanthamoebae are subjected to adverse environmental conditions, trophozoites change into pre-cyst form, which then alters into the highly protective cyst stage. Our study identified a unique, previously unreported variation in the behavior of three species of Acanthamoeba at lower temperatures. When Acanthamoeba trophozoites are placed in temperatures decreasing from 25C to 20C for 24 hours, pre-cyst stages rapidly develop into cyst forms. A unique and unreported event takes place when cyst stages are lowered from 18C to 3C. The cyst stage of Acanthamoeba reverts back to the trophozoite feeding stage. As temperatures are lowered further to -2C, all newly formed trophozoites encyst. Experiments are presently underway to determine whether the cold induced trophozoites are capable of feeding on F. tularensis. This may help clarify Francisella's existence within the environment and explain the relationship it has with Acanthamoeba, which might be used as a vehicle for surviving in the environment. This relationship represents one of the most scientifically intriguing questions to be answered regarding the epidemiology and natural transmission of tularemia.ResultsFigures 1 and 2: Trophozoite stage of Acanthamoeba with pseudopodia (a) and feeding cup (b).SIGNIFICANCE AS A BIOWEAPON: Free-living Acanthamoeba are commonly found in aquatic systems as a part of natural biofilms. Acanthamoebae exist in both the trophozoite feeding and the highly resistant, dormant cyst stage. Acanthamoeba and pathogenic bacteria are closely involved in complex symbiotic relationships2.

Francisella tularensis, the causative agent of tularemia, is classified by the CDC as a Category A biological weapons agent. This is due to its ease of dispersion through drinking water and via inhalation of aerosols. An exposure of as few as ten F. tularensis bacteria can induce severe and often fatal pulmonary tularemia.

Abd1 experimentally inoculated and cultivated F. tularensis with A. castellanii. The infectious process began when trophozoites of Acanthamoeba engulfed F. tularensis bacteria, which then replicate and grow within vacuoles, a process resembling Francisella infection in macrophages.The extreme virulence of F. tularensis makes it an exceptionally dangerous pathogen. It is considered a unique and extremely dangerous potential agent of bioterrorism. The location and form of existence of F. tularensis in the environment, as well as the nature of its relationship with Acanthamoeba currently remains unknown1. There is presently no approved vaccine for humans against tularemia. Three species of Acanthamoeba, acquired from the American Type Culture Collection, Manassas, Virginia, (A. castellanii ATCC #30234; A. astronyxis ATCC # 30137; A. culbertsoni ATCC # 30171) were grown in sterile Oxoid based culture medium (Oxoid liver digest, dextrose, proteose peptone, yeast extract, added to Page amoeba saline and supplemented with hemin solution and donor calf serum) according to techniques used by Marciano-Cabral2. Acanthamoebae were cultured using 275 mL Corning vented cell culture flasks with canted necks in individual environmental incubators (A. astronyxis and A. castellanii at 25C.; A culbertsoni at 37C.).

After 24 hours incubation, trophozoites were counted using a hemacytometer. Individual culture flasks were then placed in a refrigerated environmental chamber for 24 hours until the temperature reached 18C. Samples were taken from the flasks, and trophozoite and cyst forms were counted. The flasks were replaced in the chamber at 12C for 24 hours. The entire process was repeated 4 more times, through a final temperature of -2C.No one knows how F. tularensis survives in the natural environment. F. tularensis has been successfully cultivated within Acanthamoeba. Data from our study indicates that Acanthamoeba can excyst and revert to the trophozoite form as environmental temperatures are lowered, allowing trophozoites to potentially feed upon Francisella. Once inside the Acanthamoeba, Francisella has the potential to be retained in vacuoles as temperatures continue to lower, presenting Francisella bacteria with a means for over wintering in the natural environment. The potential for use of Acanthamoeba containing Francisella as a waterborne bio-weapon is highly significant. This work was supported in part by funds from the Virginia College of Osteopathic Medicine Interdisciplinary Research Program and by the facilities provided by Thomas J. Inzana, Ph.D, VA-MD Regional College of Veterinary Medicine, Department of Biomedical Sciences and Pathobiology. Trophozoites of Acanthamoeba (Fig. 1) typically use phagocytosis and pinocytosis to feed on bacteria and organic matter. They exhibit pseudopodia, which form a food cup (Fig. 2) for ingesting nutrients. When environmental conditions become adverse, trophozoites enter the precyst stage. If environmental conditions continue to deteriorate, pre-cysts will develop into the protective cyst stage. Acanthamoeba cysts have a wrinkled appearance composed of a double walled ectocyst and endocyst. Cyst formation occurs under conditions such as food deprivation, desiccation, pH alteration, and changes in temperature. The cyst form is resistant to biocides, chlorination, and antibiotics. Cysts can survive in a cold, moist environment (4C) for up to 24 years. Our data indicate all three species of Acanthamoeba encyst in response to depletion of nutrients in the media. When the temperature of the organism was lowered to 18C over a 24 hour period, many of the cysts returned to the trophozoite feeding stage. This represents a new, unreported finding for Acanthamoeba. Our data for A. culbertsoni, the highly pathogenic strain that grows optimally at 37C, indicated trophozoite populations increased as temperature was lowered from 18 C to 0C. Below this temperature, the organism began to revert back to cyst form. Trophozoite populations of both A. castellanii and A. astronyxis peaked at 8C and 5C respectively; both normally grow at 25 C. When temperatures remained at -2C for 72 hours, all trophozoites became cysts. This data represents new information about the lifecycle and feeding behavior of Acanthamoeba, and indicates a potential for development of this organism as a bioweapon.

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