Entamoeba histolytica-like amoebae occurring in man

Bull. Org. mond. Sante 1 1969, 40, 355-364Bull. Wid Hith Org.I Entamoeba histolytica-like Amoebae Occurring in Man MORRIS GOLDMAN' Six Entamoeba histolytica-like amoebae of the Laredo-type are compared with clas- sical E. histolytica strains with regard to morphology, temperature-tolerance, response to hypotonic solutions, antigenic make-up, pathogenicity, sensitivity to drugs, and biochemistry. Sharp discontinuities are shown to exist between the 2 groups in some parameters, sufficient, under certain conditions, to justify creation of a new species designation for the Laredo- type amoebae. Nevertheless, this action is not recommended at the present time because of the small number of" aberrant " strains so far isolated, and because of the limited com- parative studies so far carried out. Entamoeba moshkovskii is compared with the Laredo-type E. histolytica. No con- sistent distinction can be made between the 2 groups except for the sources of the strains- the former is free-living and the latter parasitic. It is therefore suggested that E. mosh- kovskii may actually be a parasite of the Laredo type, only incidentally encountered in free-living environments because of its wide temperature and tonicity tolerances. Since, here too, only limited studies are sofar available, it is suggested that the term " moshkovskii- like E. histolytica " be used until sufficient data are accumulated to assign these amoebae to a more precise location in the taxonomic scheme. In 1956, Dr F. H. Connel became aware that a strain of Entamoeba histolytica he had isolated (the " Laredo" strain) possessed the unusual capability of living and multiplying at room temperature as well as at body temperature. Between 1964 and 1966 5 more such isolations from human faeces were reported (Nelson & Jones, 1964 (no strain name); Entner & Most, 1965 (JA and AG strains); Richards, Goldman & Cannon, 1966 (strains 403 and Huff). It is the purpose of this review to attempt to answer several questions. In what ways and by how much do these " aberrant " amoebae differ from classical E. histolytica? Do the differences justify raising these strains to the status of a separate species? What is the relationship of these strains to Entamoeba moshkovskii,2 the free-living amoeba which has identical morphology to Entamoeba histolytica ? 1 Director, Department of Immunology, Bionetics Research Laboratories, Inc., Bethesda, Md., USA. ' My thoughts on the status of E. moshkovskii have benefited from an exchange of views with Dr R. A. Neal but I assume sole responsibility for the opinions expressed. DIFFERENCES BETWEEN LAREDO-TYPE AND CLASSICAL E. HISTOLYTICA 3 Temperature tolerance Table 1 shows that, whereas the range of tempera- ture under which E. histolytica trophozoites can survive in certain specified conditions is 20'C-430C, the range for Laredo-type amoebae is 0C-41'C (Siddiqui, 1963). The minimum temperature for growth of E. histolytica in continuous culture is around 30°C (Cabrera, 1958; Cabrera & Porter, 1958) whereas for Laredo-type amoebae it is 10°C (Richards, Goldman & Cannon, 1966). The normal or optimum temperature for E. histolytica is around 37°C, but is 25°C-30°C for the Laredo-type strains. As results of this nature have now been replicated by several different workers over a period of more than a Very few, if any, of the studies to be cited are precisely comparable in respect of culture media, experimental tech- niques or strains used. Nevertheless, there is, by and large, an impressive similarity in results obtained in different laboratories. For this reason techniques will be cited only incidentally in the following analyses except when they appear to have had a significant influence on results. 2305 -355- M. GOLDMAN TABLE I TEMPERATURE PREFERENCES OF CLASSICAL AND LAREDO-TYPE E. HISTOLYTICA Range of temperature tolerateda Normal or optimum temperature a Reference or which supported proliferation aRemarks E. histolytica Laredo-type E. histolytica Laredo-type Cabrera (1958) Cabrera & Porter (1958) Dreyer (1961) Siddiqui (1963) Nelson & Jones (1964) Entner & Most (1965) \Ro sas & Najarian(1965) Albach, Schaffer & Watson (1966) Richards, Goldman & Cannon (1966) 290C-42.5°C 31 .7C-41 .3°C ND 20-C-43"C 37"C 37-C-430C ND ND >27"C-35WC ND ND 10`-35"C 0"C-41 C " room tempe- rature " 20iC-<43iC 22IC-40-C No higher than 32-C 5-C-35-C 37°C 37°C ND 37-C 37-C 370C ND 37"C 35-C ND ND ND 300C "room tempe- rature" 37*C 30-C 25"C 27*C Temperature shifted gradually above and below optimum Temperature shifted In a single step above and below optimum Clone culture tested Amoebae kept at given tem- perature 72 h and subcultured back at optimum temperature Limits of temperature toler- ance not studied Temperatures shifted grad- ually and In single steps a ND = not done. a decade, there can be no doubt that these differences in temperature tolerance represent stable, valid, and rather simple characteristics for distinguishing classi- cal E. histolytica from Laredo-type amoebae. Morphology and response to hypotonic solutions The morphology of Laredo-type amoebae is known only for cultured organisms since in no instance have they been recognized as different from classical E. histolytica before being grown in the laboratory. Goldman, Gleason & Carver (1962) and Entner & Most (1965) have called attention to the fact that nuclear chromatin in the Laredo JA and AG strains is sometimes distributed unevenly in the form of one or more masses applied to the nuclear membrane. On the other hand, in a detailed study of size and nuclear morphology in a classical strain of E. histolytica (K9) and in a Laredo-type strain (Huff), before the latter was recognized for what it was, no significant differences in size or morphology were noted (Gleason, Goldman & Carver, 1963). In view of the fact that at least 4 of the 6 known strains of Laredo-type amoebae (Laredo, Huff, 403 and Nelson's strain) were initially carried in the laboratories of experienced workers as if they were typical E. histolytica strains, and since undoubted classical E. histolytica also sometimes shows uneven massing of nuclear chromatin on the nuclear membrane (Goldman, Gleason & Carver, 1962), it may be concluded that at present there are no consistent morphological criteria that can differenti- ate E. histolytica trophozoites from the Laredo-type amoebae in culture. Cultured Laredo-type trophozoites were capable of completing cycles of division, encystment and excystment in extremely hypotonic solutions, such as culture medium diluted 1: 64 with distilled water (Richards, Goldman & Cannon, 1966). Upon trans- fer to such solutions the amoebae were first immobile but typical motility was resumed within a few hours and functioning contractile vacuoles were developed. By contrast, E. histolytica did not survive even a 1: 2 dilution of culture medium for any length of time (Richards, Goldman & Cannon, 1966; Gordeeva, 1966). Such results have been reported so far for 14 strains of E. histolytica and for 5 strains of Laredo-type amoebae. In an unpublished study conducted by myself and co-workers in India, some 50 more strains of E. histolytica have also failed to grow in diluted medium. 356 ENTAMOEBA HISTOLYTICA-LIKE AMOEBAE OCCURRING IN MAN Thus, a simple culture characteristic, in addition to temperature tolerance, that serves to distinguish classical E. histolytica from Laredo-type amoebae is the reaction to hypotonic solutions. Although it is desirable that more strains be tested, this appears to be a stable and dependable differentiating charac- teristic. Antigenic make-up Analyses of the antigenic relationships between classical E. histolytica and Laredo-type amoebae have been carried out by the techniques of fluorescent antibody, precipitation in agar gel, and haemag- glutination. Details are presented below. Fluorescent antibody reactions. The fluorescence work of Goldman and co-workers in the period 1960-67 was based upon quantitative measurements of the fluorescence imparted to individual amoebae after exposing them to various fluorescein-labelled antisera. Fluorescence of amoebae was measured with specially designed microfluorimeters, and the brightness figures were taken as representing the degree to which antibody had been bound by antigens in the different amoebae studied. Details of methodology and controls must be sought in the original publications. The Huff strain, which was recognized several years later as being a Laredo-type amoeba, reacted less intensely with anti-histolytica sera than 3 strains of classical E. histolytica (Goldman, Carver & Gleason, 1960). In experiments involving cross- absorption and cross-staining, fluorescence results were interpreted as indicating that Huff was deficient in some antigens present in a classical E. histolytica strain (Goldman & Gleason, 1962). The Huff strain was also compared with E. histolytica K9 with regard to its suitability as an antigen in a fluorescent antibody test for amoebiasis (Goldman, 1966). Huff antigen yielded positive results in only 33% of proven acute cases, compared with 87.5% positives obtained with K9 antigen. In addition, the serum titres obtained with Huff organisms were lower than with K9. An early comparison of the type strain itself, Laredo, with K9 showed differences in antigenicity with 5 anti-histolytica sera when Laredo was grown at 25°C (Goldman et al., 1962). With one antiserum a clear-cut difference was also apparent when the amoebae were cultured at 37°C. In the most complete study thus far, 5 Laredo-type strains (Laredo, Huff, JA, AG and 403) were compared with 8 strains of classical E. histolytica, all 13 strains being grown at 35°C (Goldman & Cannon, 1967). The fluorimetric results indicated a distinct superiority in the ability of the E. histolytica group to react with the test reagents-a human antiserum derived from a patient with amoebic liver abscess, and a rabbit antiserum prepared against classical E. histolytica. Finally, Siddiqui & Balamuth (1965) reported that an anti-histolytica serum stained the corresponding E. histolytica strain somewhat brighter than the Laredo strain (2+ versus 1+). Precipitation in agar gel. Krupp (1966) compared Huff and Laredo with 8 strains of classical E. histo- lytica. All strains were grown at 37°C in the same all- liquid medium with the same single bacterial associ- ate. Extracts of amoebae were subjected to electro- phoresis in relatively thick layers of agar and then allowed to react with anti-histolytica sera. E. histo- lytica antigens showed 3-8 precipitin bands, Huff showed 2-3 bands, while Laredo showed 3-4 bands: I or 2 bands were also obtained with the ho-se serum component of the culture medium. Lunde & Diamond (1969) investigated 4 strains of E. histolytica growing at 35.5°C and the Laredo strain at 25°C, all from axenic cultures. As in Krupp's work, amoebic extracts were subjected to electrophoresis and allowed to react with anti- histolytica sera. At least 6-8 distinct bands were found in all the E. histolytica extracts, but only 2 were found in the Laredo antigen. Characteristically, a prominent slow-moving component present in all E. histolytica antigens was absent in Laredo. Siddiqui & Balamuth (1965) obtained a weak reaction between anti-histolytica serum and Laredo antigen in double-diffusion experiments. The re- action with E. histolytica antigen was considerably stronger. Haemagglutination. Only a single comparison has so far been carried out with this technique. Lunde & Diamond (1969) sensitized red blood cells with extracts of either axenic E. histolytica (35.5°C) or axenic Laredo amoebae (25°C). In testing a small group of human sera they obtained significantly lower haemagglutination titres with the Laredo antigens than with E. histolytica. Summary of antigenic studies. All except 2 of the studies cited above suffer from 2 important deficien- cies: no antiserum against Laredo-type amoebae was used to investigate cross-reactions from both directions, and no comparisons were run of Laredo- type organisms maintained at more than a single 357 M. GOLDMAN temperature. Thus the precise extent of antigenic dissimilarity between classical and Laredo-type E. histolytica cannot yet be detailed. These lacunae in our knowledge, however, must not be allowed to distort the otherwise clear picture that has been emerging-that while the classical and Laredo-type organisms share certain antigenic configurations they are certainly not identical. On the contrary, 3 very different immunological techniques have all shown distinct antigenic differences between the 2 groups. Unfortunately, there is no immediate likelihood of extensive antigenic studies with large numbers of strains to clarify relationships further. Studies using extracts of amoebae are almost impossible unless axenic or, at least, monoxenic cultures can be employed, but such cultures are available in only a limited number of laboratories. Fluorescent anti- body methods can cope with crude cultures but the differences encountered thus far have been so subtle as to tax the most experienced eye, and thus micro- fluorimetric methods and equipment will probably continue to be necessary. Even fewer laboratories are equipped for such studies. Thus from the practical standpoint, rapid identification and diffe- rentiation of Laredo-type strains on the basis of antigenicity is not yet feasible. Pathogenicity Information concerning the clinical status of the individuals from whom the 6 known Laredo-type amoebae were isolated is presented in Table 2. In 3 instances (Huff, Nelson's strain, and 403) the original hosts are described unequivocally as asymptomatic or cyst-passing carriers. The clinical status of the other 3 hosts is more difficult to assess either because the original descriptions are incom- plete (JA and AG), or because the patient's symptoms could be assigned as readily to the benign tumour from which he was suffering as to the amoebic infection (Laredo). At any rate, none of this latter group can be considered as showing clear-cut symptoms of amoebic invasion of tissue or of amoebic colitis. Beaver et al. (1956a) carried out experimental infections of human volunteers with the freshly isolated Huff strain. In 81 out of 130 attempts, infections were established, but no symp- toms characteristic of clinical amoebiasis were found in any of the infected volunteers (Table 3). Thus, there is an over-all impression of benignity for these strains in the human host. This in itself, however, does not provide much of a distinction from classical E. histolytica since the latter are also frequently found in asymptomatic hosts living apparently as com- mensals in the lumen of the bowel. There are 5 published accounts of attempts to infect experimental animals with 5 of the Laredo-type amoebae (all except Nelson's strain). Table 3 shows that in 4 of these experiments, involving rats, hamsters and guinea-pigs, only 12 infections were obtained out of a total of 245 animals inoculated. Furthermore, no signs of tissue invasion were seen in any instance. In contrast to this record of low infectivity and non-invasiveness is the report of Beaver et al. (1956a) which cites 17 infections out of 38 rats, guinea-pigs and dogs inoculated with the Huff strain. Of these 11 showed mild to severe TABLE 2 CLINICAL STATUS OF ORIGINAL HOSTS OF LAREDO-TYPE STRAINS Reference T Strain Clinical description Dreyer (1961); Goldman, Laredo History of diarrhoea for several years; weight loss; epigastric pain; polypoid Gleason & Carver, tumour of transverse colon (adenomatous polyp) removed surgically, followed 1962) a by complete recovery. Beaver et al. (1956b) " H " = Huff Consistent cyst-passer without symptoms of amoebiasis for 2 years. Entner & Most (1965) AG ".patient with a recurring case of mild, chronic amoebiasis which did not respond to drug therapy ". JA ".isolated in May 1963 from a young male in an institution for mental illness Nelson & Jones (1964) No name "...established ... from a cyst-bearing stool from a carrier Richards, Goldman & 403 Isolated from an asymptomatic carrier. Cannon (1966) a From unpublished data supplied by F. H. Connel. 358 ENTAMOEBA HISTOLYTICA-LIKE AMOEBAE OCCURRING IN MAN TABLE 3 INFECTIVITY AND VIRULENCE OF LAREDO-TYPE STRAINS IN EXPERIMENTAL HOSTS Reference Laredo-type strain used Experimental Routeiof io- No. infected/ Remarkshost culation a no. inoculated Beaver et al. "H "= Huff, cultured at Rats IC (t) 10/10 Mild lesions in 4, no(1956a) 370C lesions in 6 animals Guinea-pigs IC (t) 5/18 Severe lesions in 1, mild in 4 animals Dogs Oral (c) 2/10 Severe lesions in 1, mild in 1 animal Humans Oral (c) 81/130 No symptoms in all 81 infections Rosas & Laredo, cultured at 22°C Rats IC (t) 0/25 Najarian and 300C Hamsters IH (t) 0/63 (1965) Healy & Huff and Laredo, cultured Rats IC (t) 1/21 No lesions in the single Gleason at 370C infection(1966) Goldman & Huff, Laredo, AG, JA, 403 Guinea-pigs IC (t) 0/51 Cannon cultured at 350C (1967) Neal & Johnson Huff, Laredo, AG, JA, 403 Rats IC (t) 11/85 No lesion found in any (1968) cultured at 25C animals a IC = intracaecal; IH = intrahepatic; t = trophozoite; c = cysts. lesions of the intestinal tract. This difference in results of infectivity experiments is due, perhaps, to the fact that Beaver's work was performed with a relatively newly isolated strain, while the others dealt with strains maintained in vitro for a few to many years. It seems fair to conclude from these limited reports that the Laredo-type organisms are of low or even no pathogenicity to the human host, and of low or restricted pathogenicity to the common laboratory animals that are susceptible to classical E. histolytica. Nevertheless, since there is ample evidence in the literature of the existence of E. histolytica strains that are equally non-virulent in experimental and human hosts, the factor of pathogenicity in experimental animals is of limited value in differentiating the 2 groups of amoebae. It is clear that more strains of Laredo-type organisms need to be studied as soon as they are isolated before we can be certain that complete non-pathogenicity characterizes the group as a whole. Sensitivity to drugs Entner and his co-workers have compared the sensitivity to drugs of 3 Laredo-type strains (Laredo, AG and JA) and 5 classical E. histolytica (Entner, Evans & Gonzalez, 1962; Entner & Most, 1965). Determinations were made on amoebae growing in all-liquid medium at a temperature of 37°C. The 3 Laredo-type strains were all more resistant than the other strains to the following degrees: emetine- 10-fold; cycloheximide (Actidione)-20- to 50-fold; fumagillin-20- to 100-fold; carbarsone (N-carba- mylarsanilic acid)-2-fold; and chlorbetamide (Man- tomide)-2-fold. Albach, Schaffer & Watson (1966) made similar determinations on the Laredo strain versus 4 classical E. histolytica strains. In this case, however, Laredo was tested at 25°C while the others were tested at 37°C. The results showed Laredo to be more resistant to emetine, about 30- to 50-fold, and to oxytetracycline (Terramycin), about 5-fold. Sus- ceptibility to paromomycin (Humatin) was about the same as the classical E. histolytica strains. Inasmuch as in these studies each type of amoeba demonstrated reasonable uniformity of susceptibility to drugs within their respective types, and a con- sistent difference in susceptibility between the 2 types, we may conclude that the differences represent real and characteristic attributes of the 2 types of amoebae. Unfortunately, however, drug testing of the precision shown in the studies cited above 3 359 M. GOLDMAN requires special culture techniques to avoid the complications associated with indirect influences on bacteria associated with the amoebae. This imposes serious limitations on the possibility of screening large numbers of new strains in order to identify and characterize Laredo-type organisms. Biochemistry The Laredo, AG and JA strains all utilized only glucose, maltose, and galactose out of 11 sugars tested. In this respect they showed no differences from 5 strains of classical E. histolytica (Entner, Evans & Gonzalez, 1962; Entner & Most, 1965). On the other hand, analysis of the free amino-acid composition of Laredo compared with 3 strains of classical E. histolytica revealed quantitative differ- ences in 6 of the amino-acids, alanine, serine and threonine being more abundant in Laredo, while leucine-isoleucine, arginine and valine were more abundant in the other amoebae (Albach & Shaffer, 1965). Differences were also found in filtrates of the medium in which the amoebae had grown for either 72 h at 37°C (classical strains) or for 6 days at room temperature (Laredo). Nine strains of E. histolytica were compared with the Laredo and Huff strains with regard to the presence and character of the enzyme glucokinase (Reeves, Montalvo & Sillero, 1967). Each of the classical strains revealed the same 2 electrophoreti- cally distinct isoenzymes, but Laredo and Huff showed only a single band comparable in its migra- tion to the faster of the 2 isoenzymes. The biochemical evaluation of Laredo-type strains is as yet extremely sparse. However, so far it appears that the Laredo group does indeed possess at least some biochemical attributes clearly distinct from those characterizing classical E. histolytica strains. NOMENCLATURE OF LAREDO-TYPE AMOEBAE It is fashionable, in discussions of the intestinal amoebae, to refer to the " E. histolytica complex " and to emphasize the fact that this species appears to include strains showing a continuous and wide spectrum of characteristics. For this reason, the first point to be made with regard to the Laredo-type organisms is that they do not represent simply one extreme of a continuous spectrum of classical E. histolytica strains; instead they demonstrate a clear discontinuity in several attributes from the strains comprising the classical E. histolytica group. This is exemplified most obviously with regard to temperature and tonicity tolerances, since there are no known strains of E. histolytica showing characte- ristics intermediate between the Laredo and classical types. A second point is that the Laredo-type amoebae possess a cluster of common characteristics. That is, strains showing certain temperature tolerances also show certain tonicity tolerances, drug sensitivities, antigenic reactions, and biochemical characteristics. So far there are no known strains of classical E. histolytica (judged by temperature requirements) which show, for example, the resistance to drugs characteristic of Laredo. The third point to consider is that in spite of morphological identity with the classical strains, Laredo-type strains are rather easily distinguishable in culture by the simple tests of temperature and tonicity tolerances. Thus, the practical needs of the taxonomist or diagnostician are not too difficult to meet, especially in comparison with the over-all difficulties of working with the intestinal amoebae. It would appear then, from the above considera- tions, that good justification exists for acknowledging the Laredo-type amoebae as members of a species separate from E. histolytica. In fact, apart from the morphology of the mature cyst, there may already be more precisely known differences between the Laredo- type strains and E. histolytica than between the latter and Entamoeba coli. That morphological identity is not an overriding factor in retaining a single species designation when dealing with the intestinal amoebae is shown by the accepted use of separate species names to identify the morphologi- cally similar amoebae isolated from man, snakes, frogs and other hosts. When all the above is said, it is my opinion that, at present, a decisive reason for not setting up a new species to include the Laredo-type group is that too few strains have as yet been isolated, and too few laboratories have so far performed comparative studies. It is perfectly conceivable that strains may yet be located that will not show the sharp discon- tinuities we see at present; or it may be that, as studies are performed by new and different workers with new or modified techniques, distinctions may be found even among the classical E. histolytica strains of a type to parallel those shown by the Laredo-type. For these reasons I consider that the designation E. histolytica should be retained to include all strains showing the well-known morpho- logy and isolated from human sources. The qualifying 360 ENTAMOEBA HISTOLYTICA-LIKE AMOEBAE OCCURRING IN MAN term " Laredo-type " or something equivalent should be used to describe strains with characteristics similar to those of the Laredo strain of E. histolytica. I think it important to emphasize that the subject of nomenclature should remain open and flexible. Should several more Laredo-type strains be isolated, and should they all continue to show the same family of attributes we now ascribe to the Laredo strain, the case for setting up a new species will be greatly enhanced. Emile Brumpt (1949) believed in the existence of a histolytica-like species which he called Entamoeba dispar. This species, he claimed, was identical in appearance to non-invasive E. histo- lytica (which he called E. dysenteriae), was never pathogenic in man, and was only slightly infective to animals but never invasive. We should be im- pressed by the fact that this is an accurate, if in- complete, description of the Laredo-type strains now known. RELATIONSHIP OF ENTAMOEBA MOSHKOVSKII TO E. HISTOLYTICA Entamoeba moshkovskii has been isolated in vari- ous parts of the world from sewage treatment plants and from streams receiving the effluent of sewage plants (see Neal, 1966, for bibliography). Tshalaia (1941), who first described the amoeba, recognized that, in spite of its similarity in appearance to E. histolytica, it possessed 2 distinctive characteristics: it multiplied at temperatures from 10°C to 37°C, and it survived exposure to hypotonic solutions in which it formed contractile vacuoles. Thus, the species was clearly distinct from any strains of E. histolytica known at the time. With the isolation of Laredo-type strains directly from human sources, and the demonstration by Richards, Goldman & Cannon (1966) that such strains show the same temperature and tonicity tolerances as E. moshkovskii, it is clear that the status of the latter as a separate species should be re-examined. Table 4 tabulates data concerning E. moshkovskii and various Laredo-type organisms. Unlike the situation with regard to the latter and classical E. histolytica, no clear-cut distinction can be made between strains of E. moshkovskii and the Laredo group. Both are entirely similar with regard to temperature and tonicity tolerance, and to the nature of the enzyme glucokinase that both possess. With regard to pathogenicity, no laboratory infections have been obtained with E. moshkovskii, and, except for Beaver's work discussed above, only a few isolated successes have been achieved with Laredo- type strains. Antigenic studies reveal similarities and dissimilarities consistent with the distribution of reactions among different strains of a single group (see in particular Goldman & Cannon, 1967). Thus, from the standpoint of known characteristics, there is less basis for separating E. moshkovskii from the Laredo group than for separating the latter from classical E. histolytica. There remains the consideration that the amoeba called E. moshkovskii has always been isolated from the free-living state rather than directly from some animal host. However, as Neal (1953) has pointed out, the fact that he and others found the amoeba in crude sewage entering disposal plants implies that the organism was continually pouring in from outside sources. Furthermore, in Stewart & Beck's (I967) study of 67 species of protozoans, E. moshkovskii fell unequivocally into the parasitic group on the basis of the absence of nuclear DNA-histone. This is striking and unusual confirmation of the concept already voiced by de Carneri (1963), Neal (1966), and Goldman & Cannon (1967) that E. moskkovskii might actually be a parasitic species incidentally encountered in free-living environments in which it can survive because of a wide temperature and tonicity tolerance. In my opinion, the most important reasons for not at present including E. moshkovskii under the umbrella designation of " Laredo-type E. histolytica " are that too few strains have been studied in sufficient detail, and that the moshkovskii group may turn out to be a conglomerate of parasitic forms with E. histolytica-like morphology whose hosts may possibly include other species besides man. On the basis of all the above considerations, I think the designation " moshkovskii-type E. histolytica " would accurately describe and place these particular organisms in the framework of our thinking about amoebae that look like E. histolytica and form quadrinucleated cysts. With the accumulation of more data, a more precise assignment of these amoebae in the taxonomic scheme should become possible. POSTSCRIPT Since this review was written in early 1968,. 3 more papers have appeared which bear directly on the subject. Antigenic studies by Ali Khan & Meerovitch (1968), using the techniques of haemag- 361 M. GOLDMAN TABLE 4 COMPARATIVE DATA FOR E.MOSHKOVSKII AND LAREDO-TYPE E. HISTOLYTICA IN VARIOUS PARAMETERS Reference a E. moshkovskii a Findings Laredo-type E. histolytica a Temperature at which multipli- cation occurs Response to hy- potonic solutions ships Pathogenicity Biochemistry Lachance (1963) reviews previous literature Richards, Goldman & Cannon (1966) Various authors (see Table 1) Neal (1953) reviews pre- vious literature Gordeeva (1967) Richards, Goldman Cannon (1966) Zaman (1960) (immobili- zation techniques) Goldman, Carver, Gleason (1960) (FA) Goldman, Gleason & Carver (1962) (FA) Goldman & Cannon (1967) (FA) Siddiqui & Balamuth (1965) Neal (1967) reviews pre- vious literature Goldman & Cannon (1967) Various authors (see Table 3) Reeves, Montalvo & Sillero (1967) Stewart & Beck (1967) 10TC-37C (several reports of inferior growth at 37h ND ND Survival for several days with forma- tion of contractile vacuoles Permanent cultures in 1: 64 dilution of ordinary growth medium, with de- velopmnent of contractile vacuoles ND No cross-reaction with E. histolytica; strong cross-reaction with E. ranarum from frogs No specific reaction with a single anti-histolytica serum Reduced specific reaction with 4 anti- histolytica sera; no specific reaction with same serum mentioned above Specific reactions with 2 anti-histoly- tica sera Reduced reaction with anti-histolytica serum by FA, none by gel diffusion No experimental infections obtained with kittens, guinea-pigs, rats, ham- sters, or man No infections obtained with guinea- pigs ND Electrophoresis of glucokinase shows only one band, in contrast to two for classical E. histolytica Negative for nuclear DNA-histone, like all other parasitic protozoans tested. All free-living protozoans were positive; 67 species tested 1 0°C-35°C 1 0°C-37°C ND ND Complete life cycle in 1: 64 dilu- tion ot ordinary growth medium, with development of contrac- tile vacuoles ND Reduced specific reaction with 3 anti-histolytica sera Specific reactions with 4 anti- histolytica sera; reduced spe- cific reaction with sirgle serum negative for E. moshkovskii Specific reactions with 2 anti- histolytica sera Reduced reactions with anti- histolytica serum by both FA and gel diffusion ND No infections obtained wit) guinea-pigs No infections or mild infections in laboratory animals and man; see text above for details A single glucokinase band in same position as the one in E. moshkovskii ND ND = not done; FA = Fluorescent antibody. 362 Parameter ENTAMOEBA HISTOLYTICA-LIKE AMOEBAE OCCURRING IN MAN 363 glutination and precipitation in gel, have confirmed the greater reactivity with anti-E. histolytica sera of 1 strain of E. histolytica over 2 Laredo-type strains and 1 of E. moshkovskii. Reeves & Bischoff (1968) studied electrophoretic mobilities of 5 enzymes derived from 10 strains of E. histolytica, 5 Laredo- type strains, and 2 of E. moshkovskii. All Laredo- type strains and E. moshkovskii were similar to each other and distinguishable from E. histolytica in 4 or 5 out of 8 characterized bands. De Carneri (1968) compared 3 strains of E. histolytica, 2 of Laredo-type and 1 of E. moshkovskii with regard to susceptibility to 8 drugs. In contrast to work reported earlier in this paper, variability within the E. histolytica group often encompassed that found in the other amoebae. This was probably due to the fact that each culture appears to have been grown with different bacterial associates, rendering these results of limited value for characterizing the amoebae. The over-all effect of these additional studies is, thus, to confirm the earlier findings reported in the body of this paper. RESUME AMIBES SEMBLABLES A ENTAMOEBA HISTOLYTICA ISOLtES CHEZ L'HOMME L'auteur a compare six amibes semblables a Entamoeba histolytica, du type Laredo, isolees chez l'homme, avec des souches classiques d'E. histolytica sous les rapports suivants: morphologie, tol6rance aux temperatures, comportement en solutions hypotoniques, structure antigenique, pathogenicite, sensibilite aux medicaments et biochimie. Pour certains de ces caracteres, il existe entre les deux groupes des differences marquees qui, dans certaines conditions, sont suffisantes pour justifier une nouvelle designation d'espece pour les amibes du type Laredo. Toutefois, cela n'est pas recommande actuelle- ment car les souches # aberrantes )) qui ont e isolees jusqu'a pr6sent sont trop peu nombreuses et les etudes comparatives qui ont ete faites trop limitees. On a egalement compare Entamoeba moshkovskii avec E. histolytica du type Laredo. 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