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The Abstracts of the XIIth International Meeting on the Biology andPathogenicity of Free-Living Amoebae
27-30 August 2007, National Institute of Public Health,Wako-city, Japan
Keynote Lecture (Kn)-01
Free-living amebae and infection of the central nervous system
Francine MARCIANO-CABRAL(Dept. of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, USA)e-mail: fmcabral@vcu.edu
Infection of the central nervous system by free-living amebae Naegleria fowleri, several species of Acanthamoeba, and Balamuthia mandrillaris, is almost always fatal. The term amphizoic has been applied to theseamebae since they are free-living but can cause disease in humans and animals. For the most part, the mechanismsof pathogenicity and the characterization of virulence factors contributive to the pathogenic process remain to be
defined. Acanthamoeba cause Granulomatous Amebic Encephalitis (GAE), a chronic disease that is more common
in debilitated and immune suppressed individuals. A number of Acanthamoeba isolated from clinical cases ofacanthamoebiasis have been shown to harbor endosymbionts and pathogenic bacteria, indicating a potential forincreased pathogenesis. Recent results suggest that proinflammatory cytokines produced in response to Acanthamoeba and direct destruction of host cells by amebae contribute to neuropathology, in addition to proteases andphospholipases that the amebae secrete. Balamuthia mandrillaris causes encephalitis that also is a chronic progressive infection of the CNS. Balamuthia destroy mammalian cells in culture and induce proinflammatory cytokines from microglia that may contribute to neuropathology. Recent studies suggest that these amebae access thebrain by attaching to the extracellular matrix protein, laminin. Naegleria fowleri causes Primary Amebic Meningoencephalitis (PAM), a rapidly fatal infection of the CNS. N. fowleri has “developed” strategies to evade the hostimmune response; it is resistant to complement-mediated lysis and destroys antibodies. Induction of proinflammatory cytokines and lytic pore-forming proteins appear to be major factors in the pathogenesis of infection. In summary, there is an increasing awareness that free-living amebae have the potential to serve as human pathogens.
This potential may be heightened in view of the increasing number of individuals whose immune systems arecompromised due to immune suppressive therapy, infection with the human immunodeficiency virus, or use ofillicit drugs.
(Supported in part by NIH (USA) awards DA05832 and DA0527)

Kn-02
From Hartmannella to the genome project: Past and future of Acanthamoeba research
Gregory C. BOOTON(Dept. of Evolution, Ecology and Organismal Biology, The Ohio State University, USA)e-mail: booton.1@osu.edu
Found in a wide variety of ecological niches, the ubiquitous isolates of the free-living genus Acanthamoeba are
one of the most extensively studied protists in science. Since its discovery and initial placement in the genus
Hartmannella in 1930, until the recent inclusion of Acanthamoeba in a genome-sequencing project determining
the primary nucleotide sequence of the estimated 33Mb genome of Acanthamoeba, this genus has been extensively studied. Acanthamoeba has provided a model system of fundamental aspects of cell biology, for studies ofcellular movement, of cytoskeletal structures, of mechanisms and variability of pathogenicity, for phylogenetic
and evolutionary studies investigating eukaryotic evolution and protistan relationships, and recently in studies
investigating mechanisms and evolution of symbiotic events. Numerous bacterial species have been shown to be
temporary or long-term symbionts of Acanthamoeba. This relationship may offer protection and enhance thepathogenic potential of these bacteria. The understanding of these host/symbiont relationships is an active area ofcurrent investigations. In addition, the existence of gene transfer from symbionts to Acanthamoeba is also underinvestigation. The genome project is being performed on Acanthamoeba castellanii Neff, a widely studied isolate
whose mitochondrial genome has been sequenced previously. Classification of Acanthamoeba species and iso-162

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lates has been enhanced in recent years by molecular sequencing methods, resulting in a more quantitative, robust,and repeatable identification of isolates. Sequencing of the A. castellanii Neff genome will allow researchers to
expand numerous aspects of Acanthamoeba research and provide insights when genome-wide comparisons toother protists are completed. However, significant sequence variation has been observed between different Acanthamoeba species, and the sequencing of the Neff genome alone will not completely answers questions regarding
the differences between Acanthamoeba species. Nonetheless, the excitement provided by the current genome
project cannot be overstated and its significance to the current status of Acanthamoeba research will be discussed

Oral (Or)-01
Environmental behavior of FLA released by artificially heated water
Herbelin PASCALINE1*, Pougnard CLAIRE1, Lehericey EMILIE1, Loones NICOLAS1,
Pujo HERVÉ2(1EDF, National hydraulics and environment laboratory, 2Puj’eau measures, France)*e-mail: pascaline.herbelin@edf.fr
Free-living amoebae (FLA), such as the pathogenic Naegleria fowleri, can proliferate in water cooling circuits
due to the increase of temperature. Presence of N. fowleri is detected in the cooling circuits of nuclear power
plants. In order to reduce the quantity of N. fowleri released in the aquatic environment, water treatments (UV or
monochloramine) are performed on cooling circuits. These treatments reduce the amoebal concentration in thecircuit waters in order to respect the authorized values in downstream rivers. However there is still some small
concentration of N. fowleri released in the river and nothing is yet known about the long term effect of this pathogen release on the FLA community either in the water column and/or the sediment river.
The FLA could either survived and be transported in the water column and/or fall in the sediment where they
could stay alive and eventually be released in the water column under certain environmental conditions. All theses
hypothesis were studied by sampling water and sediment in plant downstream rivers (Vienne River, Loire River,Garonne River). Several campaigns were performed between 2004 and 2006 upstream and downstream of theplants. The identification and quantification of thermophilic and non thermophilic amoebae, Naegleria species andN. fowleri were performed using traditional methods of culture (at 30°C and 43°C). A flagellation test and a
ELISA assay were performed respectively to confirm the identification of Naegleria genus and N. fowleri species.
The results of the campaigns showed that some N. fowleri were detected in the river, closest to the plants but theconcentration of N. fowleri measured was always more or less equal to the detection limit. Moreover, as the distance from the plant increased, the percentage of positive samples decreased. Finally, no accumulation of N. fowleri was detected in sediments and no sign of N. fowleri transfer from sediment to water was observed.

Or-02
Selecting treatments to control pathogenic amoebae Naegleria fowleri in cooling systems:
An integrated approach for power plants
S. SOREAU*, M. Le BRUN, B. RICHARD(EDF R&D, France)*e-mail: sylvie.soreau@edf.fr
Closed loop water-cooling circuits of nuclear power plants are likely to face pathogenic free-living amobae(Naegleria fowleri) proliferations. Therefore EDF has implemented monochloramine as an effective cooling water
treatment since 1999. However, to reduce the chemical releases in the environment, alternatives are explored.
Compared to other cooling circuits, EDF nuclear power plants circuits are characterized by high water flows(several thousands m3/s), high exchange areas (several millions m2) and a river water supply without any pretreatment. Thus, the choice of the appropriate disinfection treatment is always a tricky issue. Four major criteria haveto be attained: effectiveness, absence of environmental and public health impact, preservation of equipment andthermal exchange performances as well as cost-acceptability. For these purposes, EDF R&D has developed amethodology in three phases:
First phase: static laboratory experiments assessing:Jpn. J. Protozool. Vol. 40, No. 2. (2007)163> the effectiveness of the treatment: e.g. determination of the couples biocide concentration/application time withregard to pathogens reduction.
> the behaviour of the biocide with regard to water supply quality: characterization of biocide consumption andidentification of its by-products.
These trials lead to select biocides for the second phase.
Second phase: experiments conducted with two industrial pilots simulating nuclear power plants cooling circuits(one of which being used as a reference without any treatment) to:> test the treatment in dynamic operating conditions,> identify the optimal treatment conditions with regard to effectiveness in water as well as in biofilms, biocide
consumption by surfaces, nature and quantity of by-products...
These trials lead to a first sizing on full scale and assess the nature and quantity of products released by the treatment.
Third phase: When first and second phases results are promising, complementary studies can be conducted toevaluate the ecotoxicity of the discharge.
Different treatments have been tested using this approach: chlorine dioxide, peracetic acid, hydrogen peroxide,
combination of hydrogen peroxide and UV, organic filming agent. Results are presented and compared withmonochloramine as a reference.

Or-03
Ecology of free-amoebae in real in-house water networks- Amoeba/Legionella interactions: disinfection elements –Florence MÉNARD-SZCZEBARA1*, Nelsie BERTHELOT1, Dorothée CAVEREAU1, Sandrine OBERTI1,
Yann HÉCHARD2, Virginie SARROCA2, Delphine RIVIÈRE2 , Stéphane MAZOUA2, France WALLET3(1VEOLIA Environnement, Research Centre for Water - Chemin de la digue, 2UMR CNRS, Laboratory of Chemistry of Water and Environment, 3Electricité de France, Service des Etudes Médicales, France)*e-mail: florence.menard-szczebara@veolia.com
Free amoebae are protozoa largely encountered in environmental and hydric niches. They have the ability tomultiply without the help of any host. A few species are direct human pathogens (e.g. Naegleria fowleri) and a lotof them can be reservoirs for pathogenic bacteria like Legionella pneumophila.
To evaluate the occurrence and the characteristics of free living amoebae in in-house water networks, an analytical campaign was conducted in real distribution systems (cold and hot water). Moreover, another objective wasto study the impact of curative treatments on amoebae and intra-amoebae Legionella. The applied treatments were
those usually used and accepted by French authorities on real networks to manage Legionella risk.
Methods: Analysis campaign and Amoeba identification: 36 sites were tested for the presence of amoebae (fivesampling points for hot and cold water). Total amoebae were quantified by the method (MPN) described by Pernin
et al. Identification was made by microscopic morphology determination.
Treatment assays: Different disinfection or cleaning treatments were studied in vitro on an amoeba model(Acanthamoeba castellanii) which was infected or not by Legionella pneumophila. The treatments were:> Thermal shock : : 70°C, 1h> Chlorine shock: 50 mg/l, 1 h> Chlorine dioxide: 1; 5; 10 mg/l, 1h> Basic shock: pH 12 , 1 hResults & Conclusions: In French in-house networks, the most representative amoebal genus is Hartmannella.
For several samples, Acanthamoeba were isolated.
Among installation network parameters (temperature, pipe material, presence of loops, type of hot water production), temperature (up to 60°C) seems to be the preponderant parameter controlling the presence of amoebae.
Disinfection assays show that
> Chlorine treatment permits a 2.5 Log decrease of amoebal trophozoites but does not lead to encystations whilethermal and basic treatments promote encystation.
> Our results confirm the protective effect of the amoebae for Legionella against disinfection based on oxidative
molecules.
These results may have an impact on the way to manage in-house water networks.

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Or-04
Legionella species in amoebae isolated from saline environments
R. J. GAST1*, D.M. MORAN1, M.R. DENNETT1, J. ROCCA2, L. AMARAL-ZETTLER2( Woods Hole Oceanographic Institution, 2Marine Biological Laboratory, Woods Hole, USA)*e-mail: rgast@whoi.edu
1

Amoeba cultures were enriched from sediments from four sites in the New England estuarine system of Mt
Hope Bay, and from sediments from Farmington Bay on the east side of Antelope Island in the Great Salt Lake.
Sampling sites in Mt Hope Bay included the thermal plume region of a power plant, a secondary sewage outfall, abrackish environment and a coastal marine region considered impacted by normal bay conditions. The sites in the
Great Salt Lake included a canal used by sewage treatment plants to release water into Farmington Bay, areas near
the opening in the causeway that separates the Farmington Bay from Gilbert Bay, the bathing beach in Bridger
Bay (in Gilbert Bay) and in Bear River Bay. Cultures were enriched using both minimal and non-nutrient agar
plates, made with fresh water, brackish water or saltwater. Recovered amoeba cultures were assayed for the presence of Legionella species using nested PCR and primers specific for the genus, and positive samples were thenscreened with nested primers specific for L. pneumophila. Fifty-two of 90 isolated amoeba cultures were positive
for the presence of Legionella species. L. pneumophila was detected by PCR in ten of the amoeba cultures growing on marine media. Our results show that amoebae capable of growing in saline environments harbor not only adiverse collection of Legionella species, but also species pathogenic to humans.

Or-05
N-chlorotaurine (NCT) reveales highly cysticidal activities against Acanthamoeba spp.
Ursula FÜRNKRANZ1*, Markus NAGL2, Waldemar GOTTARDI2, Martina KOEHSLER1,
Horst ASPOECK1, Julia WALOCHNIK1(1Dept. of Medical Parasitology; Clinical Institute of Hygiene and Medical Microbiology, Medical University of
Vienna, 2Dept. of Hygiene, Microbiology and Social Medicine, Division ofHygiene and Medical Microbiology Innsbruck, Medical University Innsbruck, Austria)*e-mail: ursula.fuernkranz@meduniwien.ac.at
Objectives: Acanthamoeba spp. are the causative agents of Acanthamoeba keratitis (AK) occurring mainly incontact lens wearers, and of skin lesions, granulomatous amoebic encephalitis (GAE) and other disseminating
infections in the immunocompromised host. Problems of therapy are the high toxicity of the drugs and the resistance of the cysts against them. Therefore, new anti-Acanthamoeba drugs are needed. We investigated the antiAcanthamoeba activity of N-chlorotaurine (NCT), an endogenous mild antiseptic. Tolerability of 55 mM (1%)
NCT on the human skin and up to 0.1 M (3%) in the human eye was demonstrated before.
Methods: Trophozoites and cysts were cultured according to their needs and were treated with NCT andcotreated with NCT and NH4Cl and β-alanine, respectively in different culture media. Effectiveness was observed
after 1, 6 and 24 h by microscopy and trypan blue staining.
Results: It was shown that NCT has amoebicidal and cysticidal qualities, both in phosphate buffered saline(PBS) and in specific amoebic culture medium. Already after 6 hours treatment with 10 mM NCT, trophozoites ofall strains investigated showed at least a 2 log10 reduction. A delay of excystation was observed when cysts weretreated with 55 mM (1%) NCT, whereas a complete failure of excystment was achieved by cotreatment with 1%
NCT and 1% NH4Cl. Efficacy of NCT was generally higher in PBS, than in culture medium.
Conclusion: Altogether, NCT clearly demonstrated amoebicidal activity at concentrations well tolerated byhuman tissues and might be useful as a topic drug in Acanthamoeba infections. Addition of ammonium chloride
can be considered to enhance the activity.

Or-06
Anti-Acanthamoeba efficacy and toxicity of miltefosine in an organotypic skin model
J. WALOCHNIK1*, A. OBWALLER2, F. GRUBER3, M. MILDNER3, E. TSCHACHLER3,

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165

M. SUCHOMEL4, M. DUCHÊNE5, H. AUER1( Dept. of Medical Parasitology, 3Dept. of Dermatology, 4Dept. of Decontamination, 5Dept. of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, 2Orphanidis Pharma Research, Austria)*e-mail: julia.walochnik@meduniwien.ac.at
1

Acanthamoebae are the causative agents of the so called Acanthamoeba keratitis (AK) on the one hand and ofdisseminating infections such as skin lesions, pneumonitis and the granulomatous amoebic encephalitis occurring
mainly in immunocompromised individuals on the other hand. The treatment of Acanthamoeba infections is still
problematic, due to the lack of sufficiently effective and also easily manageable drugs. In a previous study wehave shown that out of eight different alkylphosphocholines tested miltefosine (hexadecylphosphocholine) had thehighest activity against Acanthamoeba spp.
In the current study the suitability of miltefosine for the topical treatment of Acanthamoeba eye and skin infections was evaluated in vitro. The storage life and the time dependency of the efficacy of different concentrations ofmiltefosine against trophozoites and cysts of various Acanthamoeba strains were assessed in a microtiter platesystem. Moreover, the susceptibility of other important eye pathogens to miltefosine and synergistic and adverse
effects of combinations of miltefosine with other anti-Acanthamoeba substances were evaluated. An organotypicskin model was adapted for investigating the penetration of acanthamoebae into the epidermis and the humantissue tolerability against anti-Acanthamoeba effective concentrations of miltefosine. It was shown that miltefosine can be stored as a 2 mM stock solution but also as 50 µM dilution over a period of 6 months at 4°C without
any loss of activity. Moreover, acanthamoebae were shown to penetrate the skin within 48 h, while treatment withmiltefosine prevented this penetration. The toxicity of anti-Acanthamoeba effective concentrations of miltefosine
lies considerably below those of substances used for AK treatment.
Miltefosine has been successfully used for the oral and topical treatment of leishmaniasis and may therefore
also be a promising new candidate for the topical treatment of Acanthamoeba infections.

Or-07
Effect of therapeutic chemical agents in vitro and in experimental meningoencephalitis
due to Naegleria fowleri
Jong-Hyun KIM 1*, Yang-Jin LEE1, Ae-Hee YANG1, Kyung-Jin CHOI1, Anh Trinh Minh DANG1, A-Rum
SONG1, Hae-Jin SHON1, Kyoung-Ju SONG1, Daeho KWON1, Kyung-il IM2, Ho-Joon SHIN1(1Dept. of Microbiology, and Dept. of Molecular Science & Technology, Ajou University School of Medicine,
Korea, 2Dept. of Parasitology, Yonsei University College of Medicine, Korea)*e-mail: lackesis@ajou.ac.kr
Objective: Inhalation of fresh water containing the free-living ameba, Naegleria fowleri, leads to a potentiallyfatal infection known as primary amebic meningoencephalitis (PAME). Amphotericin B is an only agent withclinical efficacy in the treatment of PAME in humans, but therapy with this drug is often associated with adverse
effects on the kidney and other organs.
Methods: In an attempt to select other useful therapeutic agents for treating PAME, the amoebicidal activities ofthe drugs, such as antibacterial (clarithromycin, erythromycin, hygromycin B, neomycin, rokitamycin, roxithromycin, and zeocin), antifungal (amphotericin B), antiprotozoan (miltefosine), and anti-brain disease antipsychotic
agent (chlorpromazine) were examined in this study. Then, the in vivo efficacy of the drugs showed the effective
amoebicidal activity was investigated in a mouse model of experimental meningoencephalitis due to N. fowleri.
Results: In results, the growth of amoeba was inhibited effectively in treatment of hygromycin B, rokitamycin,
roxithromycin, amphotericin B, miltefosine, chlorpromazine, respectively. Particularly, when the N. fowleri wastreated with rokitamycin and amphotericin B, the minimal inhibitory concentrations (MIC) of the drug were 6.25 μg/
ml and 0.39 μg/ml on 2 day. Also, when the amoeba was treated with hygromycin B, rokitamycin, and amphotericin
B on 6 days, each drug showed 100% of amoebicidal activities against N. fowleri. In experimental meningoencephalitis, survival rates of N. fowleri infected mice treated with roxithromycin, amphotericin B, miltefosine,
chlorpromazine, and rokitamycin showed 25%, 40%, 55%, 75% and 80% during one month, respectively. Theiraverage of mean time to death was 17.1 days, in comparison with 11.2 days in control. In all cases, effect of drug
was the best in 3 times administrations following 3, 7, and 11 days at dose of 20 mg/kg except amphotericin B (10mg/kg).
Conclusion: Finally, rokitamycin and chlorpromazine have both in vitro and in vivo activities against N. fowleri

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and may be useful therapeutic agents for treatment of PAME, instead of amphotericin B.
Key Words: Naegleria fowleri, amphotericin B, rokitamycin, chlorpromazine, PAME

Or-08
Structure-activity relationship and selectivity of amoebocidal activity
Kazuaki HIGASHI*, Hiroshi HORI, Miyuki OTSUKA, Tadayuki ISHIYAMA(Dept. of Life Science, Faculty of Agriculture, Tamagawa University, Japan)*e-mail: khigashi@agr.tamagawa.ac.jp
At the XIth International Meeting on the Pathogenic Free-Living Amoebae, we had explained a screening
method for amoebocidal compounds from microbial metabolites; a diffusion method with impregnated paper discon agar plated for inhibition of growth of Naegleria. The active compound, which was later identified as 1-monoO-linoleoyl-grycerol, had inhibited the growth of N. lovaniensis trophozoites at MIC of 31.2µg/ml by the diffusion
method and induced cell destruction at IC50 of 8.0µg/ml in a hole-slide glass.
In the present experiment, we have examined the quantitative structure- activity relationship analysis of amoebocidal activity of this compound and its selective activity on amoeba species. Using a hole-slide glass with 10
wells with a diameter of 6mm and 0.1mm in depth, each of the wells was filled with 20µl of different compound
solutions at a final concentration of 100µg/ml in ethanol-Tween 80, and was air-dried. Then, 20µl of the amoeba
suspension (around 100cells/well) supplemented with 0.25% Trypan Blue was transferred into the wells, and thewhole-slide glass was attached firmly with a cover glass with double-sided sticky tape to make wet chambers.
Observation was made under light microscope for up to 120 min to determine the number of cell death.
It was demonstrated that around 90% of the N. lovaniensis trophozoites underwent destruction within first 10
min of incubation when they were transferred into a well precoated with 1-linoleoyl-glycerol, while those in a wellprecoated with 1-stearoul-glycerol remained intact. The trophozoites transferred in a well precoated with 1-oleoylglycerol gradually underwent destruction in 80 or more minutes of incubation. Interestingly, the trophozoites were
not affected by the compounds in the form of free acids, namely linoleic or oleic acid, or in the form of alcohols,
namely linoleyl or oleyl alcohol.
Possible amoebocidal activity of the compounds was also tested against other amoeba species such as Hartmannella sp. H1-1 and Acanthamoeba sp. AC-1 isolates both of which were originally isolated from the soil in our
university campus and have been maintained by serial passages in vitro in our laboratory. Hartmannella sp. HT-1
was slightly sensitive to 1-Linoleoyl-glycerol and 1-oleoyl-glycerol, and 20% to 40% of the trophozoites underwent destruction within 120 min of incubation. Neither of these compounds exhibited any amoebocidal activity
against Acanthamoeba species.

Or-09
Amoebic gill disease in farmed Atlantic salmon in Tasmania: Research progressPhilip CROSBIE*, Neil YOUNG, Richard MORRISON, Mark ADAMS,
Michael ATTARD, Barbara NOWAK(School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute,
University of Tasmania, Australia)*e-mail: Philip.Crosbie@utas.edu.au
Amoebic gill disease (AGD) has impacted on the marine culture phase of Atlantic salmon (Salmo salarL.) in Tasmania since the early 1980’s and remains a serious health problem in Tasmania. Some fundamental
aims of the research are to unequivocally identify the aetiological agent of the disease, refine the diseasechallenge model and to test the efficacy of vaccine candidates. It has been known for some time that thecausative agent(s) of AGD belonged to the genus Neoparamoeba, a marine Gymnamoeba, but identification
to species level was problematic with 2 species, N. pemaquidensis and N. branchiphila both being isolated
and cultured from AGD-affected salmon. However, using PCR amplification and specific oligonucleotide
probes on AGD-affected gill tissue significant progress has been made and the aetiological agent has now
been identified (Young et al., 2007). However, this new species remains to be cultured. Two AGD challenge
models have been validated and used to monitor attachment of the amoeba to the gills and the onset of pa-

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thology and to test the efficacy of prospective vaccines. The first is a short term model with potential to test
the ability of treatments to block amoeba attachment to the gills and the second a traditional challenge to a
pre-determined level of morbidity in challenged salmon. This paper will present an update of the most recent
developments.
Young, N.D., Crosbie, P.B.B., Adams, M.B., Nowak, B.F. and Morrison, R.N., 2007. Neoparamoeba perurans n.
sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar) International Journal of Parasitology (inpress)

Or-10
Another real-time PCR assay detecting Balamuthia mandrillaris DNA
Albrecht F. KIDERLEN*, Elke RADAM, Phiroze S. TATA, Astrid LEWIN(Cellular Immunology Unit P22, Robert Koch-Institute, Germany)*e-mail: a.kiderlen@rki.de
A TaqMan real-time PCR assay was established specifically targeting the RNase P gene of B. mandrillarisamoebae. The assay detected down to 0.3 amoebae-equivalents in dilutions of DNA extracted from axenic B.
mandrillaris cultures. The assay did not detect DNA from closely related Acanthamoeba species (A. hatchetti 2HH, A. lenticulata 72/2, A. castellanii 1BU), nor DNA extracted from a murine macrophage-like cellline (RAW 264.7), murine tissues (brain, lung), a human neuroblastoma cell line (Kelly), or human tissue(brain). The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ
homogenates, infected mice, and DNA preparations from 2 patients with proven balamuthiasis. This realtime PCR assay showed similar sensitivity as the one published by Qvarnstrom et al. (2006 J Clin Microbiol
44:3589) which targets B. mandrillaris 18S ribosomal DNA and it could be combined with the latter in a
duplex assay for enhanced specificity and reliability.

Or-11
Challenges in the diagnosis of balamuthiasis
G.S.VISVESVARA1*, S.YAGI2, R.SRIRAM1, F.L.SCHUSTER2( Division of Parasitic Diseases, Centers for Disease Control and Prevention, 2Viral and Rickettsial
Disease Laboratory, California Dept. of Health Sciences, USA)*e-mail: gsv1@CDC.GOV
1Balamuthia mandrillaris is a free-living ameba and is known to cause a devastating central nervous system
infection, granulomatous amebic meningoencephalitis (GAE), in humans and other animals. In some cases thedisease may disseminate to other organs including lungs, prostate, kidneys etc. Although GAE manifests as CNS
infection in many cases the infection may occur initially as a skin ulcer especially on the extremities. In SouthAmerica, especially Peru, lesions can occur on the face and torso as well, before invading the CNS. Balamuthiasis
cases have been recorded from all over the world and the outcome, almost always, is death. There are about 150
reported cases of balamuthiasis worldwide. The infection is usually identified at autopsy but in several cases it was
identified premortem in brain biopsy sections. In three instances, because of early detection, the patients havesurvived the infection with minimal or no neurological deficits after treatment with multiple drugs. Identificationof Balamuthia, especially in the biopsied brain sections can be challenging. We discuss here the difficulties involved in arriving at a diagnosis in two recent cases. Of the two cases, one was a young girl of Hispanic ethnicity
from Colorado, and the other a 35-year-old male also of Hispanic ethnicity from California. In both cases initial
examination of H&E-stained sections did not reveal the identity of the infecting organism, even though the serum
antibody titer was ≥ 64. Additionally, in the California case, a PCR of the fresh brain was positive for Balamuthia
but the immunofluorescence staining was negative. Further examination of multiple stained and unstained sections
together with immunofluorescence and PCR finally led to the correct identification of the infecting agent. It isbelieved that both patients are under treatment for balamuthiasis and may survive this infection.

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Or-12
AFLP fingerprinting reveals intraspecific genetic diversity in Naegleria fowleri
Jonas BEHETS1*, Hellemans BART1, Lieve VERELST2, Ollevier FRANS1( Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, 2LABORELEC, Belgium)*e-mail: Jonas.behets@bio.kuleuven.be
1

Genetic diversity among different N. fowleri strains, as well as other ecological relevant amoebae species wasevaluated with the AFLP method. Discriminatory and reproducible DNA profiles were found for all different
reference species. The N. fowleri strains clustered into two main groups, containing the same strains as the twogroups obtained after rDNA sequencing studies. One subcluster of N. fowleri containing discriminatory and reproducible DNA fingerprints differentiated into three intraspecific genotypes. The results indicate that the AFLP
technique is a valuable tool for (intra)specific genotyping of Naegleria sp. This molecular fingerprinting method
could be useful in tracking the source of (possible) N. fowleri contamination in aquatic habitats or even tracking
the source of human or animal infection with this pathogen.
Keywords: N. fowleri, AFLP, intraspecific genotypes

Or-13
The phylogenetic structure of Acanthamoeba as seen through the window of the small subunit
ribosomal RNA gene: Genotypes and “species” do not reflect the same images
Paul A. Fuerst*, Gregory C. Booton(Dept. of Evolution, Ecology and Organismal Biology, The Ohio State University, USA)*e-mail: fuerst.1@osu.edu
The genus Acanthamoeba contains more than 20 described species, based on morphological or cytological characteristics. Phylogenetic analysis of the nuclear small subunit ribosomal RNA gene (Rns) suggests that species do
not correspond to phylogenetic units. Previous studies identified at least 12 sequence types within Acanthamoeba(>5% pairwise divergence between Rns sequences). Many sequence types also contain significant phylogenetic
subgroups, with divergence between 1-5%, especially in type T4, the most common sequence type seen in environmental or clinical samples. Phylogenetic analysis suggests at least six significant subtypes within T4. Data
from “almost complete” Rns sequences (sequences > 2000 bases) are available for over 185 Acanthamoeba isolates, with 133 genotype T4 isolates. Examination of correspondence between sequence type and species classification indicates that sequence types contain multiple nominal species, while particular nominal species occur inmultiple sequence types. Twenty different nominal species are represented, constituting 71 sequences, while theterm Acanthamoeba sp. applies to the remaining sequences. Twelve of twenty named taxa are represented bymultiple isolates, but only three taxa have all isolates placed within a single sequence type or T4 subtype (12 A.
lenticulata isolates in type T5; 2 A. giffini isolates in T3; 2 A. lugdenensis isolates in the same T4 subtype). A.
castellani is associated with T1 and four subtypes of T4, while A. polyphaga isolates are identified as T2, T3 orfour different subtypes of T4. The disconnection between species and genotype is even more evident when examining the distribution of nominal species within sequence types. Seven sequence types (T2, T3, T5, T6, T9,T11
and five subtypes of T4) contain multiple nominal species. Only sequence type T5 contains a single species (A.
lenticulata), while other sequence types contain 2-6 nominal species each. Clearly, species nomenclature within
Acanthamoeba has little relationship to phylogenetic classification. (Supported by grants from NIH)

Or-14
Acanthamoeba spp. in tap water of houses of contact lens wearers
Patricia BONILLA-LEMUS *, Gerardo A. RAMIREZ, Muñoz CLAUDIA, Ma. del Rocio IBARRA- MONTES, Elizabeth RAMIREZ-FLORES, Ricardo ORTIZ-ORTEGA, Castro ALEJANDRO(Project of Conservation and Improvement of Environment, Lab. of Environmental Microbiology, FES-Iztacala,National Autonomous University of Mexico, Mexico)*e-mail: blemus@servidor.unam.mx

Jpn. J. Protozool. Vol. 40, No. 2. (2007)

169

Several species of Acanthamoeba can produce Acanthamoeba keratitis (Ak) and Granulomatous Amebic Encephalitis (GAE) in human and animals. A survey was carried out in the metropolitan area of Mexico city to determine the presence of Acanthamoeba in tap water of houses of contact lens wearers and its relation with the qualityof the water. One liter samples of tap water of 27 houses were taken by duplicate and the saline solution containedin the contact lens boxes were collected. A total of 108 samples were analyzed. Samples were filtered and put onto
NNE media; plates were incubated at 25 ºC and 37 ºC respectively. The isolates were identified on the basis of
their morphological features. Total (TC) and faecal coliforms (FC), water temperature, pH, dissolved oxygen(O.D.) and residual free-chlorine were measured. Forty-nine isolates of Acanthamoeba (45.3%) from the 108 water samples were obtained. The greater number was obtained from the cistern (49%) followed by the roof tanks(27%), the municipal network intakes (12%) and tap water of wash basin of the bathroom (12%). No amoeba wasisolated from the saline solutions of the contact lens boxes. FC were detected in 37% of the water samples, and TC
in 44.4%. The higher number of amoebae was isolated from the places where the water is stored, which favour theevaporation of residual free-chlorine and thus stimulate the proliferation of amoebae, bacteria and other microorganisms are stimulated. Besides, the containers are not always are cleaned regularly; therefore the possibility ofcontamination is higher. Most of the isolates were associated with the presence of O.D. (1.2 - 5.4 mg L-1), lowlevels of residual free-chlorine (below 0.2 mg L-1) and bacterial contamination. Most of isolates were non pathogenic, however the presence of Acanthamoeba in domestic tap water is a potential hazard since some species ofthese genera are able to cause Acanthamoeba keratitis and GAE. These results show that domestic tap water is an
important source of these organisms.
This work was supported by UNAM-DGAPA- PAPIIT-IN-219406.

Or-15
Characterization and identification of various Acanthamoeba isolates from the Philippines
Filipinas F. NATIVIDAD1*, Corazon C. BUERANO1,2, Ruben Lim Bon SIONG3, Samuel Alan B. INOVEJAS1,
Dennis B.BACANI1, Shinji IZUMIYAMA4, Kenji YAGITA4, Ronald R. MATIAS1, Takuro ENDO4
1( Research and Biotechnology Division, St. Luke’s Medical Center, 2Institute of Biology, College of Science,
University of the Philippines, 3St. Luke’s International Eye Institute, St. Luke’s Medical Center, Philippines,
4
Dept. of Parasitology, National Institute of Infectious Diseases, Japan)*e-mail: ffnatividad@stluke.com.ph
Six Acanthamoeba spp were obtained from different sources. Two were from hot springs, two from human nasal cavity, and two from keratitis patients. Isolates were cloned and each clone was grown either in non-nutrient
agar plates with heat-killed E. coli and/or in proteose-peptone-yeast-glucose medium after axenization. Characterization was based on (a) trophozoite and cyst morphology using phase contrast and electron microscopy, (b) temperature tolerance, (c) growth rates in agar culture (d) growth patterns, (e) ability to migrate in an “under-agarose
system, (f) osmotolerance and (g) cytopathic effects on Vero cells.
Based on cyst morphology 5 clones belong to Group II, while one clone from nasal cavity isolates is classified asGroup III. From the DNA sequence of the 18s rRNA gene, 2 isolates were identified as identical to A. castellanii,and 1 each were identical to A. lenticulata and A. triangularis. Remaining two of the Acanthamoeba isolates are yet
to be identified.

Or-16
Occurrence of Acanthamoeba spp. in the nasal cavity of selected human populationsin Metro Manila, Philippines
Corazon C. BUERANO1*, Michael Thomas T. GONZALES1, Samuel Alan B. INOVEJAS2, Kenji YAGITA3 ,Shinji IZUMIYAMA3, Ronald R. MATIAS2, Takuro ENDO3, Filipinas F.NATIVIDAD2
1( Institute of Biology, College of Science, University of the Philippines, 2Research and Biotechnology Division,
St. Luke’s Medical Center, Philippines, 3 Dept. of Parasitology, National Institute of Infectious Diseases, Japan)*e-mail: ccbuerano@hotmail.com
Free-living potentially pathogenic and opportunistic amoebae, especially those belonging to the genus Acan-

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thamoeba are ubiquitous unicellular organisms. They have been isolated from soil, dust, air, sewers, eyewash
stations, air-conditioning units, contact lenses, nasal epithelium and many other common areas. These organisms
have two life forms, an amoeboid trophozoite from and a dormant cyst form. Some Acanthamoeba species cause
fatal granulomatous amebic encephalitis (GAE), amoebic keratitis as well as cutaneous lesions and sinusitis in
humans. They may also harbor pathogenic bacteria in their cytoplasm. In this study, nasal swabs were obtained
from volunteers of different sample populations in Metro Manila, Philippines to associate presence of ubiquitous
free-living amoebae with occupational risk. The populations consisted of fifty high-rise office workers, fifty factory workers and fifty public utility jeepney (PUJ) drivers. An additional fifty-seven unemployed volunteers were
included in the study. Organisms from the nasal swabs were allowed to grow in agar plates containing heat-killed
E. coli. Plates inoculated with nasal swabs from office and factory workers as well as from the unemployed volunteers were negative for growth of amoebae. However, cultures of nasal swab samples from 2 PUJ drivers
showed trophozoites and cysts. The amoebae were cloned and labeled as Clone D-26 and Clone D-38. Morphological characterization showed that the cysts and trophozoites are from the genus Acanthamoeba. Based on Pussard and Pons classification, Clone D-26 belongs to Group III (GIII) while Clone D-38 belongs to Group II (GII).
Results of 18srDNA sequence typing/T-typing revealed that Clone D-26 is identical to isolates of A. lenticulata
which is grouped as GIII and belongs to T5. Clone D-38 is identical to isolates which include A. castellanii whichis grouped as GII and belongs to T4. This is the first report on the detection and isolation of Acanthamoeba spp.
from nasal cavities among Filipinos.

Or-17
Recent interesting identifications of free-living amoebae from the Brussels laboratory
Johan F. De JONCKHEERE(Scientific Institute of Public Health, Belgium, Present address: Unit for Tropical Diseases,
Christian de Duve Institute of Cellular Pathology, Belgium)*e-mail: jdjonckh@ben.vub.ac.be
Since the last conference two years ago I have obtained the following interesting identifications by sequencing
PCR amplified rDNA from new isolates.
Eight new Naegleria spp. from Arctic and sub-Antarctic locations constitute two new clusters in the phylogenetic tree of the genus. The total number of Naegleria spp. is now 47.
Identification of N. byersi and N. indonesiensis in samples from Mexico (collaboration with ITSON, Cd. Obregon, Mexico). Both species were previously only known from the opposite side of the world. Identification of N.
angularis in water samples from Benin in Africa (collaboration with Institute of Tropical Medicine, Antwerp,
Belgium). This species had been detected only in Peru and is one of the few Naegleria spp. with a particular cyst
morphology.
Identification of two new Tetramitus spp. isolated in Arizona (USA), T. anasazii and T. hohokami, the latter also
found later in samples from Sinoloa and Sonora (Mexico).
Identification of Vahlkampfia ciguana in Chihuahua (Mexico), species previously found only in Spain (Isle ofTenerife).
Identification of Acanthamoeba genotype T4 in a fatal encephalitis in a patient treated for cryoglobulinemia(collaboration with Gasthuisberg University Hospital, Leuven, Belgium). Identification of genotype T5 (A. lenticulata) in a fatal disseminated Acanthamoeba infection in a hart transplant patient (collaboration with Hôpital
Pitié-Salpêtrière, Paris, France).
50 % of environmental Acanthamoeba isolates from Arizona (USA) were identified as genotype T5 (A. lenticulata), the other 50% were genotype T4. The majority of Acanthamoeba isolates in samples from Chihuahua(Mexico) and Mongolia were identified as genotype T4, but in Mexico also genotypes T1 and T15 (A. jacobsi)
were identified.

Or-18
The feeding of Balamuthia mandrillaris on cultured cells
Thelma DUNNEBACKE-DIXON(Viral and Rickettsial Disease Laboratory, State of California Dept. of Health Services, USA)Jpn. J. Protozool. Vol. 40, No. 2. (2007)171*e-mail: dunnebacke@gmail.com
Microscopic observations of live cultures of Balamuthia mandrillaris on a sheet of mammalian cells showedthat individual amebas could be distinguished and followed before the occurrence of cell sheet disruption and/or
the development of lesions, areas cleared of cells bordered by a dense rim of feeding amebas. An early indicationof ameba-cell contact was the presence of small, clear vesicles (cvs) that looked like “holes” in the cytoplasm of
the cells although such cells excluded vital dyes. The cvs occurred only in cultures with amebas; they may represent a deposit of an amebic enzymes, or the removal of cellular material. Both the number of cells with cvs, thenumber of cvs per cell and the numbers of ameba increased near expanding lesions. Timed photographs showed anameba extend a pseudopodium until the tip reached into the cytoplasm of a cell where it moved about leaving
visible change in the cell when it was retracted; amebas were seen with their pseudopodia spread in the cytoplasmof several cells at the same time; an ameba within the cytoplasm of two adjacent cells was seen to move and toexit the cells. In rounded cells near the rim of a lesion, whole amebas were seen to enter, and move about within
the cytoplasm before leaving the cell. The cytoplasm was consumed first, then the denuded nuclei that no longer
excluded vital dyes; mammalian chromosomes in the form of a mitotic plate were not consumed. Cellular invasion of monkey kidney cells or CHO cells by the pseudopodia and/or the whole ameba was seen for B. mandrillaris isolates from two humans and from two soil samples. There was no indication of cellular cytopathology prior
to the invasion by amebic pseudopodia. Balamuthia mandrillaris amebas feed by the invading mammalian cells.

Or-19
Morphological characterization of inflammation during experimental primaryamoebic meningoencephalitis (PAM)
Isaac CERVANTES-SANDOVAL1,2*, Ethel Awilda GARCÍA LATORRE1, Victor TSUTSUMI2,
Mineko SHIBAYAMA2
1( Immunology Dept., National School of Biological Science-National Polytechnic Institute, 2Dept. of Experimental Pathology, Center for Research and Advanced Studies, National Polytechnic Institute, Mexico)*e-mail: cervantessi@yahoo.com.mx
Naegleria fowleri is a free-living amoeba that produces an acute and rapidly fatal infection of the central nervous system. Trophozoites reach the brain by penetrating the olfactory epithelium, passing thru the cribriform
plaque, until reaching the olfactory bulbs. Experimental studies have shown that an intense inflammatory reaction
is produced in the brain, and it is always associated with extensive tissue damage. Detailed studies of the early
inflammation stages, as well as their role in pathogenesis of the PAM have not been addressed yet. We analyzed
the morphological changes of this inflammatory process during experimental PAM production, using both opticaland electron microscopy. After three to four days post-inoculation, several N. fowleri trophozoites were observedin the olfactory bulbs. At 3 days, no inflammation or apparent tissue damage was observed. The number of amoebae increased rapidly in the next 24 h. Soon after, eosinophils and neutrophils were seen surrounding the parasites.
Ultrastructural analysis showed features suggesting leukocytes attempt to eliminate the amoebae. The inflammatory reaction was increased rapidly, with leukocytes showing important signs of damage and evident necrosis ofparenchyma. We suggest that lysis of the host polymorphonuclear cells, added to other parasite factors, may contribute in the tissue damage. To corroborate the former hypothesis, we also used knockout (KO) CD38 mice,
which have deficiencies in leukocytes chemotaxis. Their mortality rate was compared with the parental strain
C57BL/6, after a lethal challenge with live parasites. Results showed that KO mice had a statistically significant
higher survival. The histological analysis of brain tissue of KO mice showed a delayed appearance of inflammation. This may explain the slight, but significant increase in their survival rate. We conclude that the inflammatory
reaction observed during experimental PAM may contribute importantly in the tissue damage and eventual deathof mice.

Or-20
Experimental murine granulomatous amoebic encephalitis: Immunohistochemichal
characterization of early stages of the infection

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Maritza OMAÑA-MOLINA1*, Leticia VERDÍN-TERAN2, Dolores HERNÁNDEZ-MARTINEZ1,
Leticia MORENO-FIERROS2, Patricia BONILLA-LEMUS1(1Universidad Nacional Autónoma de México, Facultad de Estudios Superiores Iztacala, Laboratorio de Microbiología Ambiental, 2Universidad Nacional Autónoma de México, Facultad de Estudios
Superiores Iztacala, Unidad de Biomedicina, Laboratorio de Inmunología. México)*e-mail: maritzaomanam@yahoo.com.mx
Early stages of experimental murine granulomatous amoebic encephalitis with Acanthamoeba castellanii were
immunohistochemically characterized after 48 and 72 h post intranasal instillation of 1 × 106/20µl trophozoites. At
the end of time point interactions, mice were sacrificed; lungs, kidneys, spleens, liver and heads were fixed andprocessed for their inclusion in paraffin, sectioned to obtain 5 µm-thick slices and then treated accordingly withimmunohistochemical techniques, using IgG of rabbit anti-Acanthamoeba and goat anti IgG of rabbit conjugatedto peroxidase, finally revealed with H2O2-diaminobenzidine and counterstained with Harris hematoxylin.
The immunohistochemical analysis of mouse tissue during the initial stages of Acanthamoeba castellanii encephalitis, revealed the invasion of trophozoites through all tissues in study. The immunolocalization of amoebae
after 48 and 72h of interaction was very similar. By light microscopy the analysis of the head’s slides showed
trophozoites in contact with the surface of the mucus layer of the olfactory epithelium. In some zones, trophozoites moved through the mucus with disruption of a number of areas. Invasion of the olfactory epithelium wasobserved. Several cyst forms were observed in the brain too.
The analysis of lung, spleen, kidney and liver showed trophozoites as well as cysts in all organs. In some cases
disorganization but not inflammation of the tissues was evident. Amoebae were located near vessels, frequently
migrating through the different tissues.
The present work describes the sequence of histopathological events following Acanthamoeba infection during
the initial penetration into nasal mucosa and other tissues. It was believed that Acanthamoeba spp. reached thebrain by hematogenous dissemination but we have proven that it is able to adhere, migrate and penetrate throughthe olfactory epithelium, probably by mechanical and enzymatic processes. In contrast to Naegleria fowleri, A.
castellanii migrates and penetrates slowly explaining the chronic course of the infection.
Immunohistochemistry is a useful technique to identify free-living amoebae which are difficult to observe withconventional staining techniques.
This study was supported by UNAM-DGAPA-PAPIIT-IN219406.

Or-21
Balamuthia mandrillaris traversal of the blood-brain barrier
Abdul MATIN1*, Ruqaiyyah SIDDIQUI1, Monique STINS2, Naveed Ahmed KHAN1(1School of Biological and Chemical Sciences, Birkbeck College, University of London, UK,
2
Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, USA)*e-mail: a.matin@sbc.bbk.ac.uk
Balamuthia mandrillaris granulomatous encephalitis is a serious human disease that almost always results indeath. However, the pathogenesis and pathophysiology of Balamuthia encephalitis remains incompletely understood. An important step in Balamuthia encephalitis is amoebae invasion of the bloodstream followed by theirhaematogenous spread. Balamuthia mandrillaris entry into the central nervous system most likely occurs at the
blood-brain barrier sites. However, it is not clear how circulating amoebae cross the blood-brain barrier. Here we
studied B. mandrillaris interactions with the human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. Adhesion assays revealed that B. mandrillaris exhibit more than 90% binding to
HBMEC in a galactose-inhibitable manner. Affinity chromatography using galactose-sepharose column, a galactose-binding protein (GBP) was isolated from detergent extracts of unlabeled amoebae. The isolation of GBP fromcell surface biotin-labeled amoebae suggested its membrane-association. One dimensional SDS-PAGE confirmed
the proteinaceous nature of GBP and determined its molecular mass in the range of 100 kDa. Using Western blotting assays, we showed that B. mandrillaris inhibits HBMEC cell cycle by modulating protein retinoblastoma
phosphorylations. Furthermore, it was shown that B. mandrillaris target zonula-occludens-1 (ZO-1) and occludin.
Both ZO-1 and occludin are involved in the formation of tight junctions, suggesting that B. mandrillaris disrupt
tight junctions to induce HBMEC permeability and/or HBMEC monolayer disruptions, leading to blood-brain
barrier perturbations. Subsequently, B. mandrillaris produced HBMEC cytotoxicity. The ability of B. mandrillarisJpn. J. Protozool. Vol. 40, No. 2. (2007)173

to produce HBMEC death may also be dependent on extracellular molecules released by B. mandrillaris. To thisend, we demonstrated that B. mandrillaris ecto-ATPases hydrolyse ATP to release ADP, which may exert toxic
effects on HBMEC. In support, suramin, i.e., P2 receptor antagonist, inhibited B. mandrillaris-mediated HBMEC
death. Future work will continue to study Balamuthia encephalitis pathogenesis which should help identify potential targets for therapeutic interventions.

Or-22
A novel model for the study in vivo of central nervous system infections due to free-living
amoebaeNaveed Ahmed KHAN, Parisa Nakhostin MORTAZAVI, Mary LIGHTFOO, Ricky DUDLEY*,
Graham GOLDSWORTHY(Dept. of Biology, School of Biological and Chemical Sciences, Birkbeck College, University of London, UK)*e-mail: r.dudley@sbc.bbk.ac.uk
A whole-organism approach to the study of disease is recognised as essential to gaining a full understanding of
the inter-relationships between infectious agents and their hosts. While vertebrate model systems are seen as immediately more relevant, it is proposed here that the use of an invertebrate model at an early stage can offer several advantages in terms of speed, cost, technical convenience, and ethical acceptance. It is shown for the first timethat the African migratory locust can be used as a model to study Acanthamoeba pathogenesis. Mature adult locusts were allocated randomly in groups of ten to different treatments. In the experimental groups, each locust was
injected intra-abdominally with 10 μl of a suspension of 1 x 106 A. castellanii (a clinical isolate belonging to T4
genotype) in PYG medium. In control groups, locusts were injected with the same volume of PGY alone. Mortality was recorded every 24 h, when any dead locusts were removed to minimise cannabalism. The injection ofamoebae killed almost 100% of the locusts within 14 days. To determine whether parasites disseminate via haematogenous spread, samples of haemolymph together with various tissues were dissected and cultured on nonnutrient agar plates containing bacterial lawns. Amoebae were recovered from locust haemolymph, muscles, fatbodies, but not faeces. To determine A. castellanii association with the central nervous system, locust brains wereisolated. Briefly, the injected locusts were killed and the left side of the head was removed by a saggital cut
through the base of the left antenna using a sharp scalpel. The brain was dissected out from the right-hand side of
the head using fine sterile forceps. After several washes with PBS, the brains were incubated with pentamidine (40µM; final concentration) at 37oC for 60 min to kill extracellular amoebae. The brains were then washed a further
three times, homogenised and cultured for the presence of amoebae. Brain lysates from amoebae-infected locusts
were positive for A. castellanii, whereas brain lysates from PYG-infected locusts showed no organisms. Overall,these results suggest that locusts can be used as a model to study Acanthamoeba pathogenesis in vivo. Because
insects rely for their protection against infection on an entirely innate immune system, the use of an insect modelis particularly relevant in the study of human Acanthamoeba encephalitis, the control of which has significant
dependency on the innate immune system.

Or-23
Encystment in Balamuthia mandrillaris: A property that enhances resistance
and/or recurrence of infection
Ruqaiyyah SIDDIQUI1, Abdul MATIN1, David WARHURST2, Monique STINS3, Kwang Sik KIM3,
Naveed Ahmed KHAN1*
1( School of Biological and Chemical Sciences, Birkbeck, University of London, UK,2
London School of Hygiene and Tropical Medicine, University of London, UK,
3
Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, USA)*e-mail: n.khan@sbc.bbk.ac.uk
Balamuthia mandrillaris is an emerging protozoan pathogen that is known to infect the central nervous system
to produce fatal granulomatous encephalitis. Early diagnosis, followed by aggressive treatment using a combination of drugs is a prerequisite in successful treatment but even then, prognosis remains very poor (∼98% mortal-

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ity). In addition, amoebae can transform into dormant cyst forms, which may provide resistance and/or reactivate
following drug therapy. Here, we tested B. mandrillaris resistance to physical, chemical and radiological conditions. Following these treatments, viability was determined by culturing amoebae on human brain microvascularendothelial cells for up to 12 days. B. mandrillaris cysts were resistant to repeated freeze-thawing (5X), temperatures of up to 70°C, 0.5% SDS, 25ppm chlorine, 10µg/ml pentamidine and 200mJ/cm2 of ultraviolet irradiation.
When amoebae were cultured with primary human brain microvascular endothelial cells (HBMEC) which constitute the blood-brain barrier, cycloheximide, ketoconazole or pre-exposure of organisms to cytochalasin-D prevented amoeba-associated cytotoxicity. In an assay for inhibition of cyst production in the absence of feeder cells,
these 3 agents prevented the production of cysts. These findings indicate that the biosynthesis of proteins andergosterol and the polymerization of actin are important in cytotoxicity and encystment. Of the three targets, ketoconazole and other azoles are of most interest for further investigation, because ergosterol is not produced bymammalian cells.

Or-24
Characterization of membrane carbohydrates in pathogenic Naegleria fowleri and non-pathogenic
Naegleria gruberi
Isaac CERVANTES-SANDOVAL1,3*, José de Jesús SERRANO-LUNA2, Mineko SHIBAYAMA3(1Immunology Dept., National School of Biological Science-National Polytechnic Institute, Mexico.
Depts of 2Cell Biology, 3Experimental Pathology, Center for Research and Advanced Studies,
National Polytechnic Institute, Mexico)*e-mail: cervantessi@yahoo.com.mx
Naegleria fowleri is a free-living ameboflagellate and the etiologic agent of the primary amoebic meningoencephalitis (PAM). This is a fulminant and rapidly fatal infectious disease of the central nervous system. Knowledgeof the parasite surface molecules including glycoproteins may be an important factor in understanding the parasitemechanisms of pathogenesis. Presently, we characterized the carbohydrate residues by using different lectins, whichinteract specifically with glycoconjugates by binding to a specific carbohydrate. We analyzed the differences in themembrane glycoprotein composition between N. fowleri (pathogenic) and N. gruberi (non-pathogenic) strains, andtheir possible role in cell adhesion. The following biotinilated lectins were used: Canavalia ensiformis (Con A),
Tetragonolobus purpureas agglutinin (TPA), Helix pomatia agglutinin (HPA), Pisum sativum agglutinin (PSA) and
Galanthus nivalis lectin (GNL). Trophozoites of both strains were fixed and incubated with the specific biotinilated
lectins. After washing, amoebae were incubated with streptavidin-fluorescein and analyzed by FACS (Fluorescentactivated cell sorting). Results showed that N. fowleri expressed with a higher intensity than N. gruberi, the glycoproteins that are detected with Con A (which recognizes α-D-Glucosyl and α-D-mannosyl residues) and TPA (which
recognizes α-L-fucosyl residues). This recognition was 2.7 fold higher in N. fowleri using both lectins. The role ofthese glycolconjugates in amoebic adhesion to epithelial cells was also evaluated. Trophozoites of both strains wereinteracted with MDCK cells for 1 h in the presence of different concentrations of specific sugars. D-mannose and L-fucose inhibited 70% and 66% of N. fowleri cell adhesion, respectively. On the other hand, the same carbohydrates
inhibited 40.3% and 43% of N. gruberi adhesion to MDCK cells. The differences observed suggest that parasite
membrane glycoproteins with mannose, glucose and fucose residues may be involved in amoebic adhesion to hostcells, prior to tissue invasion.

Or-25
Acanthamoeba castellanii differentiation: A two-faced drama
Ricky DUDLEY*, Selwa ALSAM, Naveed Ahmed Khan
(School of Biological and Chemical Sciences, Birkbeck College, University of London, UK)*e-mail: ricky.dudley@btinternet.com
Acanthamoeba castellanii of the T4 genotype is an environmentally ubiquitous protozoan pathogen that cancause a rare, but fatal, encephalitis and a blinding keratitis. One of the distressing aspects in combating Acanthamoeba infections is the prolonged and problematic treatment. This is due to the ability of Acanthamoeba to
rapidly adapt to harsh conditions and switch phenotypes into a resistant cyst form, resulting in both disease resur-Jpn. J. Protozool. Vol. 40, No. 2. (2007)

175gence and longevity. One possibility of improving the treatment of Acanthamoeba infections is to inhibit the ability of these parasites to switch into the cyst form. In keeping with other protozoans, Acanthamoeba elaborate proteases, considered to be key determinants of pathogenicity and cytolysis of host cells. However, there is now
growing evidence that their localised incitement and amplification may equally be synonymous with morphological changes. Previous research has established that Acanthamoeba secretes serine proteases. Using a number ofprotease inhibitors and corroboratory evidence from short interfering RNA primers (siRNA), we have found that
serine proteases play an important role in A. castellanii differentiation. Inhibition of serine proteases attenuates A.
castellanii metamorphosis, as demonstrated by arrest of both encystment and excystment of Acanthamoeba. Further, as serine and threonine residues are important biological regulatory sites, the foregoing has led to an initialinvestigation into the contribution that phosphorylative signalling cascades play in determining Acanthamoebatransposition.

Or-26
RNA interference (RNAi) as a tool for the silencing of differentiation process in Acanthamoeba
Jacob LORENZO-MORALES1*, Jarmila KLIESCIKOVÁ2, Enrique MARTÍNEZ-CARRETERO1,Luis Miguel De PABLO3, Antonio OSUNA3, Basilio VALLADARES1
1( Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias. University of
La Laguna, Spain, 2Dept. of Tropical Medicine, 1st Faculty of Medicine, Charles University
in Prague and Faculty Hospital Bulovka, Czech Republic, 3Institute of Biotechnology,
Dept. of Parasitology, University of Granada Campus, Spain)*e-mail: JMLORENZ@ULL.ES
The genus Acanthamoeba undergoes encystation under unfavourable environmental conditions. Cellulose is amajor component of the inner cyst wall in this organism. The process of cellulose synthesis is not clearly understood yet, however in order to synthesise cellulose, these amoebae must have storage of glucose for its synthesis
regarding unfavourable conditions. It has been suggested that the glycogen, which amount is decreasing duringencystation, is the main source of free glucose for the synthesis of cellulose in Acanthamoeba. The glycogen phosphorylase is supposed to be the main tool that these amoebae use in order to maintain the free glucose levels during the encystation process.
In this study the glycogen phosphorylase of four strains, with different pathogenic levels, of Acanthamoeba wassilenced using RNA interference (RNAi) and the effects on the differentiation process in RNAi-treated and nontreated cells was evaluated at different stages using inverted and electron microscopy and by measuring the levelsof glycogen phosphorylase using northern and western blots.
Our results demonstrated that the free glucose during encystation was exclusively delivered via glycogen phosphorylase activity, which was increasing due to higher levels of mRNAs codifying this enzyme. Additionally, thisstudy highlights the potential of RNAi technology as an attractive candidate for future development of new agentsfor the treatment of Acanthamoeba infections.

Or-27
Losses of the encystment potential of Acanthamoeba spp. involve epigenetic regulation
Martina KÖHSLER1*, David LEITSCH2, Michael DUCHÊNE2, Ursula FÜRNKRANZ1,
Horst ASPÖCK1, Julia WALOCHNIK1
1( Dept. of Medical Parasitology, Clinical Institute of Medical Microbiology and Hygiene,2
Dept. of Specific Prophylaxis and Tropical Medicine, Center for Physiology andPathophysiology, Medical University of Vienna, Austria)*e-mail: martina.koehsler@meduniwien.ac.at
The extremely high abundance of Acanthamoeba in a broad range of habitats is mostly due to their ability toevade unfavourable environmental conditions by encystment. Apart from their high tolerance against desiccationand prolonged starvation, Acanthamoeba cysts show high resistance against therapeutic agents, and thus represent
a challenge in therapy of Acanthamoeba infections, because remaining cysts have been reported to lead to recurrent infections. In the laboratory, encystment of Acanthamoeba trophozoites can be induced by replacing the

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nutrient medium by a defined salt medium. However, prolonged culture of microorganisms under laboratory conditions is known to change their physiological properties. Accordingly, loss of virulence and the decrease in cellular enzyme activities have been reported for long-term cultured Acanthamoeba.
In this study, the encystment potentials of four Acanthamoeba strains of genotype T4, previously cultured axenically for different periods of time, were compared in three previously described encystment media and a correlation of strain age and loss of encystment potential could be demonstrated. However, it was shown that decreases
in the encystment potential were partly reversible by growth in vero-cell monolayers or by treatment with 5azacytidine and trichostatin A, suggesting that epigenetic mechanisms might be involved. Our long-term aim willbe the evaluation of these mechanisms on the protein level.
Adaptations to long-term culture represent a common problem and the identification of underlying mechanisms andapproaches to overcome this phenomenon could be beneficial for studies on pathogenicity or drug susceptibility
assays of microorganisms.

Or-28
Naegleria fowleri excystation process: An ultrastructural study
Bibiana CHÁVEZ-MUNGUÍA1, Maritza OMAÑA-MOLINA2*, Guadalupe CASTAÑÓN1,
Lizbeth SALAZAR-VILLATORO1, Dolores Hernández-MARTÍNEZ2, Patricia BONILLA2,
Adolfo MARTÍNEZ- PALOMO1
1( Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional. Laboratorio de Patología Experimental, 2Universidad Nacional Autónoma de México, Facultad de Estudios Superiores Iztacala. Laboratorio deMicrobiología Ambiental, Mexico)*e-mail: maritzaomanam@yahoo.com.mx
Naegleria fowleri is an amphizoic amoeba causative of human primary amoebic meningoencephalitis (PAM).
The life cycle of N. fowleri present three morphologically different phases: the trophozoite, from which may originate the flagellated form, and the cyst, which represents the resistance phase. The cyst is not considered infective,
but its importance lies in its capacity to assure the persistence of the parasite, and is therefore of epidemiologicalimportance for this microorganism. Moreover, it has been reported that the amoeba may excyst in a relatively
short time, thus giving rise to the invading trophozoite.
We have studied the structural events that occur during the Naegleria sp. excystation process by transmission
electron microscopy (TEM). The amoeba used in the present experiments was the isolate recently isolated from a
hot spring water source and associated with a PAM case in a child. Cysts were collected from an asynchronic
population of the amoeba grown on non-nutritive agar plates pre-coated with Enterobacter aerogenes at 37°C.
Cysts were induced to excyst by plating them on the fresh culture plates for 12 or 24 h to 37°C. By fluorescencemicroscopy mature cysts presented two to three ostioles, each of one sealed by an operculum. By TEM the
operculum shows two areas different in the compaction grade of their fibrillar structure. When the excystation
process was induced, small electron dense granules were associated both to the parasite plasma membrane and to
free fibrillar material present in the peritrophic space. Images suggesting the removal of the operculum by theamoeba were also found. After removal of the operculum, the presence of pseudopodia through the ostiole was
frequently seen. The cytoplasm of the pseudopodia contained vacuoles with a fibrillar material. This fibrillar content may be derived from the incorporation of the operculum into the cytoplasm. Finally, the parasite emerges
from the cyst wall. These observations suggest that in Naegleria sp. lytic products, endocytic and pseudopodic
activities may be involved in the process of excystation.

Or-29
Protein expression profile during encystment of Acanthamoeba castellanii
Sabrina BOUYER1, Marie-Hélène RODIER1, Yann HÉCHARD2*( Laboratoire de Parasitologie et Mycologie Médicale, CHU La Milétrie, 2Equipe de Microbiologie Fondamentale
et Appliquée, Université de Poitiers, France)*e-mail: yann.hechard@univ-poitiers.fr
1

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177

form that could multiply, feed and move. Cyst is the dormant form, which appears in case of adverse conditions
such as nutrient limitation. Of particular importance is the study of cyst, as this form is highly resistant to biocides
and would be responsible for amoebae persistence in the environment. In addition, Acanthamoeba cysts have beenshown to protect pathogenic bacteria such as Legionella pneumophila. Little is known about the encystment process in free-living amoeba like Acanthamoeba. Our work is aimed to decipher, at the molecular level, the mechanism of encystment in Acanthamoeba castellanii.
We first compared protein expression of A. castellanii cells in the process of encystment at different times (0h,6h, 24h and 8 days) by 2D gel electrophoresis. Several protein spots were induced or repressed during the encystment. These spots were analysed by mass spectrometry and among them six proteins were identified. We are currently measuring, by real-time RT-PCR, expression of genes encoding these proteins. The level of RNA expression
will be then compared to that of proteins. Identification of proteins related to amoebae encystment might help tounderstand this process and, later on, to struggle against encystment.

Or-30
Characterization of encystation-mediating factor, serine proteinase of Acanthamoeba
Eun-Kyung MOON, Dong-Il CHUNG, Yeon-Chul HONG, Hyun-Hee KONG*(Dept. of Parasitology, Kyungpook National University School of Medicine, Korea)*e-mail: hhkong@mail.knu.ac.kr
The genus Acanthamoeba is an opportunistic protozoan parasites causing granulomatous encephalitis (GAE) or
amebic keratitis. Proteinases are reported to play roles in various biologic actions of Acanthamoeba including hosttissue destruction, pathogenesis and digestion. Serine protease of Acanthamoeba is known to role in parasite
physiology and in host-parasite interactions. In the present study, we purified and characterized a serine proteinase
mediating the encystation of Acanthamoeba. By the Differential Expressed Gene analysis, we identified 16 genes
expressed higher in cyst than in trophozoite. One of those, a serine proteinase was confirmed the highly expressedmRNA level during encystation by real time PCR analysis. Encystation of Acanthamoeba was inhibited by serine
proteinase inhibitor, PMSF. Chemically synthesized small interfering RNA (siRNA) of the encystation mediating
serine proteinase (EMSP) was significantly reduced the encystation efficiency in Acanthamoeba. Western blot
analysis detected the serine proteinase of 33 KDa mature form in encysting amoebae but not in trophozoite. A
EMSP-EGFP fusion protein localized at the vesicle-like structure in the amoeba. Using Lyso Tracker analysis,
these vesicular structures were confirmed to be lysosomes rather than secretory vesicles. These data show that
EMSP may play an important role in the differentiation of Acanthamoeba by being partly responsible for autolysis.

Or-31
Encystment in Acanthamoeba spp.: Application of two-dimensional gel electrophoresis forthe elucidation of stage-specific proteins involved in the developmental process
David LEITSCH1*, Martina KÖHSLER2, Andrea DEUTSCH3, Günter ALLMAIER3, Michael DUCHÊNE1, Julia
WALOCHNIK2(1Dept. of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology, 2Departmentof Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna,
3
Institute of Chemical Technologies and Analytics,
Technical University of Vienna, Austria)*e-mail: david.leitsch@meduniwien.ac.at
The capability to encyst is Acanthamoeba’s most effective measure to withstand adverse conditions and thus
has medical as well as environmental implications. The cyst stage of Acanthamoeba is remarkably resistant tochemotherapeutic agents, which often leads to recurrence of disease in patients who were supposedly treated successfully against Acanthamoeba keratitis. Moreover, Acanthamoeba cysts often harbour pathogenic bacteria, including legionellae, mycobacteria, Listeria monocytogenes and others, thereby rendering them a potential risk topublic health.

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It is, therefore, important to gain a better understanding of the encystment process in Acanthamoeba spp., its
underlying mechanisms and its interdependence with endocytobiotic bacteria. We applied two-dimensional gel
electrophoresis (2D PAGE) in order to monitor and identify the differentially expressed proteins in the encystingcell. Samples of encysting Acanthamoeba spp. cultures were taken at several time-points until the mature cyst
stage was attained. The protein expression patterns of encysting cells were found to differ dramatically from thetrophozoite stage, already at a remarkably early stage of development. A large number of protein spots was isolated from 2D gels and analysed by mass spectrometry.
In the future, the identified proteins will be studied at the genetic level in order to identify regulatory DNA sequences that are involved in directing encystment and in order to isolate and to define encystment specific transcriptional regulators of Acanthamoeba. The ultimate goal of our study will be the establishment of a molecularmodel of encystment in Acanthamoeba spp., which enables to draw comparisons to other encysting protozoa andto assess the impact of endosymbiotic bacteria on encystment by RNA profiling and proteomics.

Poster (Po)-01
An update on the Acanthamoeba DNA sequence database at the Ohio State University:
A resource for the study of clinical and environmental isolatesPaul A. FUERST*, Gregory C. BOOTON(Dept. of Evolution, Ecology and Organismal Biology, The Ohio State University, USA)*e-mail: fuerst.1@osu.edu
The genus Acanthamoeba has a worldwide distribution, inhabiting a wide range of environmental niches. It has
been isolated from soil, fresh and saltwater, humans, domestic and feral animals. The genus includes opportunistic pathogens responsible for the sight threatening disease Acanthamoeba keratitis (AK) in otherwise healthy individuals. Acanthamoeba is also responsible for life threatening infections in immunocompromised patients. Variation in the pathogenicity of Acanthamoeba strains has been observed, but the relevance of these results to humandisease is unclear. Lack of a reliable subgeneric classification system has complicated analysis. Acanthamoeba
traditionally has been classified using morphological markers such as cyst morphology and trophozoite size andshape. Questions regarding the reliability of morphological methods led our laboratory to examine molecularmethods to determine whether more definitive subgeneric classifications could be accomplished. Our initial studies focused on the nuclear small subunit ribosomal RNA gene (Rns). Subsequent studies added mitochondrial locias additional markers and as a test of the consistency of conclusions drawn from the Rns studies. The data collected over the previous decade now allow us to quickly examine a clinical or environmental sample using molecular methods and determine and classify the sequence genotype for an unknown specimen. Twelve Rns sequence types were originally identified and designated T1-T12. Several more may exist. The database currently
includes ~190 “nearly complete” sequences (defined as sequences of 2000 or more nucleotides). Of these, 133(70%) are classified as sequence type T4. In addition, more than 200 isolates have been examined for which partial Rns sequences have been reported, including 135 isolates with sequences of 400-1000 nucleotides and 20 isolates with sequences of 1000-2000 nucleotides. While mitochondrial rns sequences have been less extensively
analyzed, there are over 80 sequences that have been reported, spanning almost all nuclear sequence types.
(Supported by grants from NIH)

Po-02
Construction of EST database for comparative gene studies of Acanthamoeba
Ying-Hua XUAN1, Young-Sun YUN1, Joung-Ok KIM1, Yong-Seok LEE2, Se-Won KANG2, Weon-Gyu KHO2,
Dong-Il CHUNG1, Yeon-Chul HONG1, Hyun-Hee KONG1*
1( Dept. of Parasitology, Kyungpook National University School of Medicine, 2Dept. of Parasitology
and Malariology, PICR, College of Medicine and Frontier Inje Researchfor Science andTechnology, Inje University, Korea)*e-mail: hhkong@mail.knu.ac.kr
The genus Acanthamoeba is the causative agent of granulomatous amoebic encephalitis and amebic keratitis.

Jpn. J. Protozool. Vol. 40, No. 2. (2007)179

But little information has been reported on the genome of Acanthamoeba up to now and moreover, specific database for Acanthamoeba gene has yet to be constructed. Three kinds of cDNA library with Acanthamoeba of different virulences or different developmental stages were constructed. A total of 2,864 ESTs from 3,115 random
selected clones from the cDNA libraries was generated and subjected for BLASTx homology search. Among the
ESTs that have significant matches, 1,607 ESTs were found to be identified as unique genes, and 1,574 genes
were found in Acanthamoeba for the first time. Each ESTs data was classified by KOG analysis, predicting function. Comparing EST data by KOG analysis between the two species, actin related proteins, subtilisin-like serine
proteinase in A. healyi ESTs show higher percentage than A. castellanii ESTs. Comparing EST data by KOG
analysis between the two developmental stages of A. castellanii, cullin4, heat shock protein, calreticulin, ubiquitin
conjugation enzyme, proteasome, and serine type endopeptidase was found in only cyst ESTs. Being based on
result of Acanthamoeba ESTs data with download data related with Acanthamoeba from NCBI, database of Acanthamoeba called 'Acanthamoeba EST DB' was constructed. The sequence data of 'Acanthamoeba EST DB' is consisted of 36,227 nucleotide and 285 amino acid data. The contents of 'Acanthamoeba EST DB' are 'Home','BLAST_NT', 'BLAST_AA', 'BLAST_results', '2-sequences', and 'statistics'. In 'BLAST_results', each gene has
links for the significant information including sequence data, gene orthology annotations, relevant references anda BLAST result. Using various blast program, nucleotide analysis in 'BLAST_NT', protein analysis in'BLAST_AA', and alignment of two sequence in '2-Sequence' are possible. As first attempt in the Acanthamoeba,'Acanthamoeba EST DB' would make the gene study easy, providing many new opportunities for the scientificcommunity.

Po-03
Genotyping of Acanthamoeba strains based on mitochondrial DNA RFLPs and
18S rDNA sequences
Kenji YAGITA1*, Shinji IZUMIYAMA1, Mako OMURA1, Yosuke KAMEOKA2, Takuro ENDO1( Dept. of Parasitology, National Institute of Infectious Diseases, 2Laboratory of Genetic Resources,
National Institute of Biomedical Innovation, Japan)*e-mail: kyagi@nih.go.jp
1

The genus Acanthamoeba is distributing in a variety of environment, including soil, fresh and saltwater, house
dust, and contact-lens solution and others. Recent molecular studies of this genus have revealed that DNA analyses, such as mitochondrial (mt)DNA RFLP typing or 18SrDNA sequence typing, are more reliable than morphology of the cyst or the trophozoites for the identification of the species. Interestingly, most of the keratitis isolates
can be assigned to major 7 mtDNA RFLP types or have an 18SrDNA sequence of T4 type in different studies.
Since little is known about the relationships between mtDNA RFLP types and 18SrDNA sequence types, we extensively studied to know whether they are closely related or not. Ninety-nine isolates of keratitis isolates andsome of the authentic strains were analyzed both with mtDNA RFLPs and 18SrDNA sequences. Mitochondrial
DNA RFLPs were obtained by BglII and EcoRI digestion of whole mtDNA extracted from the cultures. Sequencesof 18SrDNA were obtained according to the published methods (Booton GC et al, 2005, JCM). Reference sequences of T-types based on the 18SrDNA sequencing in Genbank were involved for phylogenetic analysis. Major
7 mtDNA RFLP types which encompass most of the human isolates had T4 sequence. More precisely, however,these major mtDNA RFLP types except for JAC/E4 distributed in different sub-clusters within T4 clade, namely
most of the 18SrDNA sub-clusters consist of multiple mtRFLP types.

Po-04
Keratitis by Acanthamoeba spp, a great threat to users of contact lenses
Berbeli ASTORGA*(Laboratory of Environmental Parasitology, Institute of Public Health of Chile)*e-mail: berbeli.astorga@gmail.com
Background: Most of the information described on kinds of free living amoebas (FLA) pathogenic to humans
refers to Naegleria fowleri, Acanthamoeba spp, Balamuthia mandrillaris and Sappinia diploidea. The first published case of keratitis caused by Acanthamoeba was in 1973, but its association with users of contact lenses wasrecognized only in 1984. In Chile, the first isolation of FLA from environment was carried out in 1981 and it cor-

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responded to Acanthamoeba palestinensis; later in 1993 the first isolation from a patient was carried out and it
corresponded to Acanthamoeba polyphaga. It came from a sample of corneal scraping of a patient with severebilateral keratitis. Both species were typed at the Center for Disease Control, Atlanta, United States (G. Visvesvara).
Objective: To report the isolations of Acanthamoeba spp. from samples of patients with keratitis.
Material: Sixty-eight samples of corneal scraping or corneal biopsy; the patients included 48 females and 20
males with ages between 20 and 60 years.
Methods: The samples were cultivated in non-nutrient agar covered with an emulsion of Escherichia coli, andwere incubated at 37ºC and at room temperature. Observation of the culture was done every 24 hours for 7 days.
Results: Positive samples showed filamentous trophozoites and cysts of smooth walls with polygonal enclosure.
Eighteen out of 68 patients (26 %) were positive, 16/43 from female patients and 2/20 from male patients. Ten out
of 18 patients were users of contact lenses, 1 did not use contact lenses and 7 patients the information.
Conclusions: With the results obtained, it is recommended to detect the presence of this protozoan parasite in
keratitis patients who do not respond to the usual treatment procedures and in those from which no other pathogenic
agents have been isolated.

Po-05
Evaluation of potential keratopathogenicity of Acanthamoeba isolated from contact lens storage
cases and domestic tap water
Sun-Joo LEE1, Hae-Jin JEONG1,2, Jeong-Hwan KIM1, Ying-Hua XUAN 3, Keun-Hee LEE1,
Sang-Kyun PARK1, Sun-Hee CHOI1, Dong-Il CHUNG3, Hyun-Hee KONG3,
Mee-Sun OCK4, Hak-Sun YU1,2*(1Dept. of Parasitology, School of Medicine, Pusan National University, 2Pusan National University Hospital
Medical Research Institute, 3Dept. of Parasitology, School of Medicine Kyungpook National University,4
Dept. of Parasitology, Kosin University College of Medicine, Korea)*e-mail: hsyu@pusan.ac.kr
In a previous study, we reported on the contamination rate of free living amoeba, including Acanthamoeba,
isolated from contact lens storage cases (CLSC) and domestic tap water in Korea. In an effort to evaluate the potential kerato-pathogenicity of 5 isolates from CLSC and 17 isolates from domestic tap water, we have conducted
an investigation into the morphological features, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) phenotypes, 18S rDNA sequences, and drug sensitivities of these isolates, and have compared
the results with those of 20 amoebic keratitis (AK) isolates from Korea, as well as 14 reference strains. Cysts from
22 isolates obtained from CLSC and domestic tap water showed typical characteristics of morphological group 2.
A total of three and five mtDNA RFLP patterns generated by Eco RI were found in 5 of the isolates from CLSC
and 17 of the isolates from domestic tap water respectively. The mtDNA RFLP patterns of four of the five isolates
from the CLSC were found to be identical to those of the isolates from domestic tap water of students who had
contaminated CLSC. The majority had mtDNA RFLP patterns identical to those of AK isolates in Korea. The
results of 18S rDNA sequencing analysis were also shown to coincide with the results of mtDNA RFLP analysis.
KA/WP12 was determined to be profoundly sensitive to chlorhexidine (MCC; 6.25 μg/ml), and KAWP2 was themost sensitive strain to polyhexamethylene biguanide (PHMB) (MCC; 4.69 μg/ml). Some difference in the cytopathic effects of isolates against human corneal epithelial cells was observed according to their mtDNA genotypes.
In conclusion, domestic tap water may constitute a source of Acanthamoeba contamination of CLSC, and mostisolates from CLSC and domestic tap water appear to be potentially keratopathogenic.

Po-06
Granulomatous amoebic encephalitis due to Acanthamoeba sp. Case report
Patricia BONILLA-LEMUS1*, Felix ROSITAS-NORIEGA2, Maritza OMAÑA-MOLINA1,
Leticia MORENO-FIERROS4, Ma. Dolores HERNANDEZ-MARTÍNEZ1, Leticia VERDIN4,
R.GARZA-GARZA3, C.SAENZ2
1( CyMA Project-FES-Iztacala-UNAM, 2Hospital Christus Muguerza Monterrey, Nvo. Leon,
3
Hospital Universitario “José Eleuterio Gonzalez” Universidad Autónoma de Nuevo León,

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4

181

UBIMED, FES-Iztacala-UNAM, Mexico)*e-mail: blemus@servidor.unam.mx

A 41-year-old men resident of Villa Santiago, N. L., Mexico initiated the sickness with clinical history of fivemonths of partial convulsive crisis. Signs and symptoms most important were: consciousness deteriorated, somnolence, severe headache, vomiting and convulsive crisis. Nuclear magnetic resonance imaging showed a right frontal-parietal tumor of the brain. Neurosurgery was practiced and showed a right frontal lesion with neoplastic aspect.
Histopatology analysis showed subacute cerebral abscess with granulation tissues and necrosis. Amoebic trophozoites were observed and morphologically identified as Acanthamoeba sp. The patient was treated with rifampin,
trimethoprim-sulphamethoxazole, fluconazole and metronidazole. The neoplasic process was not demonstrated.
Because of patient´s neurological status continued declining and convulsive crisis persisted, a second surgery was
performed. Samples of cerebral tissue were inoculated in NNE media with Enterobacter sp. and incubated at 25 °C
for 7 days. The culture was negative for free-living amoebae. Besides, representative samples of the brain were
stained with H & E and Gomori trichromic tincion. Amoebae seen in tissue sections from the patient were identified
as Acanthamoeba sp., and Amphotericin B was added. Samples were fixed and processed for their inclusion inparaffin and sectioned to obtain 5 μ m-thinck slices and then processed for immunohistochemistry using polyclonal
serum of rabbit anti-Acanthamoeba and goat anti-IgG of rabbit conjugated to peroxidase and revealed with H2O2
diaminobenzidine. Samples were counterstained with H & E.
Two months after, the patient showed signs of increased intracranial pressure and new surgery was performed.
Findings were compatible with Glioblastoma multiforme and cerebral abscess with numerous trophozoites ofAcanthamoeba were observed. The patient died after 3 months after diagnosis of EAG.

Po-07
Possible role of immunoglobulin IgA in the protection against experimental N. fowleri meningoencephalitis
Saúl ROJAS-HERNÁNDEZ1*, Adriana JARILLO-LUNA1, Leticia MORENO-FIERROS2,
Marco A. RODRÍGUEZ-MONROY2, Rafael CAMPOS- RODRÍGUEZ 1
1( Dept. de Investigación y Posgrado, Escuela Superior de Medicina, Instituto Politécnico Nacional,
2
Inmunidad en Mucosas UBIMED, FES-Iztacala, Universidad Nacional Autónoma de México, Mexico)*e-mail: saulrohe@yahoo.com.mx
We have reported that intranasal administration of Cry1Ac protoxin alone or with amoebal lysates increases
protection against Naegleria fowleri meningoencephalitis in mice. Our data indicated that IgA responses elicitedin the nasal mucosa seem to be related with protection and suggest they may participate in the initial defense
against this parasite. In the present work we performed an immunohistochemical analysis of N. fowleri and IgA atearly times after an intranasal lethal challenge with N. fowleri in mice, that previously had been immunized by the
intranasal route with Cry1Ac alone or with amoebal lysates and in control mice. Our results showed that intranasal
immunization provoke changes in the olfactory epithelia of mice immunized with any of the three distinct treatments tested, and was clearly appreciated an increase in areas with metaplasia towards respiratory characteristics.
Moreover we found a markedly increased localization of IgA in the epithelia with metaplasia of immunized mice,
as well as IgA interacting with trophozoites. These data suggest that the increased IgA induced in the nasal mucosa
by intranasal immunization, probably impedes amebic adhesion and subsequent invasion of the nasal epithelia. In
support to this notion in immunized mice, trophozoites were mainly detected in the nasal lumen while in unimmunized mice they were detected in the lumen as well as invading the nasal mucosa. Present results suggest that immunization provokes cellular changes in the olfactory epithelia which may allow a major secretion of IgA, andmay contribute to the higher protection against N. fowleri observed in mice intranasally immunized and challenged with this parasite.

Po-08
Role of antibody responses in the protection against experimental N. fowleri meningoencephalitis
in STAT6-deficient mice

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María M. CARRASCO-YÉPEZ1*, Saúl ROJAS-HERNÁNDEZ1, Luis I. TERRAZAS2,Marco A. RODRÍGUEZ-MONROY3, Leticia MORENO-FIERROS3(1Dept. de Investigación y Posgrado, Escuela Superior de Medicina, Instituto Politécnico Nacional, 2Inmunidad
de parásitos UBIMED, FES-Iztacala, Universidad Nacional Autónoma de México,3
Inmunidad en Mucosas UBIMED, FES-Iztacala, Universidad Nacional Autónoma
de México, Mexico)*e-mail: cayem07@yahoo.com.mx
We have reported that intranasal administration of Cry1Ac protoxin alone or with amoebal lysates increases
protection against Naegleria fowleri meningoencephalitis in mice. In that study we proposed that adaptive immune
mechanisms such as antibodies (IgG and IgA) appear to play an important role in the protection against the infection. The aim of the present study was to investigate whether antibody responses were essential for resistance to N.
fowleri. STAT6-deficient mice (STAT6-/-) were used and compared with similarly treated wild-type (STAT6+/+)
mice. The mice were immunized by the intranasal route with a combination of N. fowleri lysates and Cry1Ac, andsubsequently challenged with lethal doses of trophozoites of N. fowleri. STAT6+/+ mice had a 100% protection,
whereas no protection was observed in STAT6-/- mice. Moreover, a significantly higher level of titers of Th2associated immunoglobulin G1 (IgG1) as well as interleukin-4 (IL-4) were found in STAT6+/+ mice, whereas in
STAT6-/- mice significantly more IL-12 and IFN-γ as well as significantly higher levels of titers of Th1-associatedimmunoglobulin G1 (IgG1) were observed. These findings suggest that the humoral response is critical for defense against N. fowleri infection because STAT-6+/+ mice showed a 100% protection and a Th2- response, while
STAT6-/- mice showed a cellular response that apparently does not participate in the protection against N. fowleri.

Po-09
Molecular identification of Naegleria spp. isolated in Mexico
Fernando LARES VILLA1*, Eunice GUZMÁN FIERROS1, Johan F. De JONCKHEERE2(1Dept. of Agronomic and Veterinary Sciences, Technological Institute of Sonora, Mexico,
2
Scientific Institute of Public Health, Belgium)*e-mail: flares@itson.mx
In the genus Naegleria 47 species have been described which can be identified by rDNA sequencing. Only fourof these had been identified in Mexico until now: the human pathogen N. fowleri and the closely related nonpathogenic N. lovaniensis, the mouse pathogenic N. australiensis, and N. mexicana, which is known to occur only in
Mexico until now.
In the present study we isolated in 2004-2005 Naegleria strains from different states of Mexico in an attempt toidentify as much different species as possible. The isolates were identified by sequencing the PCR amplified ITS15.8S-ITS2 and comparing the sequences to those of established species. Nine isolates were identified as N.
lovaniensis, five as N. tihangensis, two as N. pagei, and one each as N. clarki, N. indonesiensis, N. byersi and
N. americana. Of these, N. lovaniensis is the only one that had been identified previously in Mexico. One of the
new N. lovaniensis isolates is unique as it is the first time a one bp deletion is observed in the ITS2 of this species.
N. tihangensis is closely related to the pathogenic N. australiensis, differing from each other only by an indel of 2
bp in the ITS2. The isolation of N. indonesiensis and N. byersi is important because they had been reported previously as having a restricted geographic dispersal.

Po-10
Molecular identification of Acanthamoeba sp. Isolates inan Acanthamoeba keratitis outbreak
Gregory C. BOOTON1*, Charlotte E. JOSLIN2, Megan SHOFF1, Elmer Y. TU2,
Daryl J. KELLY1, Paul A. FUERST1
1( Dept. of Evolution, Ecology and Organismal Biology, The Ohio State University,2
Dept. of Ophthalmology and Visual Sciences, University of Illinois, USA)*e-mail: booton.1@osu.edu

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183

Objectives: An increase in Acanthamoeba keratitis (AK) cases has been documented in the Chicago, Ill., USA
area. Epidemiological analysis indicates this is a significant increase in cases compared to historical numbers. It
was hypothesized that the increased infections may be due to changes in water treatment. Alternatively, a more
pathogenic strain of Acanthamoeba may be responsible for the increase in AK cases. Here we use genotypic data
to test the hypothesis that a new, or more pathogenic known genotype of Acanthamoeba, is the cause of the AK
surge.
Methods: Previous sequence analysis of the 18S ribosomal RNA gene (18S rDNA) of Acanthamoeba isolates
resulted in the placement of Acanthamoeba strains into 15 different genotypic classes. Most cases (~97%) of AK
are associated with a single genotype (T4) of Acanthamoeba. Rarely, AK cases are associated with other genotypes. In this study we determine the genotypes of 23 Acanthamoeba sp. isolates from the Chicago AK outbreakby sequencing a highly informative region of the 18S rDNA.
Results: DNA sequencing shows these isolates are predominantly genotype T4 (91%), whereas the remaining
isolates were genotype T3 (9%). Both genotypes have previously been observed in AK cases. In addition, DNA
sequences are overwhelmingly similar to previously sequenced isolates.
Conclusion: There is no support for the hypothesis that cases of AK in this outbreak are the result of infection
by a new Acanthamoeba genotype. High sequence similarity between these isolates and the 18S rDNA database
does not support the hypothesis that these isolates represent more pathogenic Acanthamoeba of known genotypes.
Lastly, results lend support to the hypothesis that increased AK cases are due to changes in water treatment, permitting increased bacterial colonization of the water, increased Acanthamoeba grazing, and ultimately an increasein AK cases due to an increased abundance of Acanthamoeba in the water supply.
Support: USA NIH grant EY09073

Po-11
Selective grazing of free-living naked amoeba on methane-oxidizing bacteria
Jun MURASE1*, Peter FRENZEL2(1Graduate School of Bioagricultural Science, Nagoya University, Japan, 2Dept. of Biogeochemistry, Max Planck
Institute for Terrestrial Microbiology, Germany)*e-mail: murase@agr.nagoya-u.ac.jp
Biological methane oxidation at the oxic-anoxic interface in wetland soils and sediments is a key process inmethane cycle, preventing large amounts of this greenhouse gas from escaping into the atmosphere. Methane oxidizing bacteria (MOB) is the only group of aerobic bacteria that oxidize methane and assimilate methane-C.
However, the fate of MOB-biomass is largely unknown. Here we demonstrate that a microbial food web can be
driven by methane. Soil microcosms, in which a thin layer of water-saturated rice field soil was incubated under
opposing gradients of oxygen and 13C-labelled methane, revealed 13C-enriched “heavy” RNA which could be affiliated to MOB, to the scavenging prokaryotic Myxobacteria, and to protistan grazers including Lobosea andHeterolobosea. Amoeba-derived RNA demonstrated preferential enrichment with 13C among the group. Different
protistan communities developed depending on methane concentrations. Amoeba isolated from the soil demonstrated selective grazing on MOB. These results indicated that amoeba may shape the community structure of
MOB and vice versa. Recent work using MOB as a model has shown that microbial community structure mattersfor ecosystem functioning. Our study suggests that not only microbial, but also protistan diversity and microbeprotist interactions have to be considered to understand and control methane oxidation in wetland soils.

Po-12
A mycophagous amoeba-flagellate isolated from a Phytophthora ramorum-infectedlesion of California bay laurel
Emi YAMAMOTO1, Mark MAZZOLA2, Michael F. COHEN1*(1Sonoma State University, Dept. of Biology, 2USDA Agricultural Research Service,
Tree Fruit Research Laboratory, USA.)*e-mail: cohenm@sonoma.edu

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An amoeba-flagellate, termed strain ANN04, was isolated from hyphae of Phytophthora ramorum originatingfrom an infected California bay (Umbellaria californica) leaf lesion. P. ramorum, an oomycete, is the causative
agent of “Sudden Oak Death”, a disease syndrome currently afflicting forests along the coasts of Central andNorthern California. ANN04 was separated from P. ramorum by culturing on heat-killed bacteria and the coculture reestablished on a separate P. ramorum strain. Trophozoites (~10 μm in length) show putative feeding
behavior on hyphae and sporangia of P. ramorum. Higher densities of trophozoites and cysts could be observed byco-culturing with an ascomycete, termed strain F1, which was also isolated from the same infected bay leaf lesion
as ANN04. Further studies to determine the feeding range of strain ANN04 are underway. Characteristic of Naegleria spp., suspension of ANN04 trophozoites into dilute medium induced a temporary flagellated stage. However, to our knowledge, there are no reports of mycophagy by Naegleria. To better establish the taxonomic identityof strain ANN04 we will be conducting rDNA and actin gene sequence analysis. The potential application of
ANN04 as a biocontrol agent will be discussed in the context of studies by Old and Chakraborty [1] on the apparent role of mycophagous amoebae in suppression of soil-borne Phytophthora diseases.
[1] Old, K.M. and Chakraborty, S. 1986. Mycophagous soil amoebae: Their biology and significance in the ecology of soil-borne plant pathogens. In: Patterson, D.J., & Corliss, J.O. eds, Progress in Protistology 1:163-194.

Po-13
Presence of free-living amoebae in two habitats with different conditions of oxygen
Elizabeth RAMIREZ*, Esperanza ROBLES, Luis CHIQUILLO(National Autonomous University of Mexico, FES Iztacala, Project of Conservation and
Improvement of Environment, Mexico)*e-mail: erf@servidor.unam.mx
Free-living amoebae (FLA) are aerobic organism and as such, it is supposed cannot exist as the trophozoite
stage in environments with low oxygen content; however, cysts of Acanthamoeba have been isolated from anaerobic material such as faeces. The aim of the investigation was to compare the population of free-living amoebae of
two habitats with different conditions of oxygen. Samples of wastewater and sludge were taken from an anaerobic
digester tank used as treatment of domestic wastewater. Samples were seeded on non nutritive agar with Enterobacter aerogenes and the cultures were incubated at 30ºC. Dissolved oxygen concentration of wastewater was inan average of 0.6 mg/L and the sludge was anoxic. Seventeen species of free-living amoebae of 6 genera wereisolated from sludge and wastewater. Eleven species of amoebae were isolated from sludge; while 16 species wereisolated from wastewater. Both habitats shared 10 species of amoebae: Acanthamoeba astronyxis, Acanthamoeba
tubiashi, Acanthamoeba quina, Acanthamoeba triangularis, Acanthamoeba royreba, Acanthamoeba lenticulata,
Hartmannella vermiformis, Vahlkampfia avara, Vahlkampfia aberdonica, Dactylamoeba stella and Mayorella
microeruca; and 6 species were found only in wastewater: A. mauritenesis, A. polyphaga, A. lugdunensis, Vannella lata, Vannella platypodia y Vannella simplex. Higher number of FLA was found in sludge (16,800
NMP/100 mL) than in wastewater (2,028 NMP/100 mL).
In anoxic condition (sludge) the richness of species was low, but the number of amoebae was higher; while in
low condition of dissolved oxygen (wastewater), the richness of species was higher, but the number of amoebae
was low.
Cyst forming amoebae like Acanthamoeba, Hartmannella and Valhkampfia were more frequent; however some
cyst not-forming amoebae like Dactylamoeba, Mayorella y Vannella were also present. This finding indicates thatthese amoebae can support hostile condition of oxygen in vegetative form. This would be especially significant totheir survival and distribution in the environment.

Po-14
Prevalence of Acanthamoeba in Chicago area tap water
Megan E. SHOFF1*, Charlotte E. JOSLIN2A, B, Gregory C. BOOTON1, Elmer Y. TU2A,Paul A. FUERST1(1Dept. of EEOB, The Ohio State University, 2University of Illinois, AOphthalmology/Visual Science,
B
School of Public Health, Division of Epidemiology and Biostatistics, USA)Jpn. J. Protozool. Vol. 40, No. 2. (2007)

185*e-mail: shoff.4@osu.edu
Objectives: A significant recent increase in Acanthamoeba keratitis (AK) has been documented in the greater
Chicago, Ill. area. It was hypothesized that changes in water treatments may have led to an increase in Acanthamoeba in the water supply. This study was done to identify the prevalence of Acanthamoeba in tap water in the
Chicago area.
Methods: Over the course of 12 months (June 2006-June 2007), water samples were collected from sites in thegreater Chicago area. Water was sampled in all cases using sterile swabs from the inside surface film of the lavatory cistern reservoir tank. Further, 50mL of tank water serving the lavatory was also sampled. In all cases, theultimate source of water was cold municipal mains water. The presence of amoebae in samples was assayed using
an enrichment cultivation method appropriate for Acanthamoeba. Amoebae were identified based on diagnostic
features discernable by light microscopy and select samples were genotyped.
Results: Over 130 households’ samples were processed and amoebae were noted in over half of these. Acanthamoeba were found in a higher percentage of homes than in previous US studies. Amoebae (regardless of genus)
were also present in a higher percentage of samples than found in previous US studies.
Conclusions: Tap water in the home has been associated with corneal infection in contact lens wearers who
have exposed their lenses to tap water. The recent outbreak of AK in the Chicago area prompted a study of the
tapwater in that area. Initial findings reported here indicate a much higher incidence of Acanthamoeba in the tap
water of Chicago than was observed in S. Florida, possibly a link to the higher incidence of AK. These initial
findings support the hypothesis that the new water treatments may be permitting increased biofilms which result in
an increase of Acanthamoeba.
Support: NIH grant EY09073, NIH 15689, Prevent Blindness America, Midwest Eye-Banks, UIC Campus
Research Board, AOF AAO William C. Ezell Fellowship

Po-15
Identification of endosymbiont in Acanthamoeba isolated from domestic tap waterSeon-Hee CHOI1, Ying-Hua XUAN2, Keun-Hee LEE1, Sang-Kyun PARK1, Sun-Joo LEE1,
Dong-Il CHUNG2, Hyun-Hee KONG2, Mee-Sun OCK3, Hae-Jin JEONG1, Hak-Sun YU1*
1
( Dept. of Parasitology, School of Medicine, Pusan National University, 2Dept. of Parasitology,
School of Medicine Kyungpook National University,3Dept. of Parasitology,
Kosin University College of Medicine, Korea)*e-mail: hsyu@pusan.ac.kr
The genus Acanthamoeba is an amphizoic amoeba which is a causative agent of amoebic keratitis, pneumonitis
and granulomatous amoebic encephalitis (GAE) in immunocompromised host. Also the important feature of thegenus is that it is well-known to function as a carrier or vehicle for some pathogenic bacteria. In this study weinvestigated presence of endosymbiont from 16 Acanthamoeba isolates from domestic tap water samples. We
identified endosymbiont from 5 (31%) isolates (KA/PW3, KA/PW4, KA/PW8, KA/PW9, and KA/PW12) using
orcein stain methods and assumed their species using 16s rDNA sequencing analysis. To determine two or moredifferent endosymbionts live in one Acanthamoeba isolates, we analyzed 16S rDNA PCR products using denaturing gradient gel electrophoresis method. Ultra-structure analysis of endosymbiont achieved using transmission
electron microscopy. The 16s rDNA sequence from endosymbionts of KA/PW3, KA/PW4 were similar to Candidatus Amoebophilus asiaticus, their similarity were 96.7% and 95.4% respectively. The endosymbionts of KA/
PW8, KA/PW12 have 95.4% and 95.3% 16s rDNA sequence similarity to Candidatus Odyssella thessalonicensis,
respectively. In this study, uncommon endosymbiont which is very similar to Methylophilus sp. was found from
KA/PW9 isolates. This is firstly report that Methylophilus isolated from Acanthamoeba. The morphologic characteristics of endosymbionts in KA/PW3, KA/PW4, KA/PW8, and KA/PW12 were rod shape (1.40 - 1.80 μm ×0.20 - 0.30 μm) and they were located sporadically in cytoplasm. But endosymbionts of KA/ PW9 were amorphous (1.50 μm × 0.20 μm) and distributed with groups each other in the vesicles of cytoplasm.
In the near future, we will evaluate the pathogenicity of endosymbiont, include Methylphilus which is first reported as endosymbiont in this study.

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Po-16
Detection of four adenovirus serotypes within water isolated strains ofAcanthamoeba in the Canary Islands, Spain
Nieves M. CORONADO-ÁLVAREZ1, Jacob LORENZO-MORALES1*, Carmen M. MARTÍN- NAVARRO1,
Nuria TEIGELL-PÉREZ1, M. Gabriela CABRERA-SERRA1, Enrique MARTÍNEZ-CARRETERO1,
Sutherland K. MACIVER2, Basilio VALLADARES1
1( Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias. University of La Laguna, Spain,
2
Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Scotland)*e-mail: JMLORENZ@ULL.ES
We surveyed 236 potentially pathogenic Acanthamoeba strains, isolated from water sources in the Canary Islands, for the presence of human adenoviruses (HAdV) using a PCR based typing assay. A total of 34 of thesestrains were found to be positive for adenovirus belonging to four different HAdV serotypes (HAdV-1, 2, 8, and37). We found that HAdV-2 was the most frequency encountered serotype amongst the Acanthamoeba strains andtheir identification was confirmed by a nested PCR specific for this serotype. We showed that Acanthamoeba
genotype T4 was highly associated with serotype HAdV-2, while Acanthamoeba genotype T3 was most often
associated with adenovirus serotypes related to ocular diseases. Based on these data, we suggest that Acanthamoeba should be considered as a potential reservoir and perhaps even a transmitter of adenoviruses to humanand other secondary hosts.

Po-17
Novel culture medium for Naegleria fowleri
Tatsuru HARA, Toshihide FUKUMA(Dept. of Parasitology, Kurume University School of Medicine, Japan)*e-mail: thara@med.kurume-u.ac.jp
Background: For axenic cultivation of Naegleria fowleri, some culture media were reported up to now. Most ofthese culture media are excellent for growth of Naegleria fowleri, but they have a few disadvantages. Therefore
we developed a novel culture medium for Naegleria fowleri axenic cultivation.
Method: In this study, Naegleria fowleri YT9611 strain, originally isolated from a patient with a fatal case of
primary amoebic meningoencephalitis, was used. Commercially available basal media for mammalian and insect
cells with or without fetal bovine serum were tested at 37°C and 40°C.
Result: Results from short-term trial at 37°C indicated that the IPL-41, insect cell culture medium, supplemented with fetal bovine serum is suitable for Naegleria fowleri cultivation. Interestingly, growth rate of Naegleria fowleri in case of diluted IPL-41 condition was faster than in undiluted IPL-41. As a result of examination in aseries of mixed ratio, the best ratio of IPL-41 : fetal bovine serum : water was 1:1:8 (named IFW-118) on growthrate of Naegleria fowleri. Under axenic cultivation with IFW-118 at 37°C and 40°C, cell shape and motility oftrophozoites were normal. Part of trophozoites transiently transformed into flagellates and cysts without mediumchange. At present, we are continuing long-term cultivation of Naegleria fowleri using IFW-118 at 40°C over one
year. Further information will be presented.

Po-18
Application of flow cytometry for detection and quantification of Naegleria fowleri amoebae
in cooling water systems
Melanie Lorthioy*(EDF Generation, Ceidre, Dept. Etudes/APC, France)*e-mail: Melanie.lorthioy@edf.fr
Since 1978, the water used for cooling in French power plants has been tested for the presence of pathogenic
amoebae and especially Naegleria fowleri (Nf), the causative agent of Primary Amoebic Meningoencephalitis

Jpn. J. Protozool. Vol. 40, No. 2. (2007)

187(PAM). The replacement of brass condensers by stainless steel condensers, to reduce corrosion and copper discharges, have resulted in increase of Nf developments. In some cases, during summertime, densities could reachup to 10000 Nf.l-1 in power plants cooling water with stainless steel condensers. Consequently, to reduce the release of amoebae in the rivers, continuous chlorinations of the closed-loop cooling systems were implemented inthese power plants.
Traditional methods for detection and quantification of Nf in water samples are growth- based and results require several days to obtain the result. A new method based on the ChemScan scanning cytometer was evaluated.
It is based on the fluorescent labelling of Nf initially concentrated on a filter membrane and automatically detected
and enumerated by a laser-scanning instrument This rapid method (results in the daytime) was evaluated for thetesting of water used for cooling French power plants.
During 2 years, more than 950 water samples were tested by both methods. Results on river water samples
showed that, for concentrations of Naegleria superior to 200 Nf.l-1, ChemScan method appeared to give the same
trends of Nf developments as traditional standard plate count method (up to 60% of results were strongly correlated). For concentrations under 200 Nf.l-1, ChemScan and count plate method are not so well correlated due tolow volumes filtered and scanned (10 to 25 mL) with the ChemScan. In this case, a pre-filtration method should
be adapted to increase analysed volumes and detection limit.
In conclusion, ChemScan method, by giving significant results in daytime, is a very useful tool for evaluation ofa treatment efficiency and risk assessment.

Po-19
Quality assessment of the molecular tools used for the diagnosis of Acanthamoeba
Pablo GOLDSCHMIDT1*, Helene YERA2, Sandrine DEGORGE1, Cathy SAINT-JEAN1,
Fouad ZEKHNINI1, Laurence BATELLIER1, Laurent LAROCHE3, Christine CHAUMEI1(1Laboratoire du Centre National d’Ophtalmologie des Quinze-Vingts, 2Laboratoirede Parasitologie Mycologie, AP-HP Hopital Cochin, Universite Rene Descartes,
3
Service 5 du Centre Nationald’Ophtalmologie des Quinze-Vingts, Paris France)*e-mail: pablogol@aol.com
Aims: Early and sensitive diagnosis of Acanthamoeba infections may reduce the risks for the clinical condition
from worsening. We studied the yields of Acanthamoeba DNA that can be obtained by means of different procedures after introduction of a labile DNA tag (internal control) in the specimens.
Methods: Acanthamoeba cysts mixed with a tag virus - (seal herpes virus, DNA extraction-yield marker and
PCR inhibition detector) - were processed according to different DNA preparation methods: heat, Proteinase K(ProtK), alkali lysis, QIAmp kitR, MagNA Pure (DNA Mini kit, MagNA PureR Nucleic Acid isolation kit), ProtK+ QIAmp and ProtK + MagNA Pure. In each extract the parasite-DNA loads were assessed by real-time PCR.
Results: Heat and NaOH did partially hydrolyze the nucleic acids in the samples and therefore, did not allow the
DNA extraction yield assessment. Treatment with Prot K was not able to eliminate the PCR inhibitors. Both, theQIAmp and the MagNA Pure partially improved the levels of detection and eliminated the inhibitors. Significant
increase in positive results (more than 10X) was obtained just by adding a ProtK pre-treatment before the commercial extraction kits.
Conclusion: The Acanthamoeba cysts do not release DNA for amplification if samples are processed with methods used for other cells and viruses. To minimize false negative Acanthamoeba diagnosis the cysts should be efficiently lysed without affecting the DNA structure and inhibitors should be eliminated. Significant increase in thedetection rates were obtained by adding a ProtK treatment (10min at 56°C) before conducting the commercial
procedures. ProtK + MagNA Pure produced in 30 min the highest level of positive results followed by ProtK +QIAmp (150 min).

Po-20
Aquaporin is responsible for high water permeability of the contractile vacuole membranein Amoeba proteus
Eri NISHIHARA1*, Etsuo YOKOTA2, Teruo SHIMMEM2, Seiji SONOBE2( Dept. of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental University,1

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2

Graduate School of Life Science, University of Hyogo, Japan)*e-mail: eniskid@tmd.ac.jp

To control the cell volume, unicellular organisms living in fresh water have evolved an organelle, the contractile
vacuole (CV) complex. Amoeba proteus has a large CV that moves with the cytoplasmic streaming. Understanding of mechanism of water uptake is still poor. The aim of the present study is to elucidate the mechanism of water
uptake by CV in A. proteus.
To evaluate water permeability of CV membrane, CV was isolated from A. proteus. When the medium containing cells between the slide glass and the cover glass was soaked up with a piece of filter paper, cells were pressed
between two glasses. They became flattened and finally CVs were released into the bathing medium. When theisolated CV was incubated in a hypertonic medium, its volume quickly decreased within 10 sec. This suggested
that the CV membrane is semi-permeable and that water should be collected along the osmotic gradient in vivo.
From the rate of osmotic volume change, the water permeability of the CV membrane was calculated to be 0.94
μm/ sec. This high value suggested the presence of water channel in the CV membrane.
We cloned an aquaporin gene ApAQP from A. proteus. ApAQP encodes a protein consist of 295 amino acids,
which shows six putative transmembrane domain and two NPA motifs. Using Xenopus oocytes, we demonstrated
that the ApAQP functions as a water channel. Immunofluorescence microscopy with an anti-ApAQP antibody
revealed that ApAQP was localized on the CV membrane and the vesicles around CV. This is the first success in
explaining high water permeability of the CV membrane by aquaporin. On the other hand, we found that VATPase was highly concentrated on the vesicle membranes around CV. These findings suggest that vesicles are
involved in generating the osmotic gradient via the activity of V-ATPase and that water moves into the vesicles
along the osmotic gradient through aquaporin.

Po-21
The heat shock protein 70 of pathogenic Naegleria fowleri is necessary forrophozoite growth and cytotoxicity
Kyoung-Ju SONG1*, Ae-Hee YANG1, Hae-Jin SHON1, Kyung-Jin CHOI1, Anh-Trinh Minh DANG1,
A-Rum SONG1, Jong-Hyun KIM1, Yang-Jin LEE1, Daeho-KWON1, Kyung-Il IM2, Ho-Joon SHIN1(1Dept. of Microbiology and Molecular Science & Technology, Ajou University School of Medicine,2
Dept. of parasitology, Yonsei University School of Medicine, Korea)*e-mail: skjyl261@hanmail.net
OBJECTIVES: To evaluate the role of heat shock 70 protein (Hsp70) in the pathogenicity of N. fowleri, wecloned, characterized a constitutive and inducible heat shock 70 gene from pathogenic N. fowleri, and named as
Nf-cHSP70. To investigate role of Nf-cHSP70, we inhibited the Nf-cHsp70 protein with benzylidene lactam compound, KNK437, which has effect on Hsp70 synthesis and knock-downed Nf-cHsp70 gene with antisense oligimers which has designed with a start region specific antisense oligonucleotides (24 mers) and modified with phosphorothioate.
METHODS: 1. Amoeba : Trophozoites of N. fowleri (Carter NF69; ATCC No. 30215) were axenically cultured
in PYG medium at 37˚C.
2. Proliferation inhibition assay: To evaluate the effect of KNK437 (Hsp70 inhibitor) on proliferation of N. fowleri, the trophozoites of N. fowleri were treated with various concentration of KNK437 and incubated at 37˚C for
24hrs. Proliferated cells were counted by hemocytometer after trypan blue stain.
3. Phosphorothioated Oligomers: The target sequence for antisense oligomer (24mer) corresponded to the startregion of Nf-cHsp70.
4. AS-Oligomer transfection: To evaluate the gene specific inhibition effect of AS-Oligomers, the trophozoites ofN. fowleri were transfected with various oligomers modified with phosphorothioated and complexed with PEI(polyethylenimine) as transfection reagent.
RESULTS: KNK437 inhibited the induction of N. fowleri HSP70 in a dose-dependant manner. After treatment
with 300µM of KNK437, the proliferation of N. fowleri was reduced to 79.4% compared with untreated control(100%). Hsp70-knock downed N. fowleri with antisense oligomer showed reducing proliferation to 70% comparedwith untreated control. Hsp70 inhibited N. fowleri showed reducing cytotoxicity against CHO target cells as to50%.

Jpn. J. Protozool. Vol. 40, No. 2. (2007)

189

CONCLUSION: AS Oligomers directed against Nf-cHsp70 caused a dramatic and sequence specific decrease oftarget protein level, resulting in the inhibition of growth and cytotoxicity on target cells of pathogenic N. fowleri.
Our study suggests that Nf-cHsp70 is essential proliferation and pathogenicity of N. fowleri.

Po-22
In vitro activity of perifosine against the trophozoite stage of Acanthamoeba
Fernando LANCEL1, M. Gabriela CABRERA-SERRA1, Jacob LORENZO-MORALES1*, Carmen Ma MARTÍNNAVARRO1, Nieves María CORONADO-ÁLVAREZ1, Efraín ALAMANCA -CAPUSIRI2,
José E. PINERO1, Basilio ALLADARES1(1University Institute of Tropical Diseases and Public Health of the Canary Islands, University of
La Laguna, Spain; 2Instituto de Investigaciones Fármaco Bioquímicas, Facultad de Ciencias
Farmacéuticas y Bioquímicas, Universidad Mayor de San Andrés, Bolivia)*e-mail: JMLORENZ@ULL.ES
Perifosine is a novel alkyl-phospholipid that has displayed significant antiproliferative activity in vitro and invivo in several human tumor model systems and has recently entered phase II clinical trials. This drug was previously used against Leishmania promastigotes. In this study, a modification of a previous colorimetric assay for theassessment of antiamoebic drugs was developed and also the activity of perifosine was tested, against the trophozoite stage of Acanthamoeba castellanii Neff, a type strain, and a clinical isolate of Acanthamoeba previously
identified as A. polyphaga.
A previously developed colorimetric 96-well microtiter plate assay, based on the oxido-reduction of Alamar
Blue® Assay, was optimized for the determination of drug efficacy against the trophozoites of Acanthamoeba spp.
The drug used for the optimization of the protocol was the alkyl-phospholipid perifosine.
The obtained results demonstrated that the newly optimized assay is more comparable to a manual counting
than the previously developed one and also the activity of perifosine was demonstrated against Acanthamoeba
spp, using the developed protocol.
The activity of perifosine is demonstrated for the fist time against the trophozoite stage of Acanthamoeba spp.
The newly optimized assay offers a number of advantages such as being less labour-intensive, faster and morereliable than those previously described, as well the results can be read in a non-subjective manner.

Po-23
Acanthamoeba encystation versus „toxic reaction“: differences in morphology,
cellulose synthesis and resistanceJarmila KLIEŠČIKOVÁ*, Eva NOHÝNKOVÁ
(Dept. of Tropical Medicine, 1st Faculty of Medicine, Charles University in Prague, Czech Republic)*e-mail: jarmila_k@email.cz
Under unfavourable environmental conditions Acanthamoeba spp. undergoes encystation. The mature cyst is
characterized by the presence of double-layered wall – exo and endocyst; both of them are containing cellulose.
Encystation is a relatively slow process continuing for 48 hrs, when the first mature cysts are observed in-vitro. In
contrast to encystation, usage of different solvents leads to „toxic reaction“, where acanthamoebae form singlelayered pseudocysts with minor content of polysaccharide with beta linkages. Cellulose synthesis during encystation and toxic reaction was detected by calcofluor staining. The formation of exocyst takes 24 hrs. During the next
24 hrs endocyst is formed and first mature cysts are observed. Toxic reaction is a rapid process where trophozoites
detach and round up within 30 min of exposure and patchy cellulose remnants on the cell surface appear 1 hr after
induction. Single-layered pseudocyst is formed within 2 hrs. To test the sensitivity of cysts, pseudocysts and trophozoites, acanthamoebae were exposed 0.5% SDS as well as different range of pH (3-11) and temperature (4065°C). In contrast to mature cysts, trophozoites and single-layered pseudocysts are not resistant to the SDS treatment. Exposure to different pH revealed that in contrast to mature cysts, pseudocysts were sensitive to acidic pH(3-6) and within 24 hrs of exposure most cells underwent lysis. Mature cysts were shown to be resistant to 5 min
incubation in 65°C in contrast to pseudocysts (resistant to 55°C for 10 min) and trophozoites (resistant to 45°C for190

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10 min). „Toxic reaction“ is a rapid reaction of acanthamoebae to the agents, which might act as amoebicidal.
However, even if the speed of the response is suitable, single-layered wall of pseudocysts provides only relative
protection against different unfavourable conditions and mature cysts are the most important resistant forms ofacanthamoebae.

Po-24
Gene profile of encysting Acanthamoeba castellanii
Eun-Kyung MOON, Dong-Il CHUNG, Yeon-Chul HONG, Hyun-Hee KONG*(Dept. of Parasitology, Kyungpook National University School of Medicine, Korea)*e-mail: hhkong@mail.knu.ac.kr
The members of the genus Acanthamoeba are the causative agent of GAE and amebic keratitis. The trophozoite,
the vegetative state of Acanthamoeba transforms to the cyst, the resistant form under harmful environments suchas starvation, cold and by which poor responses in medical treatment are resulted in. To investigate the factors
mediating encystations, expressed sequence tag (EST) of encystations-induced A. castellanii was analyzed andcompared to that of trophozoite. Total 1,021 and 905 ESTs were generated from the cyst and trophozoite cyst
cDNA libraries of A. castellanii Castellani. We compared the predicted proteins from the ESTs of the cyst and thetrophozoite by reciprocal BLAST analysis, KOG (euKaryotic Orthologous Groups) assignment, and gene annotation. Various genes were identified in the cyst ESTs. In addition to previously reported cyst specific genes such as
cyst specific protein 21, protein kinase C, enolase, proteasome and heat shock protein, we identified several genes
like as cullin 4, caleticulin, autophage protein 8 and ubiquitin-conjugating enzymes seems to be related to encystation. Five kinds of proteinases including subtilisin-like serine proteinase, cysteine proteinase, peptidase M28, serine-type endopeptidase and aspartyl aminopeptidase were detected in cyst ESTs. The information on the encystation mediating factors identified by ESTs opens the way for further study on differentiation and resistance of cystforming pathogenic protozoa.

Po-25
Variabilities in protein profiles and immunoreactivities among Acanthamoeba genotypes
Wilawan PUMIDONMING1*, Martina KÖHSLER1, David LEITSCH2, Julia WALOCHNIK1(1Dept. of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology,
Medical University of Vienna, 2Dept. of Specific Prophylaxis and Tropical Medicine,Center for Physiology and Pathophysiology, Medical University of Vienna, Austria)*e-mail: pumidonming@yahoo.com
Acanthamoeba is a free-living protozoan found in a wide variety of habitats. Acanthamoeba has also been isolated from humans and animals. Several representatives of the genus Acanthamoeba are known as causative
agents of Acanthamoeba keratitis (AK), a sight-threatening disease of the eye, and granulomatous amoebic encephalitis (GAE), an infection predominantly occurring in the immunocompromised host. Recently, fifteen genotypes (T1-T15) of Acanthamoeba have been established. Most clinical isolates group into genotype T4 but also
genotype T3, T5, T6, T11, T12 have each caused individual cases. Previous studies have indicated physiologicaldifferences between pathogenic and non-pathogenic Acanthamoeba strains. However, there is little information ondifferences between genotypes.
The aim of this study was to compare protein profiles and immunoreactivities between Acanthamoba strains ofdifferent genotypes, and between pathogenic and non-pathogenic stains within genotype T4, the genotype most of
all linked with disease. Altogether, thirteen strains, both pathogenic and non-pathogenic, from seven genotypes(T3, T4, T5, T6, T9, T11, T12) of Acanthamoeba were studied for their protein profiles and immunoreactivities
with IgA, IgG, IgM. It was shown that protein profiles and immunoreactivities are notably different among genotypes. Moreover, differences were also observed between pathogenic and non-pathogenic strains within genotype
T4.
Key words: Acanthamoeba, genotypes, protein profiles, immunoreactivities

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2D-page protein profiles of pathogenic and non-pathogenic Naegleria species (II)Mako OMURA*, Rieko FURUSHIMA-SHIMOGAWARA, Kenji YAGITA, Shinji IZUMIYAMA,
Takuro ENDO
(Dept. of Parasitology, National Institute of Infectious Diseases, Japan)*e-mail: omura@nih.go.jp
Naegleria fowleri is a thermophilic free-living amoeba that causes primary amoebic encephalitis in humans,
while N. lovaniensis, the morphologically identical species, is not. A two-dimensional (2-D) gel analysis was employed to compare total proteins in N. fowleri to those of N. lovaniensis in order to identify proteins that may link
to its pathogenesis. Protein was extracted from log-phase growing amoebae and recovered by TCA precipitation.
The results of 2-D PAGE image analysis showed that 228 protein spots were common in N. fowleri strains and sowere 246 protein spots in N. lovaniensis strains. A comparison of the average gel showed that 88 protein spots
were common between two species. The number of species-specific protein is 102 proteins in 5 strains of N. fowleri. Among the species-specific spots of N. fowleri, 63 spots were analyzed N-terminal and/or internal amino acidsequences along either with Cleveland peptide mapping or with EST-IT tandem MS followed by similarity searchfor known proteins against NCBInr database using Mascot search engine. In addition, some of the MS/MS spectra
have also been de novo sequenced by means of PEAKS Studio v2.4, revealing many peptide candidates from each
spot. The latter method showed an ultimate advantage for the identification of the proteins from the internal sequences of protein spots especially those whose N-terminal is blocked like many of those of N. fowleri.
According to amino acid sequences of about 20 residues, 14 spots were identified with high confidence as
cyclophilin, nucleoside diphosphate kinase, NADP-isocitrate dehydrogenase, and MP2CL5, N. fowleri HSP70,
albumin, actin, and others. We partially cloned the genes of 2 protein spots, tentatively designated as #15(24.1kDa, pI6.5) and #35 (50.9 kDa, pI 16.7), that are specific to N. fowleri by means of degenerate PCR. The
respective PCR products had approximately 250 bp and 400 bp in sizes. From the amino acid sequence predictedfrom the amplified DNA, #15 was identified with high confidence as Thioredoxin peroxidase (22.3 kDa,
Q6DV14) with 80% (54/67) homology. Similarly the predicted amino acid sequence of #35 showed 55% (67/120)
homology to that of glutamate dehydrogenase (55.0kDa, Q54KB7). The 2D-PAGE database will be updated spotby spot without delay for public access.

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Observations on Acanthamoeba isolates from the Philippines
Samuel Alan B. INOVEJAS1, Dennis B. BACANI1, Lindsay Sydney N. FAJARDO1, Corazon C. BUERANO2,
Michael Thomas T. GONZALES2, Jay-R T. SORREDA2, Ruben Lim Bon SIONG3, Ronald R. MATIAS1*(1Research and Biotechnology Division, St. Luke's Medical Center, 2Institute of Biology, College of Science,
University of the Philippines, 3St. Luke's Intl. Eye Institute, St. Luke's Medical Center, Philippines)*e-mail: rrmatias@stluke.com.ph
Acanthamoeba sp., a free living amoeba that is distributed worldwide, is considered to be one of the mostprevalent protozoa distributed in the environment. The organism can be found almost everywhere, from natural ortreated water, seawater, swimming pools, air-conditioning units, soil and contact lenses. Being an opportunistic
pathogen, it can also be isolated from humans. The organism exists as an actively dividing trophozoite and as adormant cyst. In our study we isolated Acanthamoeba sp. from various sources. Four were human isolates and two
were from hot springs. Two of the four human isolates were isolated from corneal specimens, and two came fromnasal swabs. The isolates were cultured on non-nutrient agar and incubated for 36 hours at 37oC. The trophozoites
vary in size ranging from 25–40µm and feed on bacteria and yeast in the environment. There are several ways thetrophozoites acquire food either through pseudopod formation and phagocytosis or by food cup formation. The
amoeba moves in a sluggish movement with the formation of a hyaline pseudopodium. The cysts measure 15–20µm in diameter and are doubled-walled and usually polygonal and spherical in shape.
In higher magnification, trophozoites of acanthamoeba possess a trilaminar plasma membrane that surrounds
the cytoplasm. The trophozoite serves as the feeding stage of the amoeba. It also comprises of a nucleus, vacuoles,
mitochondria, ribosomes and endoplasmic reticulum. The amoeba has a double-walled wrinkled cyst that is com-

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posed of an ectocyst and an endocyst. The cyst is formed during adverse conditions like food deprivation, change
in pH and temperature. Cysts are resistant to chlorination, and are able to survive at low temperatures ranging
from 0–2˚C. Excystation and encystations of the amoeba occur when conditions are favorable. This paper describes both the trophozoite and cyst stages of the organism.