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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, VirginiaMaryland 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
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.
Results
Discussion
Growth Rate of A. astronyxis Trophozoites at
Low Temperatures
5.00E+06
4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
-2
0
5
8
12
18
25
Temperature in Degrees Celsius
Growth Rate of A. castellanii Trophozoites at Low
Temperatures
7.00E+06
Number of Amoeba
We 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 25˚C to 20˚C for 24 hours,
pre-cyst stages rapidly develop into cyst forms. A unique and
unreported event takes place when cyst stages are lowered
from 18˚C to 3˚C. The cyst stage of Acanthamoeba reverts
back to the trophozoite feeding stage. As temperatures are
lowered further to -2˚C, 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.
Introduction
Number of Amoeba
Abstract
6.00E+06
5.00E+06
4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
-2
0
5
8
12
18
25
Temperature in Degrees Celsius
Growth Rate of A. culbertsoni Trophozoites at Low
Temperatures
Experimental Design
5.00E+06
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 18°C. Samples were taken from
the flasks, and trophozoite and cyst forms were counted.
The flasks were replaced in the chamber at 12°C for 24
hours. The entire process was repeated 4 more times,
through a final temperature of -2°C.
Number of Amoeba
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
25°C.; A culbertsoni at 37°C.).
Conclusion
4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
-2
0
5
8
12
18
25
37
Temperature in Degrees Celsius
Trophs per flask
Cysts per flask
References
1. Abd et al. 2003. “Survival and Growth of Francisella tularensis in
castellanii”. Applied and Environmental Microbiology. Vol. 69
No. 1 p. 600-606.
Figures 1 and 2: Trophozoite stage of Acanthamoeba with
pseudopodia (a) and feeding cup (b).
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 (4°C) 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
18°C 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 37°C, indicated trophozoite populations
increased as temperature was lowered from 18 °C to
0°C. Below this temperature, the organism began to
revert back to cyst form. Trophozoite populations of
both A. castellanii and A. astronyxis peaked at 8°C and
5°C respectively; both normally grow at 25 °C. When
temperatures remained at -2°C 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.
Acanthamoeba
2. Francine Marciano-Cabral and Guy Cabral. 2003. “Acanthamoeba species. as Agents
of Disease in Humans”. Clinical Microbiology Reviews. Vol.16 No.2, p. 273–307.
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.