© 1999 by the Infectious Diseases Society of America. All rights reserved.Free-living amoebae cause three well-defined disease entities: a rapidly fatal primary meningoencephalitis, a chronic granulomatous amoebic encephalitis (GAE), and a chronic amoebic keratitis. GAE occurs in immunocompromised persons. Recently, another type of free-living amoeba, Balamuthia mandrillaris, has been shown to cause GAE. The finding that this amoeba has caused infection in some healthy children has raised the possibility that humans may lack immunity to B. mandrillaris. Human serum was examined for the presence of surface antibodies specific for this amoeba by immunofluorescence. Sera from adults contained titers of 1/64–1/256 of anti–B. mandrillaris antibodies (IgM and IgG classes), which did not cross-react with other amoebae. Cord blood contained very low antibody levels, but levels similar to those in adults were seen in serum of 1- to 5-year-old children.Z. Hua Huang, Antonio Ferrante, and Rodney F. Carte

Carter, R.

Ferrante, A.

Huang, Z.

The Journal of Infectious Diseases

Adelaide Research & Scholarship

1305Serum Antibodies to Balamuthia mandrillaris, a Free-Living Amoeba RecentlyDemonstrated to Cause Granulomatous Amoebic EncephalitisZ. Hua Huang,1 Antonio Ferrante, andRodney F. CarterDepartment of Immunopathology, Women’s and Children’s Hospital,and Department of Paediatrics, University of Adelaide,South AustraliaFree-living amoebae cause three well-defined disease entities: a rapidly fatal primary me-ningoencephalitis, a chronic granulomatous amoebic encephalitis (GAE), and a chronic amoe-bic keratitis. GAE occurs in immunocompromised persons. Recently, another type of free-living amoeba, Balamuthia mandrillaris, has been shown to cause GAE. The finding that thisamoeba has caused infection in some healthy children has raised the possibility that humansmay lack immunity to B. mandrillaris. Human serum was examined for the presence of surfaceantibodies specific for this amoeba by immunofluorescence. Sera from adults contained titersof 1/64–1/256 of anti–B. mandrillaris antibodies (IgM and IgG classes), which did not cross-react with other amoebae. Cord blood contained very low antibody levels, but levels similarto those in adults were seen in serum of 1- to 5-year-old children.Since the original postulation by Fowler and Carter in 1965[1] that free-living amoebae present in soil and water were re-sponsible for a rapidly fatal meningoencephalitis in children inSouth Australia, there have been worldwide reports of fatalcases of this disease and diseases caused by other types of free-living amoebae. Naegleria fowleri was named as the agent re-sponsible for the rapidly fatal primary amoebic meningoen-cephalitis [2]. Acanthamoeba species cause two otherwell-de®ned disease entities. They are responsible for the de-velopment of a granulomatous amoebic encephalitis (GAE)and a chronic amoebic keratitis [2].In its life cycle, N. fowleri displays cyst, trophozoite, andtransient ¯agellate stages. The persistence of speci®c stages de-pends on temperature, nutrients, and salt concentrations in theenvironment. The trophozoite stage is the infective form of thisamoeba, as it is for Acanthamoeba species which do not havea ¯agellate stage. N. fowleri and Acanthamoeba species are ubiq-uitous in nature (e.g., N. fowleri has been isolated worldwidefrom a variety of sources, such as public water supplies, swim-ming pools, freshwater lakes, and thermally polluted waters).Presented in part: 8th International Congress of Infectious Diseases. Bos-ton, 15±18 May 1998 (abstract 92.009).This work was approved by the Adelaide Women's and Children's Hos-pital's Ethics Committee (REC699/9/99).Financial support: Women's and Children's Hospital Clinical ResearchFellowship (to Z.H.H.).1 Present af®liation: Section of Endocrinology and Metabolism, RushPresbyterian and St. Luke's Medical Center, Chicago, Illinois.Reprints or correspondence: Professor A. Ferrante, Director of Immu-nopathology, Dept. of Immunopathology, Women's and Children's Hos-pital, 72 King William Rd., North Adelaide, South Australia 5006(aferrant@medicine.adelaide.edu.au).The Journal of Infectious Diseases 1999;179:1305–8q 1999 by the Infectious Diseases Society of America. All rights reserved.0022-1899/99/7905-0040$02.00Most cases of Naegleria meningoencephalitis involve childrenwith a history of swimming in public pools or freshwater lakes[2]. The organism invades via the olfactory neuroepithelium,directly entering the central nervous system (CNS), where theamoebae cause rapid destruction of the olfactory bulbs and awidespread meningitis and hemorrhagic, necrotizing encepha-litis. In contrast, Acanthamoeba species usually invade the CNSby a hematogenous spread, producing a GAE. Invasion of theCNS follows the establishment of the organism in primary sites,such as the skin, eyes, and respiratory tract [2]. Patients exhib-iting GAE do not have an associated history of swimming, areusually either debilitated and chronically ill or undergoing im-munosuppressive therapy, or are patients with AIDS [2]. Incontrast, chronic keratitis due to Acanthamoeba species occursin healthy individuals who may have experienced minor traumato the eye or who wear soft contact lenses [2].In the community, naturally acquired immunity to Naegleriaand Acanthamoeba species plays an important role in restrictingthe number of cases of primary amoebic meningoencephalitisand GAE [3]. Naegleria species does not spread hematogen-ously [1, 3] and has a restricted invasion route. In contrast,Acanthamoeba species spreads hematogenously, owing mainlyto a depressed defense system [2, 3]. A number of the com-ponents of the immune system are responsible for the immunitydisplayed against these organisms. These include the ability ofthe amoebae to activate complement through the alternativepathway (not requiring antibody), the ability of neutrophils andmacrophages to kill the amoebae, and the presence of anti-bodies against these amoebae [4±8].Another free-living amoeba, Balamuthia mandrillaris, wasdiscovered recently and has been shown to cause a fatal GAE[2, 9]. Of interest, many cases previously diagnosed as GAEhave now been retrospectively characterized as granulomatousBalamuthia encephalitis [2]. Although it has been assumed that1306 Huang et al. JID 1999;179 (May)Balamuthia species, like Acanthamoeba species, infect only im-munocompromised persons (including AIDS patients), infec-tion with this amoeba of 2 US children [10] and 2 Australianchildren [11, 12] possessing apparently normal immunity in-dicates that Balamuthia species may be more virulent thanAcanthamoeba species.The question of virulence is a crucial one since infectionscaused by B. mandrillaris are believed to also occur in healthychildren; thus, this organism is of great concern to our com-munity. Not only do we not know the environmental distri-bution of the organism, but very little is known about naturalhost defenses against the amoeba. Knowledge as to whetherwe possess ªnaturalº immunity to Balamuthia species will beof major importance in the education of parents and the com-munity regarding the dangers of this amoeba.Materials and MethodsSerum samples were randomly collected from healthy adult vol-unteers. In addition, serum samples were available from 1- to 5-year-old children who had undergone testing for immunoglobulinde®ciency and were found to be normal. These subjects were allSouth Australian residents. Umbilical cord blood was collected asthe source of neonatal serum.B. mandrillaris (leptomyxida ameba: CDC, V039), which wasisolated from the brain of a mandrill baboon [9], was obtainedfrom F. L. Schuster (Brooklyn College, Brooklyn, NY) [13]. Thisspecies has been well characterized, grown axenically, and used toraise antisera for diagnosing human cases of B. mandrillaris me-ningoencephalitis [9]. The amoeba was cultured axenically accord-ing to the method of Schuster and Visvesvara [13]. In brief, B.mandrillaris was maintained in growth medium containing biosatepeptone (Becton Dickinson Microbiology Systems, Cockeysville,MD), yeast extract (Sigma, St. Louis), Torula yeast RNA (Sigma),and ox liver extract (Oxoid, Hampshire, UK) supplemented with10% fetal calf serum for 5±7 days. When cultures reached con¯u-ence, they were chilled on ice for 5±10 min to detach the amoebae.After three washes with Hanks' balanced salt solution (HBSS), theamoebae were ®xed with 1% formaldehyde and stored at 47C foruse in experiments. N. fowleri and Acanthamoeba culbertsoni weresimilarly cultured in Fulton's medium A supplemented with 10%fetal calf serum and trypticase soy broth as described previously[4, 5].For immuno¯uorescence staining to detect anti±Balamuthia an-tibodies, the amoebae were incubated with serum at different di-lutions for 1 h at 47C, as indicated in the results [14, 15]. The cellswere washed, and 50 mL of ¯uorescein isothiocyanate (FITC)±labeled anti-human immunoglobulin (IgG1IgA1IgM) antisera(1/20 dilution; Behring, Marburg, Germany) was added. To ex-amine for antibody class, we added 10 mL of FITC-labeled anti-human IgM monoclonal antibodies (PharMingen, San Diego) or10 mL of phycoerythrin-labeled anti-human IgG monoclonal an-tibodies (PharMingen) for 1 h at 47C. After incubation, the cellswere washed and ®xed with 1% formaldehyde±Isoton II (CoulterElectronics, New South Wales, Australia) solution. Anti-amoebaantibodies were quanti®ed by ¯ow cytometry (FACScan with LysisII software; Becton Dickinson Immunocytometry Systems, Moun-tain View, CA). In addition, the cell preparations were added tomicroscope slides in liquid phase and examined by use of a ¯uo-rescence microscope.ResultsHuman serum contained anti±Balamuthia antibodies, as de-termined by the indirect immuno¯uorescence test. When theamoebae were viewed with a microscope under UV light, in-tense surface staining was evident (data not presented). Initialstudies demonstrated that the amount of ¯uorescence could bequantitated by ¯ow cytometry analysis. Flow cytometry of 50serum samples from adults showed that all containedanti±Balamuthia antibodies but with different levels of activity(data not presented). The titer ranged from 1/64±1/256, and theantibodies were IgM and IgG types. Placental transfer ofanti±Balamuthia antibodies was evident from the presence ofthese antibodies in cord blood (®gure 1). However, the levelswere much lower than those in adult serum (titer of <1/4; ®gure1). The anti±Balamuthia antibody levels in sera from the groupof 1- to 5-year-old children were similar to those in sera fromthe adults (®gure 1).To determine whether the antibodies against B. mandrillariswere speci®c for this amoeba or cross-reactive with Naegleriaor Acanthamoeba species, we examined the effects of serumadsorption with either of these amoeba on the serum titer ofthe anti±Balamuthia antibodies. B. mandrillaris (1 mL) wasmixed with either N. fowleri ( ), A. culbertsoni ( ),7 72 3 10 2 3 10or HBSS at 47C for 4 h, and the amoebae were removed bycentrifugation. This adsorbed serum was then tested for thepresence of antibodies to the 3 types of amoebae. Serum ad-sorbed with either N. fowleri or A. culbertsoni retained amountsof anti±Balamuthia antibodies equivalent to those of the non-adsorbed serum (table 1); however, the adsorbed serum hadlost 198% of the antibodies to either Naegleria or Acanthamoe-ba species (table 1). When human serum was adsorbed withB. mandrillaris (at one-half dilution), 89% of the anti±B.72 3 10mandrillaris antibody activity was lost. This showed that theantibodies binding to B. mandrillaris were speci®c for thisamoeba.DiscussionThe ability of amoebae that freely live in soil and fresh waterto cause disease in humans is of major concern to the com-munity, particularly as there is no effective treatment of CNSinfection caused by these organisms, and infection is invariablyfatal. We now present evidence that human serum containsantibodies to another type of amoeba, B. mandrillaris, that wasJID 1999;179 (May) Antibodies to Balamuthia mandrillaris 1307Figure 1. Levels of antibodies to Balamuthia mandrillaris in serumfrom adults, neonates, 1- to 3-year-old children (1±3Y), and 3- to 5-year-old children (3±5Y) as total immunoglobulin (Ig) and IgG andIgM classes. Serum was analyzed by indirect immuno¯uorescence tech-nique, using ¯ow cytometry at one-fourth serum dilution. Levels foradults and neonates were compared by use of Wilcoxon's signed ranktest: * ; ** .P ! .05 P ! .01Table 1. Serum anti-Balamuthia antibody levels after adsorption withother pathogenic free-living amoebae.Serum treatmentaAntibody titers toB. madrillaris N. fowleri A. culbertsoniNonadsorbed 1/256 1/64 1/256A. culbertsoni±adsorbed 1/256 ND !1/4N. fowleri±adsorbed 1/256 !1/4 NDNOTE. done.ND 5 nota Serum was adsorbed with Acanthamoeba culbertsoni or Naegleria fowleri andthen tested for antibody levels to Balamuthia madrillaris, A. culbertsoni, and N.fowleri by indirect immuno¯uorescence assay and ¯ow cytometry.recently shown to be responsible for a GAE. This amoeba isbelieved to invade the CNS secondary to the colonization ofprimary sites, such as the skin, and invasion of the CNS followsthe hematogenous spread of the amoeba. In many cases, suc-cessful invasion has been associated with immunosuppressionin the infected individual. Previous studies with N. fowleri andAcanthamoeba species have shown that immunity to these spe-cies results from the collaborative action of phagocytic cellsand antibody [3]. It is likely that the antibodies to B. mandril-laris also play an opsonization role. Preliminary studies haveshown that unlike Naegleria and Acanthamoeba species, B.mandrillaris was not killed (lysed) by human serum, althoughthis serum did promote adherence of neutrophils to the amoeba.It would be interesting to compare the ability of sera to promoteneutrophil-mediated killing of the amoeba.Any invasion from primary sites probably requires some de-pression of immunoglobulin levels or decreased phagocytic cellfunction (or both). The reasons why young, healthy childrencontracted Balamuthia infections that developed into a GAEremain unclear [10±12], but in one case, the child exhibitedfacial lesions that were colonized by the organism [12]. Anexamination of the serum of these patients for levels of anti±Balamuthia antibodies might provide some insight. However,initial studies with serum from our patient [11] who died ofB. mandrillaris GAE showed an increase in antibody titer of1/1024 ( /64; /1024). This is a 4-fold increase inIgM 5 1 IgG 5 1antibody titer relative to the titer found in control children ofsimilar age, suggesting that the child responded to the amoebaantigens. Whether this represents a protective response or notremains to be established. The rise in the titer, which is wellabove the range in noninfected children, could provide a meansof diagnosing the disease.Similar to the previous ®ndings with Acanthamoeba and Nae-gleria species, anti–Balamuthia antibodies were present in theIgM and IgG classes [15]. The serum was also found to beopsonic in attracting neutrophils to bind to B. mandrillaris andkilling the amoeba (data not presented); this ®nding could beexplored as another marker of immunocompetence. Of interest,the antibodies to this amoeba were speci®c because they couldbe adsorbed by B. mandrillaris but not Naegleria or Acanth-amoeba species. Since the antibody levels were very low in neo-nates, the ®ndings suggest that these levels substantially increasewith age, probably as a result of environmental exposure to theamoeba in the soil.The present studies were done with a B. mandrillaris isolatethat was recovered from a baboon that died from GAE [9].The isolate is the most characterized isolate, which led to thediscovery that it causes human GAE and can successfully becultured axenically. Indeed, antisera raised against this isolatehave been used to diagnose human cases of B. mandrillaris GAE1308 Huang et al. JID 1999;179 (May)[9]. It is therefore unlikely that our ®ndings are not represen-tative of human isolates. Our studies have also been con®nedto the sera of residents of South Australia; studies on sera froma wider international community may add further knowledgeto our understanding of natural immunity to B. mandrillaris.References1. Carter RF. Primary amoebic meningoencephalitis: an appraisal of presentknowledge. Trans R Soc Trop Med Hyg 1972;66:193±213.2. Martinez AJ, Visvesvara GS. Free living, amphizoic and opportunistic ame-bas. Brain Pathol 1997;7:583±98.3. Ferrante A. Free living amoebae: pathogenicity and immunity. Parasite Im-munol 1991;13:31±47.4. Ferrante A, Mocatta T. Human neutrophils require activation by mononu-clear leucocyte±conditioned medium to kill the pathogenic free-livingamoeba, Naegleria fowleri. Clin Exp Immunol 1984;56:559±66.5. Ferrante A, Abell TJ. Conditioned medium from stimulated mononuclearleukocytes augments human neutrophil-mediated killing of a virulentAcanthamoeba. Infect Immun 1986;51:607±17.6. Ferrante A, Rowan-Kelly B. Activation of the alternative pathway of com-plement by Acanthamoeba culbertsoni. Clin Exp Immunol 1983;54:477±85.7. Ferrante A, Rowan-Kelly B. The role of antibody in immunity against ex-perimental naegleria meningoencephalitis (ªamoebic meningitisº). Im-munology 1988;64:241±4.8. Ferrante A, Carter RF, Lopez AF, et al. Depression of immunity to Naegleriafowleri in mice by selective depletion of neutrophils with monoclonal an-tibody. Infect Immun 1988;56:2286±91.9. Visvesvara GS, Schuster, FL, Martinez AJ. Balamuthia mandrillaris, N. G.,N. Sp., agent of amebic meningoencephalitis in humans and other animals.J Eukaryot Microbiol 1993;40:504±14.10. Rowen JL, Boerr CA, Vogel H, Baker CJ. Balamuthia mandrillaris: a newrecognized agent for amebic meningoencephalitis. Pediatr Infect Dis J1995;14:705±10.11. Hansman D, Kernick C, Abbott K, et al. Granulomatous amoebic enceph-alitis caused by Balamuthia mandrillaris: a case report from South Aus-tralia. In: abstracts of the 7th International Conference on Small Free-Living Amoeba (Adelaide, Australia, January 1996). Salisbury, SouthAustralia: Australian Water Quality Centre, 1996:4.12. Reed R, Cooke-Yarborough CM, Jaquiery A, et al. Notable cases: fatalgranulomatous amoebic encephalitis caused by Balamuthia mandrillaris.Med J Aust 1997;167:82±4.13. Schuster FL, Visvesvara GS. Axenic growth and drug sensitivity studies ofBalamuthia mandrillaris, an agent of amebic meningoencephalitis in hu-mans and other animals. J Clin Microbiol 1996;34:385±8.14. Ferrante A, Thong YH. Antibody-induced capping and endocytosis of sur-face antigens in Naegleria fowleri. Int J Parasitol 1979;9:599±601.15. Cursons RTM, Brown TJ, Keys EA, et al. Immunity to free-living amoebae:role of humoral antibody. Infect Immun 1980;29:401±7.

Serum antibodies to Balamuthia mandrillaris, a free-living amoeba recently demonstrated to cause granulomatous amoebic encephalitis

Z. Hua Huang

Antonio Ferrante

Rodney F. Carter

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Serum Antibodies to
                    Balamuthia mandrillaris,

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Serum antibodies to Balamuthia mandrillaris, a free-living amoeba recently demonstrated to cause granulomatous amoebic encephalitis

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