key: cord-010530-w8ir0e07
authors: MOURA, HERCULES; WALLACE, SARA; VISVESVARA, GOVINDA S.
title: Acanthamoeba healyi N. Sp. and the Isoenzyme and Immunoblot Profiles of Acanthamoeba spp., Groups 1 and 3
date: 2007-05-01
journal: J Protozool
DOI: 10.1111/j.1550-7408.1992.tb04853.x
sha: 
doc_id: 10530
cord_uid: w8ir0e07

ABSTRACT Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti‐Acanthamoeba antisera. One of the human strains (CDC:1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni, Lilly A‐1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba, it was considered as a strain of A. royreba. Polyacrylamide gradient gel electrophoresis appears to be a powerful technique for the study of isoenzymes and antigens of Acanthamoeba.

M water and soil amebae that occur world-wide. During the last decade, these amebae have been isolated with increasing frequency from contact lens paraphernalia as well as from human tissue such as corneal scrapings and the central nervous system [9, 22] . Acanthamoeba can be readily recognized because of the striking morphologic characteristics of the trophozoites and cysts. Based on the size and morphologic features of cysts, Pussard & Pons [ 151 established 18 species and placed them in three groups. Group 1 now consists offour species (A. astronyxis, A. comandoni, A. echinulata, and A. tubiashi) characterized by large trophozoites and cysts (2 18 pm) [ 14, 191 . Species in groups 2 and 3 are smaller ( 5 18 pm) but can be separated from each other on the basis of cyst morphology. Group [14, 191. Although cyst characteristics make genus identification easy, differentiation to the species level is difficult. This is especially true for members of groups 2 and 3 which cannot always be identified by species on a morphologic basis alone. As a result, the most recent identification attempts have been based on both morphologic and biochemical criteria such as isoenzymes, antigens, protein profiles, and restriction fragment-length polymorphism analysis of DNA resolved by electrophoretic methods [2, 6, 14, 191 . Cladistic analysis of the data has also been used to elucidate the affinities of the various species of Acanthamoeba [4] .

This paper presents the methods used to isolate Acanthamoeba from the CNS of humans and identify them to the species level based on morphologic characteristics and isoenzyme and antigenic profiles. We also describe the usefulness of polyacrylamide gradient gel electrophoresis (PAGGE) in the resolution of isoenzymes and antigens of groups 1 and 3 Acanthamoeba spp. and its applicability to the taxonomy of this protozoan.

MATERIALS A N D METHODS Case 1. A 7-year-old girl from Barbados with headache, weakness of the right upper and lower extremities, several episodes of vomiting, and positive Babinski sign on the right was admitted to the hospital. Computed tomography (CT) showed I To whom correspondence should be addressed. a large mass in the left parietal area. A craniotomy was performed, and histopathology of the excised tumor-like mass revealed amebic trophozoites [ 131. A small piece of the biopsied brain tissue was frozen and sent to the Centers for Disease Control, where Acanthamoeba (CDC: 1283:V013) was isolated on non-nutrient agar plates seeded with Escherichia coli and MRC human lung cell culture [ 131. Based on cyst morphology and reactivities of the amebae in the brain sections with the rabbit anti-A. palestinensis serum in the indirect immunofluorescence (IIF) test, the amebae were identified at that time as A. palestinensis [ 131.

Case 2. A 34-year-old man with AIDS was admitted to the hospital with numerous painful, widely distributed skin lesions, hemiparesis of the right side, and clonic movement of the right arm [23] . A CT scan showed three walnut-sized, lucent lesions in the left cerebral cortex. Histopathology of the biopsied left frontal brain revealed acute necrotizing encephalitis but no microorganisms. The patient died the next day. At autopsy, amebic trophozoites and cysts were seen in the brain as well as in the skin lesions [23] . Amebic organisms from brain tissue were isolated in human embryonic kidney (HEK) cell culture and identified as A. culbertsoni (CDC:0884:V021) on the basis of morphology and reactivities of the amebae in the tissue sections with rabbit antiserum to A. culbertsoni in the IIF assay [23] . Acanthamoeba strains. The 13 strains ofAcanthamoeba used in this study are listed in Table 1 , along with the history of their isolation and growth temperature. They include the five species belonging to group 3; four species belonging to group 1; A. castellanii, a representative of group 2; two strains of amebae (CDC: 1283:VO 13 and CDC:0884:V02 1) isolated from the brain tissue of patients with Acantharnoeba encephalitis; and a strain of ameba (CDC-Fish-SK) with a history of isolation from a fish in South Korea. This isolate was submitted to one of us (GSV) by Dr. James Yang on agar plate with E. coli and subsequently axenized at the Centers for Disease Control. All strains o f amebae were grown initially on non-nutrient agar plates with E . coli and then axenically grown in a proteose peptone-yeast extractglucose medium as described previously [20] , except for the addition of 5% fetal bovine serum, in 150-cmZ Falcon plastic tissue culture flasks. Preparation of cell pellets. Trophozoites in log-phase ofgrowth were harvested after 2 4 days of axenic culture at the optimum temperature ( Table 1 ). The supernatants were discarded, and 50 ml of WB saline [20] was added to the flasks containing attached trophozoites. After the flasks were chilled on ice for 5-10 min, the solution was transferred to 40-ml Falcon plastic Obtained from Dr. Marc Pussard. Obtained from Dr. James Yang.

tubes, which were then centrifuged at 563 g for 10 rnin at 4" C. The cells were washed twice as above and counted using a hemocytometer. To every 2 x lo8 cells, 0.8 ml ofenzyme-stabilizing solution containing 1 mM each ofdithiothreitol, 6-amino caproic acid, and disodium EDTA [ 161 was added, and the pellets were stored in liquid nitrogen until used.

Extract preparation and SDS-treatment. Acantharnoeba cell pellets were retrieved from liquid nitrogen and processed. For enzymatic studies, amebae were lysed by three cycles of freezing and thawing, 5 rnin per cycle, in a dry ice-methanol slurry and a 37" C water bath. The suspension was centrifuged at 24,000 g for 30 min at 4" C, and the water-soluble supernatant containing native proteins was aliquoted and stored in liquid nitrogen. For antigenic studies, the pellets were sonicated using a W-375 Sonifier (Heat Systems-Ultrasonics Inc., Plainview, NY) at 70% power, 10% duty cycle, pulse mode, for 3 rnin at 4" C in a solution (TBE) containing 8 1.2 mM Tris(hydroxymethy1) aminomethane (Tris), 23 mM boric acid, and 1.5 mM EDTA. The sonicated extracts were centrifuged at 24,000 g for 30 rnin to remove particulate matter and cell debris. The supernatants were collected, aliquoted, and stored in liquid nitrogen. A protein determination was done for each extract using the method of Bradford [ 11. The dye reagent was purchased from Bio-Rad Laboratories (Richmond, CA), and a suspension of albumin and globulin from Sigma (St. Louis, MO) was used as protein standard.

For isoenzyme analysis, equal volumes of a solution containing 80 pg bromophenol blue in 30% sucrose were added to each extract of native proteins just before electrophoresis. For silver staining and immunoblots, the extracts were treated with a solution containing 10% sodium dodecyl sulphate (SDS), 9 M urea, and 0.05 M Tris hydrochloride, pH 8, to obtain a final concentration of 2.5% SDS, 2.25 M urea, and 1 pg ofprotein/pl. Tracking dye solution (50 mg bromophenol blue, 8 ml glycerol, 1 ml 0.05 M Tris hydrochloride, pH 8.0, and 1 ml deionized water) was used in a 3% concentration after the samples were heated for 15 rnin at 65" C in a water bath.

High-resolution PAGGE and buffer systems. All PAGGE reagents were purchased from Bio-Rad, except where noted. Conditions for PAGGE were as previously reported [ 1 1, 181, with some modifications. Briefly, for isoenzyme electerophoresis we used 3-20% gradient gels with a 3% stacking gel; the gels measured 200 x 180 x 1.5 mm. For silver staining and immunoblot procedures, we used 5-20°/o gradient gels with a 3% stacking gel; the gels measured 80 x 180 x 0.75 mm.

A continuous and a discontinuous buffer system were used in the study. The continuous system, TBE, was used for enzymes. The discontinuous system, used for SDS-treated proteins, consisted of TBE in the lower chamber and a freshly prepared solution of 4 1 mM Tris, 40 mM boric acid, and 0.1% (w/v) SDS in the upper chamber.

A Pharmacia vertical electrophoresis apparatus GE-2/4 LS (Pharmacia LKB Biotechnology, Piscataway, NJ) was used for isoenzyme electrophoresis, which was performed at least four times for each strain using extracts prepared from different harvests to check for fidelity and reproducibility of the patterns. Optimum concentration of proteins necessary for the visualization of particular enzymes was also determined. For enzyme separation, the gels were pre-electrophoresed for 45 min at 250 V constant. Constant amounts ofprotein for each enzyme ( Table  2) were loaded and electrophoresed at 250 V maximum. After 60 rnin the voltage was increased to 500 V maximum. The power was turned off after 3 h, when the blue tracking dye could no longer be seen at the bottom of the gel.

In the discontinuous system, the SDS-treated proteins were loaded at a concentration of 0.25 pg/mm of lane width for silver stain and 0.75 pg/mm lane width for immunoblot. Unstained molecular weight standards, a 1:l mixture of high molecular weight standards (Bio-Rad) and low molecular weight standards (Pharmacia) were used to calibrate relative molecular weights of the resolved bands. For immunoblots, 0.5 pl/mm of prestained protein standards (Bethesda Research Laboratories, Gaithersburg, MD) was also used. A constant current of 7.5 mA per gel was applied during the first 20 rnin and then increased to 15 mA per gel. Voltage was allowed to float, and the wattage was limited to 25 W per gel. The power was turned off after 1 h and 30 rnin of electrophoresis, 15 rnin after the tracking dye migrated away from the gel. A constant temperature of 4" C for enzymes and 7" C for SDS-treated proteins was maintained during electrophoresis.

Developing solutions for enzymes. All reagents used for isoenzyme study were purchased from Sigma, except where noted. The enzymes used in this study are listed in Table 2 along with the amount of protein loaded and details for developing solutions [5, 10, 1 I]. All enzymes except ES were developed in the dark at 37" C. After enzyme development, the gels were immersed in a solution of 20% methanol and 5% acetic acid and photographed immediately using a Polaroid camera, model 545. Substrate control gels for each enzyme tested were immersed in the same developing solution, but substrate was omitted.

Silver stain and immunoblot assays. Separated SDS-treated proteins were either silver stained [ 181 or electrophoretically transferred to Immobilon membranes with a 0.2-pm pore size (Millipore Corporation, Bedford, MA). Electrophoresis was performed using a Trans-blot cell and a power supply model 200/ 2.0 (Bio-Rad) at a constant voltage of 90 V for 1 h and 30 min. The current increased from 1.05 A at the beginning to 2.0 A at the end of electrophoresis and the buffer temperature increased from 10" C at the beginning to 35" C at the end [17] . After A. astronyxis, A . comandoni, A. castellanii, A.  culbertsoni, A . palestinensis, A. royreba, A . lenticulata, and CDC: 1283:VO 13) were produced in rabbits by multiple intravenous injections of washed trophozoites and cysts from culture as described previously [21] .

Isolation of ameba from brain tissue. Agar plates inoculated with thawed and minced brain tissue from Case 1 revealed growth of amebae within 72 h and the amebae began to differentiate into cysts after 7 days. However, in the MRC lung cell culture, amebic growth became noticeable only after 9 days of brain tissue incubation, when foci of cleared plaques began to appear in the monolayer. The cell culture was totally destroyed within 15 days, resulting in a monolayer of amebae and cysts. Morphologic analysis of the trophozoites and cysts indicated that the amebae belonged to group 3. The trophozoites (Fig.  1A ) measured 2 0 . 4 4 2 pm long (mean, 32.6 fim) and 16.7-24 pm wide (mean, 18.9 pm) when grown on an agar plate. Feeding form was variable in shape and size, with a wide, clear, hyaline anterior zone of protoplasm. Cysts (Fig. 1 B) were oval to round, with a relatively thick, gently rippled ectocyst and a well-developed round or irregular endocyst. The cysts ranged from 10.5 to 18.0 pm (mean, 14.1 pm) in diameter.

Isoenzymes. Isoenzyme profiles for 8 of the 12 enzymes studied are shown in Fig. 2 and 3 , and the corresponding lane number for each species is given in Table 1 . All substrate control gels (not shown) showed no visible enzymatic activity except where noted for MDH (Fig. 3D ). All isozyme patterns were consistent in all runs.

Among the four species ofgroup 1, A. astronyxis, A. comandoni. and A. tubiashi exhibited distinctly different patterns for all 12 enzymes. Acanthamoeba echinulata also had unique profiles for eight isoenzymes: ES, PGM, ACP, Gd, ME, IDH (Fig. 2, 3) , GPI and ALP (not shown) but resembled A. comandoni in the HK, MDH (Fig. 2, 3) , LAP, and ADH (not shown) patterns.

All five species included in group 3 showed unique ES, PGM, ACP, Gd, LAP, ALP, and ME patterns (Fig. 2,3) . Acanthamoeha culbertsoni, A. palestinensis, and A. pustulosa had unique HK profiles, but the HK profiles for A. Ienticulata and A. royreba were similar (Fig. 2) . Acanthamoeba culbertsoni, A. lenticulata, and A. royreba had unique IDH, MDH, ADH, and GPI isozyme profiles that differed not only from one another but also from those of A. palestinensis and A. pustulosa. whose IDH, MDH (Fig. 3) , ADH, and GPI (not shown) patterns were similar. CDC: 1283:VO 13, which was originally identified as A. palestinensis [ 131, exhibited unique isoenzyme patterns for all the tested enzymes except MDH (Fig. 2, 3) . However, it resembled A. lenticulata in its MDH isoenzyme patterns. Based on its trophic and cyst morphology and isoenzyme patterns, CDC: 1283:V013 was named A. healyi in honor of Dr. George R. Healy, Chief (retired), Protozoal Diseases Branch, Division of Parastitc Diseases, Centers for Disease Control.

The HK, ACP, Gd, MDH, and GPI enzyme patterns of CDC: 0884:V02 1 were very similar to those of A. culbertsoni. Though slight differences were noticed in the PGM, IDH, ALP, and LAP profiles of the two strains, the overall patterns of these enzymes were similar. However, the ME and ES isoenzyme patterns of CDC:0884:V021 were distinctly different from those of A . culbertsoni. Based on these observations, CDC:0884:V02 l is being considered A. culbertsoni pending further studies, Isoenzyme patterns of CDC-Fish-SK were similar to those of A. royreba for all the enzymes tested (not shown). CDC-Fish-SK was also morphologically very similar to A. royreba.

SDS-PAGGE of Acunthamoebu polypeptides. SDS-PAGGE and silver staining of proteins extracted from whole trophozoites revealed very complex profiles, with multiple major bands ranging from 14.4 to 200 kDa for each of the 13 strains examined (Fig. 4A) . Despite the overall similarity noticed in the protein profiles of the 13 strains, members of group 1 could be easily distinguished from those of the other two groups. For example, members of groups 2 and 3 had a dense, dark staining band at about 44-46 kDa which was either absent or faint in group 1 organisms. Further, group 3 as well as A. castellanii had multiple dark staining bands in the region between 25 and 100 kDa that were either faint or not present in group 1 species. The most similar silver-stained protein profiles were those for A. palestinensis-A. pustulosa. those for A. royreba and CDC-FISH-SK in group 3 and those for A. tubiashi-A. comandoni-A. echinulata in group 1. Acanthamoeba astronyxis (group 1) and A. castellani (group 2) each presented remarkably unique profiles. A careful analysis ofthe silver-stained gel revealed that many of the strains possessed unique bands, e.g. A. healyi at 146. Immunoblot assays. Figure 4B-7 show the immunoblot patterns obtained when the separated Acanthamoeba polypeptides were reacted with rabbit antisera to A. palestinensis (Fig. 4B) , '4. culbertsoni (Fig. 5A) , A . royreba (Fig. SB) , A. healyi n. sp. (Fig. 6A) , A. astronyxis (Fig. 6B) , A. castellanii (Fig. 7A) , and A. comandoni (Fig. 7B) . Hyperimmune rabbit antisera reacted extensively with the polypeptides of each of the 13 strains studied; however, the reactivity was most prominent in the homologous extracts. Major antigens were detected between 20 and 116.5 kDa. In these homologous reactions, the staining intensity was so great that some individual bands could not be easily distinguished in the photographs (Fig. 4-7) . It is obvious from these studies that sera made against the group 3 species reacted extensively with the polypeptides of group 3 organisms, whereas they reacted moderately with those of A. castellanii (lane 8 ) and very little with the polypeptides of group 1 species. Only A. astronyxis (lane 9), among the group 1 species, appeared to react moderately with the group 3 and group 2 antisera. Conversely, sera made against group 1 species reacted prominantly with the antigens of group 1 organisms and sparsely with those of group 2 and group 3 organisms. All four species of group 1 produced a characteristic dense, dark staining band at about 30 kDa when reacted with the anti-A. astronyxis and anti-A . comandoni sera. Anti-A. castellanii serum reacted much more . AS a result, etiologic agents are being isolated with increasing frequency. Since it is not always possible to identify the infective agent to the species level by morphologic criteria alone, other nonmorphologic criteria need to be used. Isoenzyme electrophoresis is one such nonrnorphologic trait that has been used by others [3, 6, 251 . We have found that, for native proteins, high-resolution PAGGE appears to be a useful technique for The differences we observed in the separated proteins strongly support the existence of the three morphologic groups, as reported before [7, 15, 191. Further, the immunoblot profiles clearly indicate that group 1 species share only a few antigens with those of group 2 and group 3 species and hence are distinctly different from them. However, A. castellanii, a representative of group 2, shares a considerable number of antigens with those of group 3 species (Fig. 4B-6A ) but few with those of group 1 species (Fig. 6A , 7B) indicating closer relationship with the group 3 strains rather than group 1 species.

An Acanthamoeba species (CDC: 1283:VO 13) isolated from a brain biopsy specimen had been previously identified as A . palestinensis based on its morphologic characteristics and reactivity of the amebae in tissue sections with anti-A. palestinensis serum [13] . Our more detailed study involving morphologic, enzymatic, and antigenic parameters clearly indicates that although this strain bears some morphologic resemblance to A. palestinensis, it is nevertheless very different from A. palesti-nensis and from all other species of Acanthamoeba. Hence it has been designated A. healyi, a new species. CDC:0884:V02 1, the human brain isolate that was previously identified as A. culbertsoni [23] , has been confirmed as A. cul- Fig. 6 . Immunoblot profiles of SDS-treated PAGGE separated proteins of 13 Acunthamoeba strains. Numbers in the horizontal axis and molecular mass markers (MW) are the same as those in Fig. 4 . The proteins were transferred to Immobilon membranes and treated with a 1 : 1,000 dilution of A) rabbit anti-CDC: 1283:VO 13 (A. heulyi n. sp.) and B) antid. astronyxis sera.

bertsonibecause of its morphologic, enzymic, and antigenic similarity to the Lilly A-1 strain of A. culbertsoni.

We believe this is the first report of the isoenzyme make-up and antigenic characteristics of A . tubiashi which had not been cultivated axenically before. Our studies confirm the uniqueness of A . tubiashi, not only at the morphologic level, as noted previously [S], but also with regard to its isoenzyme patterns and antigenic profile. This is also the first time that axenically cultivated A . echinulata has been used to examine its isoenzyme and antigenic profiles. By unequivocally demonstrating the unique isoenzyme and protein profiles ofA. echinulata and clearly showing its differences from those of A . comandoni, our results support the existence of four species in group 1 rather than three as reported previously [3, 61. Fig. 7 . Immunoblot profiles of SDS-treated PAGGE separated proteins of 13 Acanthamoebu strains. Numbers in the horizontal axis and molecular mass markers (MW) are the same as those in Fig. 4 . The proteins were transferred to Immobilon membranes and treated with a 1: 1,000 dilution of A) rabbit anti-A. custellanii and B) A. comandoni sera.

In group 3, A. culbertsoni, A. palestinensis, A. lenticulata, and A. royreba have been well characterized on the basis of morphologic [ 14, 151, isoenzyme [3, 6, 71, and immunoprecipitation [24] studies. However, A. pustulosa has been identified as an invalid synonym ofA. palestinensis, based on their lack ofpathogenicity to mice, morphologic similarity and identity of bands obtained for LAP, MDH, GPI, PGM, PE, and ADH [7, 141. However, in our studies, A. pustulosa and A. palestinensis could be easily distinguished based on their HK, ES, PGM, ACP, Gd, and ME profiles even though they had similar MDH, ADH, IDH, and GPI profiles. Costas and Griffiths [3] also differentiated the two based on their ES and ACP profiles. Further, in reactions with the anti A. palestinensis serum in the immunoblot assay (Fig. 4B ) distinct differences were noted in the antigenic pattern of A. pustulosa from that of A. paleststinensis. W e therefore believe that A. pustulosa is a valid species.

A careful review of the isozyme data on groups 1 and 3 Acanthamoeba suggests that members of these groups can easily be differentiated from one another based only o n HK, ACP, and ES profiles, which will reduce the investigator's time, effort, materials, and expense. If, however, numerical analysis of data is planned or other strains are isolated in the future that cannot be differentiated by these three enzymes, then other enzymes such as PGM, ME, and Gd m a y be used.

Profiles of SDS-treated and silver-stained polypeptides of Acanthamoeba species belonging t o group 1 a n d to group 3 exhibited great within-group similarity, as evidenced by a large number of comigrating bands. Nevertheless, unique bands detected for each species allowed them to be differentiated, even though the unique bands were neither numerous nor prominent. O u r results confirm the differences reported previously in the protein profiles of Acanthamoeba using agarose isoelectric focusing [6].

When probed with seven different polyclonal rabbit sera, immobilon membranes containing the electrotransfer peptides also confirmed the antigenic similarity among the species, as has been reported before for six different species of Acanthamoeba [25] based on immunoelectrophoresis patterns. However, we were able to recognize unique bands for each strain examined by probing with different antisera.

With PAGGE, a powerful technique for the separation of enzymes and antigens, the bands resolved are sharp and clear a n d thus facilitate the comparison of strains. I n conclusion, we have used this technique to resolve the unique isoenzyme and antigenic patterns of two strains ofkanthamoeba isolated from human brain tissues and classify one as A. healyi n. sp. and the other as A. culbertsoni.

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Analysis of mitochondria1 DNA variation as an approach to systematic relationships in the genus Acanthamoeba

The esterases and acid-phosphatases of Acanthamoeba (Amoebida, Acanthamoebidae). Protistologica

A molecular approach to the phylogeny of Acanthamoeba

Isoenzvme Datterns ofDathoEenic and 6. DeJonckheere, J. F. 1983. Isoenzyme and total protein analysis by agarose isoelectric focusing, and taxonomy of the genus Acanthamoeba

Amphizoic Amoebae: Human Pathology. Piccin Nuova Libraria

a new species of fresh-water amoebida

Naegleria and Acanthamoeba infections: review

Isoelectnc focusing of some enzymes from Echinococcus granulosus (horse and sheep strains) and E. multilocularis

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naegleria species

Isoenzyme analysis of Babesia microti infections in humans

Granulomatous brain tumor caused by Acanthamoeba. Case report

A New Key to Freshwater and Soil Amoebae

Morphologie de la paroi kystique et taxonomic du genre Acanthamoeba (Protozoa, Amoebida)

Electrophoretic isoenzyme patterns of the pathogenic and nonpathogenic intestinal amoebae of man

The enzyme-linked immunoelectrotransfer blot

Enzyme-linked immunoelectrotransfer blot technique (Western blot) for human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-I II/ LAV) antibodies. Immunology Series no. I5 Procedural Guide. US. Department of Health and Human Services

Comparative studies on related free-living and pathogenic amebae with special reference to Acanthamoeba

Leptomyxid ameba, a new agent of amebic meningoencephalitis in humans and animals

Acanthamoeba meningoencephalitis in a patient with AIDS

Acanthamoeha royreba sp. n. from a human tumor cell culture

non-pathogenic Naegleria spp. using agarose isoelectric focusing

The authors thank D. M. Moss, Centers for Disease Control, for helpful suggestions; Dr. Marc Pussard, Station d e Recherche sur la Faune du Sol, Dijon Cedex, France, for providing A. pustulosa (Ge3a) and A. echinulata (378); and Dr. James Yang, Toronto General Hospital, Toronto, Ontario, for providing the CDC-Fish-SK strain. Dr. Hercules Moura is a recipient of a scholarship from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil.