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Vol. 58, No. 6

IMMUNITY, June 1990, p. 1802-1806

0019-9567/90/061802-05$02.00/0 Copyright C) 1990, American Society for Microbiology

Pathogenic and Nonpathogenic Strains of Entamoeba histolytica Can Be Differentiated by Monoclonal Antibodies to the Galactose-Specific Adherence Lectin WILLIAM A. PETRI, JR.,'* TERRY F. H. G. JACKSON,2 VINODH GATHIRAM,2 KENDRA KRESS,' LINDA D. SAFFER,1 TERRI L. SNODGRASS,' MARTIN D. CHAPMAN,' ZVI KEREN,3 AND DAVID MIRELMAN3

Departments of Medicine and Microbiology, University of Virginia, Charlottesville, Virginia 22908'; Research Institute for Diseases in a Tropical Environment, Durban, South Africa2; and Department of Biophysics, Weizmann Institute, Rehovot, Israel3 Received 11 January 1990/Accepted 15 March 1990

Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patients with invasive disease have characteristic isoenzyme proffles (pathogenic zymodemes), suggesting a role for parasite factors in determining the severity of infection. A galactose-specific cell surface lectin from a pathogenic zymodeme was shown to mediate in vitro adherence to human colonic mucins and contact-dependent killing of target cells. Six nonoverlapping antigenic determinants were identified on the 170-kilodalton heavy subunit of the pathogenic lectin. Anti-lectin monoclonal antibodies (MAb) directed against epitopes 1 and 2 enhanced adherence whereas MAb to epitopes 3 through 6 either inhibited or had no effect on adherence. We tested 50 pathogenic and nonpathogenic strains for reactivity to these anti-lectin MAb by radioimmunoassay. MAb to epitopes 1 through 6 reacted in the radioimmunoassay with all 16 pathogenic zymodeme strains tested. In contrast, only MAb to epitopes 1 and 2 bound to the lectin from nonpathogenic strains. Western immunoblots with anti-lectin antibodies showed that the 170-kilodalton heavy subunit was present in the nonpathogenic amebae. Adherence of the nonpathogenic SAW 760 strain to human erythrocytes was enhanced by MAb to epitope 1 and blocked by galactose, confirming the presence of a functionally active lectin. A lectin radioimmunoassay based on MAb to epitopes 1 and 3 proved to be a simple and rapid method to distinguish pathogenic from nonpathogenic amebae in culture. Further exploration of the functional consequences of the antigenic differences demonstrated for the lectin may lead to a better understanding of its role in pathogenesis.

Entamoeba histolytica infects an estimated 480 million individuals annually, of which only 10% develop colitis or liver abscess (18). E. histolytica strains cultured from patients with invasive disease have been classified into pathogenic zymodemes on the basis of their distinctive hexokinase and phosphoglucomutase isoenzymes. Pathogenic zymodeme amebae are indistinguishable from the nonpathogenic zymodeme E. histolytica by the morphologic criteria currently used to identify E. histolytica in clinical specimens (14). Isoenzyme analysis has been performed on a research basis on over 3,000 isolates. A nonpathogenic zymodeme has never been cultured from a patient with invasive amebiasis, and only rarely have pathogenic zymodemes been isolated from asymptomatically colonized individuals (1215). The reasons for the apparently great difference in virulence of these E. histolytica zymodemes and the search for practical clinical tests to distinguish them are areas of intense investigation. The galactose-binding lectin has been purified from a pathogenic zymodeme (axenic strain HM1:IMSS) of E. histolytica. It is a 260-kilodalton (kDa) heterodimeric glycoprotein consisting of a 170-kDa heavy subunit linked by disulfide bonds to a 35-kDa light subunit (7-9). This lectin mediates in vitro adherence to human colonic mucin glycoproteins, suggesting that it is involved in the initial colonization and invasion of the colon (3). Six antigenically and functionally distinct epitopes have been mapped on the *

heavy subunit with monoclonal antibodies (MAb) (W. A. Petri, Jr., T. L. Snodgrass, K. Chadee, and M. D. Chapman, J. Immunol., in press). Here we used these MAb to demonstrate that the lectin is present in nonpathogenic zymodemes but that it contains only two of the six epitopes identified on the pathogenic lectin. MATERIALS AND METHODS Cultivation and harvesting of E. histolytica. Axenic E. histolytica, pathogenic strain HM1:IMSS, was grown in TYI-S-33 medium (Trypticase yeast extract [BBL Microbiology Systems, Cockeysville, Md.], iron, and serum) with 100 U of penicillin per ml and 100 ,ug of streptomycin sulfate (Pfizer, Inc., New York, N.Y.) per ml at 37°C in 250-ml plastic tissue culture flasks (8, 10). Amebae were harvested after completion of log-phase growth at 72 h by centrifugation at 150 x g for 5 min at 4°C and washed twice in ice-cold 75 mM Tris (Sigma Chemical Co., St. Louis, Mo.)-65 mM NaCl (pH 7.2) (8, 10). Nonaxenized pathogenic and nonpathogenic strains were grown in TYSGM-9 medium (2) containing rice starch in the presence of bacterial flora. In a few cases nonpathogenic amebae were grown in Robinson medium or TYI-S-33 containing bacterial associates (2, 11). The bacterial associates were not identified in any of the cultures. The amebic strains were classified into pathogenic and nonpathogenic zymodemes by thin-layer starch gel electrophoresis followed by visualization of the hexokinase and phosphoglucomutase isoenzyme bands (13). Amebae were washed in 75 mM Tris-65 mM NaCl (pH 7.5), pelleted, and

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lyophilized to transport them to the United States for radioimmunoassay (RIA). Pathogenic amebae utilized included HM1:IMSS, SAW 755, Rhaman, Thailand 078 and 090, NIH 303 and 200, and nine new isolates from South Africa. Nonpathogenic amebae tested included WI-1285:I, CDC: 0784:4, SAW 760 and 1734, and over 20 new isolates from South Africa, plus the Laredo strain. Purification of the lectin. Amebic trophozoites harvested from a 72-h culture (250-ml flasks) were preincubated on ice in 5 ml of 75 mM Tris-65 mM NaCl with a 1:1,000 dilution of diisopropylfluorophosphate (Sigma) before solubilization in 10 ml of 150 mM NaCl-50 mM Tris (pH 8.3)-0.5% Nonidet P-40 (Sigma)-5 mM EDTA (Sigma)-2 mM phenylmethylsulfonyl fluoride. The solubilized amebae were sedimented by centrifugation (10,000 x g) for 10 min, and the supernatant was applied at 4°C to a MAb affinity column consisting of 2 mg each of protein A-purified anti-lectin MAb H85, 7F4, 5B8, 3F4, and 6D2 immobilized on 1 to 2 ml of Affi-Gel 10 (Bio-Rad Laboratories, Richmond, Calif.). The supernatant was recirculated through the column with a peristaltic pump overnight, and the column was then washed with 15 ml of solubilization buffer followed by 15 ml of phosphate-buffered saline (PBS) (pH 7.5). The bound amebic lectin was eluted with 10 ml of 0.2 N acetic acid (pH 2.5), immediately frozen, and lyophilized to remove the acetic acid (8). MAb. The production and epitope mapping of murine anti-lectin MAb are reported elsewhere (8; Petri et al., in press). The epitope specificities and designations of the MAb used were as follows: epitope 1, 3F4; epitope 2, 8A3; epitope 3, 7F4; epitope 4, 8C12; epitope 5, 1G7; epitope 6, H85. The MAb were purified from 50% ammonium sulfate fractions of ascites by protein A affinity chromatography or preparative isoelectric focusing (4). Purified MAb were labeled with 125I by the chloramine T technique to a specific activity of 20 to 40 ,uCi/,ug (4). RIA of the lectin. Polyvinyl chloride microdilution plates (Dynatech Laboratories, Inc., Alexandria, Va.). were coated with 1 ,ug of anti-lectin MAb 3F4 (epitope 1) per well in 0.1 M bicarbonate buffer (pH 9.6) overnight at 4°C, and residual binding sites were blocked with 1% bovine serum albumin (BSA) in PBS (pH 7.2) containing 0.05% Tween 20. Affinity-purified lectin (1 to 50 ng per well) or amebae solubilized in 50 mM Tris-250 mM NaCl-0.5% Nonidet P-40 (pH 8.0) were incubated in the antibody-coated wells for 2 h at room temperature. After the wells were washed, immobilized lectin was quantified by adding 105 cpm of 125I-labeled anti-lectin MAb 7F4 (epitope 3) per well for 4 h at room temperature (8). Protein concentrations were determined by the BCA protein assay (Pierce Chemical Co., Rockford, Ill.) with BSA as the standard. Recognition of pathogenic and nonpathogenic amebae by

RIA. The ability of 125I-labeled MAb directed against epitopes 1 through 6 to bind to the lectin from the different E. histolytica strains was tested by RIA. Polyvinyl chloride microdilution plates were coated with MAb 3F4 (epitope 1) and blocked as described above. Serial dilutions of solubilized amebae were added to the wells and incubated for 2 to 4 h at room temperature. The plates were then washed, and 1 X 105 to 2 x 105 cpm of 125I-labeled MAb specific for epitopes 2 through 6 was added for an additional 2 to 4 h of incubation. After further washing the plates were dried, and the radioactivity in individual wells was counted in a gamma counter. Standard curves for the lectin were determined with each of the 125I-labeled MAb, and final results are expressed as picograms of lectin detected per 103 amebae. Western immunoblots. Trophozoites were incubated with

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1:1,000 diisopropylfluorophosphate on ice and solubilized in 50 mM Tris-150 mM NaCl containing 0.5% Nonidet P-40, 5 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride. Solubilized amebae (105 per lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis with ,Bmercaptoethanol on an 8% polyacrylamide gel and transferred to Immobilon-P membranes (Millipore Corp., Bedford, Mass.) at 0.5 A for 1 h. The Immobilon membrane was then blocked with 3% BSA-0.05% Tween 20 in PBS overnight. The primary antibody was diluted in 1% BSA-PBSTween 20 (1:2,000 dilution for polyclonal antisera; 1:100 for MAb ascites) and incubated for 1 h at room temperature. The Immobilon was then washed four times with PBSTween 20, with the first wash for 10 min at 50°C and additional washes at room temperature. The secondary antibody (anti-mouse immunoglobulin G-alkaline phosphatase conjugate) was added for 1 h diluted (1:7,500) in BSA-PBS-Tween 20, and then the membrane was washed three times at room temperature in PBS-Tween 20 before it was developed with the Protoblot system (Promega Biotec Corp., Madison, Wis.). Adherence of trophozoites to erythrocytes. Packed human erythrocytes (group A) were diluted 1:5,000 in PBS (pH 7.1). They were counted and then centrifuged at 150 x g for 5 min, and the pellet was suspended in M199 medium (GIBCO Laboratories, Grand Island, N.Y.) containing 25 mM N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Sigma)5.7 mM cysteine -0.5% BSA (M199S). Nonpathogenic strain SAW760 amebae grown with bacterial associates from patient flora in TYI-S-33 were centrifuged at room temperature at 150 x g for 5 min and washed once in M199S, and the pellet was suspended in M199S to give a concentration of 105 amebae per ml. Adherence was measured in the presence or absence of 55 mM galactose (Pfansteil) or MAb 3F4 (10 ,ug per 104 amebae). The trophozoites and the erythrocytes were centrifuged together in Eppendorff tubes (1 x 103 amebae and 2 x 104 erythrocytes) at 150 x g for 5 min at room temperature and then incubated at 37°C for 20 min. They were fixed in PBS-1.5% formaldehyde for 10 min at room temperature and washed twice with PBS. The erythrocytes were stained with a freshly prepared solution of 3,3-diaminobenzidene (2 mg/ml)-0.2% H202 in 50 mM Tris (pH 9.7) at 37°C for 30 min (17). They were washed twice in PBS, part of the supernatant was removed, and the pellet was suspended with a Pasteur pipette. Adherence was expressed as the number of amebae having at least three adherent erythrocytes, with at least 40 amebae counted per determination. RESULTS RIA using epitope 1 and 3 MAb is specific for pathogenic E. histolytica. We previously reported the development of a two-site MAb-based assay for the 170-kDa subunit of the galactose lectin. In this assay the lectin is captured by epitope 1 MAb 3F4 coated on microdilution wells and detected by 1251I-labeled epitope 3 MAb 7F4 (8). The amount of radioactive 7F4 bound per well is linearly related to the amount of lectin added to the well from 1 to 25 ng per well. The RIA is capable of detecting as few as 30 pathogenic strain HM1:IMSS amebae or 0.5 ng of antigen added to a well (Fig. 1). In contrast, no lectin could be detected by this RIA in the nonpathogenic zymodeme CDC:0784:4, even with 1,000 amebae per well (Fig. 1). The failure of this RIA to detect lectin in CDC:0784:4 was not due to the different culture conditions required for the nonpathogenic zymo-

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FIG. 1. RIA of the galactose-N-acetylgalactosamine adherence lectin. Microdilution wells were coated with anti-lectin MAb 3F4 (epitope 1). Known amounts of the purified lectin or detergentsolubilized E. histolytica trophozoites (0, pathogenic zymodeme strain HM1:IMSS; 0, nonpathogenic zymodeme CDC:0784:4) were added to the 3F4-coated wells. The amount of lectin bound to the 3F4-coated wells was quantitated with 125"-labeled anti-lectin MAb 7F4 (epitope 3).

deme. No significant difference in lectin content was detected when pathogenic HM1:IMSS amebae were grown in TYI-S-33 medium without bacterial associates or TYSGM-9 medium with bacterial associates from the CDC:0784:4 culture (7.7 or 4.8 pg per ameba, respectively). A total of 18 pathogenic and 32 nonpathogenic zymodeme strains were assayed with the epitope 1- and 3-based RIA. Lectin was detected in all of the pathogenic strains but none of the nonpathogenic strains (Fig. 2). The absolute quantity of lectin detected per ameba varied greatly among the pathogenic amebae; however, the mean lectin concentration of the pathogenic amebae remained significantly different from the undetectable lectin concentration in the nonpathogenic amebae (P < 0.01). Amebae with lower lectin concentrations generally were the strains that had been frozen, lyophilized, and sent from South Africa to the United States before RIA, so that the lower lectin concentrations detected could have been due to instability in the lectin during transport. No correlation could be made between lectin concentration and culture medium (TYI-S-33, TYSGM-9, Robinson medium) used. RIA using epitope 1 and 2 MAb detects pathogenic and nonpathogenic E. histolytica. The inability of the RIA with epitopes 1 and 3 to detect lectin could reflect either an absolute lack of lectin or a loss of MAb binding to the nonpathogenic lectin. The two-site MAb-based RIA was therefore modified so that 1251I-labeled epitope 2 MAb 8A3 was used to detect lectin captured in the wells coated with epitope 1 MAb 3F4. This RIA had a sensitivity similar to that of the epitope 1 and 3 assay, with the amount of radioactive 8A3 bound per well linearly related to pathogenic lectin concentrations under 50 ng per well (data not shown). The assay in this configuration detected lectin in the nonpathogenic zymodemes as well as in the pathogenic zymodemes, indicating that the lectin was present but no longer recognized by epitope 3 MAb. The amount of lectin per ameba showed variations that were generally the same as those of the pathogenic amebae (Fig. 3). Western blots of nonpathogenic amebae with anti-lectin antibodies. To confirm that the lectin heavy subunit was

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088888o Oooo88oo 0888808 Zymodeme Z=nod FIG. 2. RIA with MAb to epitopes 1 and 3 of the galactoseN-acetylgalactosamine (Gal/GalNAc) lectin performed on pathogenic and nonpathogenic E. histolytica. Solubilized amebic proteins were added to wells coated with anti-lectin MAb 3F4 (epitope 1). The amount of lectin bound to the wells was quantitated with 251I-labeled anti-lectin MAb 7F4 (epitope 3). Symbols: 0, pathogenic zymodeme; 0, nonpathogenic zymodeme. The broken line represents the limit of sensitivity for the RIA. present although antigenically altered in nonpathogenic zymodemes, solubilized amebae were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed

with anti-lectin antibodies. Heavy-chain-specific polyclonal antibody (8) and epitope 1 MAb 3F4 detected the 170-kDa heavy subunit in the nonpathogenic zymodeme strain SAW 760 (Fig. 4). This 170-kDa band comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the pathogenic lectin heavy subunit (data not shown). Antigenic conservation of epitopes 1 through 6. The binding of radiolabeled MAb to all six of the epitopes on the 170-kDa subunit was measured by RIA. All six epitopes defined by the MAb were present in 16 of 16 pathogenic zymodemes tested (Table 1). These pathogenic strains included HM1: IMSS, SAW 755, Rhaman, Thailand 078, Thailand 090, and nine new isolates from South Africa (seven from liver abscess patients and two from asymptomatically colonized individuals). Epitopes 1 and 2 were present and epitopes 3 through 6 were absent in all of the 27 nonpathogenic isolates tested, including strain SAW 1734 and 26 recent isolates (assayed within several weeks of initial culture) from colonized individuals in South Africa. Nonpathogenic strains CDC 0784:4 and SAW 760 reacted with MAb against epitopes 1 and 2 but not with MAb against epitope 3; MAb against epitopes 4 through 6 were not tested on these two strains.

VOL. 58, 1990

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FIG. 3. RIA with MAb to epitopes 1 and 2 of the galactoseN-acetylgalactosamine (Gal/GalNAc) lectin performed on pathogenic and nonpathogenic E. histolytica. Solubilized amebic proteins were added to wells coated with anti-lectin MAb 3F4 (epitope 1). The amount of lectin bound to the wells was quantitated with 1251I-labeled anti-lectin MAb 8A3 (epitope 2). Symbols: 0, pathogenic zymodeme; 0, nonpathogenic zymodeme. The broken line represents the limits of sensitivity for the RIA.

Adherence of nonpathogenic amebae to erythrocytes. The of a functional galactose lectin in the nonpathogenic zymodeme strain SAW 760 was confirmed by measuring adherence at 37°C to erythrocytes. The number of trophozoites forming rosettes with human blood group A erythrocytes was enhanced almost threefold by anti-lectin MAb to epitope 1; the 3F4-enhanced adherence was inhibited 95% by 55 mM galactose. The number of amebae forming erythrocyte rosettes was increased from 19.1 + 1.2% to 53.1 5.2% with 10 ,ug of epitope 1 MAb 3F4 per ml. Galactose (55 mM) decreased amebic adherence to erythrocytes in the presence of 3F4 MAb to 2.9 3.1%. presence

±

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has not been useful in clinical practice. Generally several days of laboratory work are required to determine the zymodeme after already an average of 2 days spent to establish a culture from the specimen. The rapidity of the anti-lectin RIA was demonstrated by its accurate assignment of zymodemes to 35 fresh clinical isolates from South Africa. We have adapted the RIA to a more practical enzyme-linked immunosorbent assay format and plan to test the feasibility of direct testing of stool specimens, with the hope of eliminating even the 2 days required to establish cultures. Strachan et al. have reported a MAb to an undefined antigen which by immunofluorescence distinguished pathogenic from nonpathogenic amebae (15). The RIA that we have developed is quantitative, is for a well-defined antigen, and is easier to perform and automate than immunofluorescence. DNA probes that differentially hybridize to pathogenic and nonpathogenic zymodemes have been developed by Garfinkel et al. (6) and Tannich et al. (16). Only with TABLE 1. Summary of anti-lectin MAb reactivity with pathogenic and nonpathogenic zymodemes of E. histolytica

Zymodeme

Immuno-

DISCUSSION MAb against the amebic lectin have proven to be an excellent means to distinguish pathogenic from nonpathogenic zymodemes of E. histolytica. The data presented here show a perfect correlation of MAb reactivity with zymodemes for all 50 strains tested, including isolates from three continents. The remarkable antigenic conservation of the epitopes defined by the murine MAb on the lectin from pathogenic zymodemes offers the promise of worldwide applicability of this antigen detection test. Although zymodeme classification has been the standard epidemiologic tool for over a decade, it is a lengthy and complex procedure that

Epitopea MAba globulin G isotypea

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Pathogenic'

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a Data from Petri et al. (in press). b Sixteen isolates were tested by RIA. c Twenty-seven isolates were tested by RIA. d Adherence of pathogenic strain HM1:IMSS to human colonic mucins and Chinese hamster ovary cells (Petri et al., in press).

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further testing and development will it be determined whether antigen detection or DNA probes are better suited for the diagnosis of amebiasis. What is clear is that these recent developments in diagnosis offer significant advantages over the presently cumbersome microscopic identification of E. histolytica performed in clinical microbiology laboratories today. With the additional ability of these new tests to determine whether the infecting E. histolytica is pathogenic or nonpathogenic, potentially toxic and expensive anti-amebic chemotherapy could be reserved for the minority of individuals who are infected with pathogenic zymodemes. Previous work has demonstrated the central role of the galactose lectin from pathogenic zymodemes in the initiation of contact-dependent cytolysis and binding to colonic mucins (3, 10). The presence of a similar lectin in nonpathogenic zymodemes, which colonize but do not invade humans, had not been demonstrated until the present report. Lectin heavy-chain-specific antiserum, which completely blocks the adherence of pathogenic zymodeme amebae to target cells, cross-reacted with an identical-size 170-kDa protein in nonpathogenic zymodemes. MAb mapping by RIA indicated that heavy-chain epitopes 1 and 2 but not epitopes 3 through 6 were present in nonpathogenic E. histolytica. Adherence assays demonstrated galactose-inhibitable adherence to human erythrocytes for the nonpathogenic SAW 760 strain. On this basis we conclude that the galactose lectin is present but antigenically altered in the nonpathogenic zymodemes. The MAb used in these studies define six antigenically and functionally distinct regions of the heavy subunit of the pathogenic lectin. MAb to epitopes 1 and 2, which are conserved in pathogenic and nonpathogenic amebae, enhance adherence to colonic mucins and CHO cells by 300 to 500%. Earlier studies showed that epitope 1 and 2 MAb enhancement of adherence was due to a direct activation of the galactose-binding ability of the lectin (Petri et al., in press). We have speculated that the enhancing epitopes could facilitate colonization of the colon in the presence of an anti-lectin antibody response. The conservation of these adherence-enhancing epitopes on all 50 strains of E. histolytica tested lends further support for their potential role in immune evasion by the parasite. The lack of epitopes 3 through 6 did not affect the galactose-inhibitable adherence of strain SAW 760 to erythrocytes. The antigenic differences demonstrated in the lectin therefore may -not result in functional changes in the lectin, at least as can be measured by our in vitro adherence assays of one nonpathogenic strain. This is not unexpected, since the activity of the lectin may be essential for colonization of the large bowel via binding to colonic mucins and apparently is also required for the attachment and ingestion of bacteria (1, 3). ACKNOWLEDGMENTS We thank Henry Mathews of the Centers for Disease Control and Louis Diamond of the National Institutes of Health for their invaluable assistance in culturing nonpathogenic amebae and Doris Estes for excellent secretarial assistance. William A. Petri, Jr., is a Lucille P. Markey Scholar. This work was supported by the Lucille P. Markey Charitable Trust, the Thomas F. and Kate Miller Jeffress Memorial Trust, a National Cancer Institute Cancer Center Support Grant, the U.S. Army Division of Chemical Research Development and Engineering, and Public Health Service grants AI-26649 and AI-24687 from the National Institutes of Health. Work at the Weizmann Institute was supported by a grant from the John D. and Catherine T. MacArthur

INFECT. IMMUN.

Foundation, and work at the Research Institute for Diseases in a Tropical Environment was supported by the Medical Research Council of South Africa.LITERATURE CITED 1. Bracha, R., and D. Mirelman. 1983. Adherence and ingestion of Escherichia coli serotype 055 by trophozoites of Entamoeba histolytica. Infect. Immun. 40:882-887. 2. Burchard, G. D., and D. Mirelman. 1988. Entamoeba histolytica: virulence potential and sensitivity to metronidazole and emetine of four isolates possessing nonpathogenic zymodemes. Exp. Parasitol. 66:231-242. 3. Chadee, K., W. A. Petri, Jr., D. J. Innes, and J. I. Ravdin. 1987. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica. J. Clin. Invest. 80:1245-1254. 4. Chapman, M. D., P. W. Heymann, and T. A. E. Platts-Mills. 1987. Epitope mapping of two major inhalant allergens, DerPI and DerFI from mites of the genus Dermatophagoides. J. Immunol. 139:1479-1484. 5. Diamond, L. S., D. R. Harlow, and C. C. Cunnick. 1978. A new medium for axenic cultivation of Entamoeba histolytica and other entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72:431-432. 6. Garfinkel, L. I., M. Giladi, M. Huber, C. Gitler, D. Mirelman, M. Revel, and S. Rozenblatt. 1989. DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect. Immun. 57:926-931. 7. Petri, W. A., Jr., J. Broman, G. Healy, T. Quinn, and J. I. Ravdin. 1989. Antigenic stability and immunodominance of the Gal/GalNAc adherence lectin of Entamoeba histolytica. Am. J. Med. Sci. 296:163-165. 8. Petri, W. A., Jr., M. D. Chapman, T. Snodgrass, B. J. Mann, J. Broman, and J. I. Ravdin. 1989. Subunit structure of the galactose and N-acetyl-D-galactosamine inhibitable adherence lectin of Entamoeba histolytica. J. Biol. Chem. 264:3007-3012. 9. Petri, W. A., Jr., R. D. Smith, P. H. Schlesinger, C. F. Murphy, and J. I. Ravdin. 1987. Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica. J. Clin. Invest. 80:1238-1244. 10. Ravdin, J. I., and R. L. Guerrant. Role of adherence in cytopathogenic mechanisms of Entamoeba histolytica. Study with mammalian tissue culture cells and human erythrocytes. J. Clin. Invest. 68:1305-1313. 11. Robinson, G. L. 1968. The laboratory diagnosis of human amoebae. Trans. R. Soc. Trop. Med. Hyg. 62:285-294. 12. Sargeaunt, P. G., and J. E. Williams. 1979. Electrophoretic isoenzyme patterns of the pathogenic and non-pathogenic intestinal amoeba of man. Trans. R. Soc. Trop. Med. Hyg. 73: 225-227. 13. Sargeaunt, P. G., J. E. Williams, and J. D. Grene. 1978. The differentiation of invasive and non-invasive Entamoeba histolytica by isoenzyme electrophoresis. Trans. R. Soc. Trop. Med. Hyg. 72:519-521. 14. Sargeaunt, P. G., J. E. Williams, and R. A. Neal. 1980. A comparative study of Entamoeba histolytica (NIH 200, HK9 etc), "E. histolytica like" and other morphologically identical amoeba using isoenzyme electrophoresis. Trans. R. Soc. Trop. Med. Hyg. 74:469-474. 15. Strachan, W. P., P. L. Chiodini, W. M. Spice, A. H. Moody, and J. P. Ackers. 1988. Immunologic differentiation of pathogenic and nonpathogenic isolates of Entamoeba histolytica. Lancet i:561-563. 16. Tannich, E., R. D. Horstmann, J. Knobloch, and H. H. Arnold. 1989. Genomic differences between pathogenic and nonpathogenic Entamoeba histolytica. Proc. Natl. Acad. Sci. USA 86:5118-5122. 17. Trissl, D., A. Martinez-Palomo, M. de la Torre, R. de la Hoz, and E. Perez de Suarez. 1978. Surface properties of Entamoeba. Increased rates of human erythrocyte phagocytosis in pathogenic strains. J. Exp. Med. 148:1137-1145. 18. Walsh, J. A. 1986. Problems in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Rev. Infect. Dis. 8:228-238.

Pathogenic and nonpathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin.

Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patien...
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