Louis S. Diamond

A Redescription of Entamoeba Histolytica Schaudinn, 1903 (Emended Walker, 1911) Separating It From Entamoeba Dispar Brumpt, 1925

ABSTRACT. Explaining the low incidence of invasive disease (10%) in humans infected with Entamoeba histolytica has occupied the attention of generations of both clinical and nonclinical investigators. One possible explanation would be the existence of two morphologically identical species—one an invasive pathogen, the other noninvasive. This was first proposed by Brumpt in 1925, but his explanation was virtually ignored until 1978 when the first of several publications appeared suggesting that E. histolytica did indeed consist of two species. We have reexamined Brumpts claim in light of recent biochemical, immunological and genetic studies and conclude that the data derived from these investigations provide unequivocal evidence supporting his hypothesis. With this in mind, we redescribe the invasive parasite retaining the name Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911), and set it apart from the noninvasive parasite described by Brumpt, Entamoeba dispar Brumpt, 1925.

Methods for Cultivation of Luminal Parasitic Protists of Clinical Importance

SUMMARY Cultivation of luminal protistan parasites has a long history. In this review we discuss the methods and media that are most widely used for the establishment and maintenance of the following organisms in culture: Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, Dientamoeba fragilis, Blastocystis hominis, and Balantidium coli. While cultivation is of limited importance in the diagnostic laboratory, it is essential to most research laboratories, and it is toward the latter that this review is primarily aimed.

Ribosomal RNA genes of ‘pathogenic’ and ‘nonpathogenic’ Entamoeba histolytica are distinct

Most infections with Entamoeba histolytica are asymptomatic. Two forms of the organism can be distinguished biochemically, and this finding has been explained by two distinct hypotheses: (1) there are two morphologically indistinguishable species, one of which causes disease; (2) there is one species which exists in two interconvertible forms, one of which causes disease. Knowledge of which hypothesis is correct has major implications for evaluation and treatment of carriers. We have studied the ribosomal RNA genes of the two forms hypothesizing that, if E. histolytica is one species, there should be no differences between them. We have found that the ribosomal RNA genes of the two forms are quite distinct, which supports the hypothesis that E. histolytica is two species.

Axenic Cultivation of Entamoeba histolytica.

Entamoeba histolytica (National Institutes of Health strain 200) has been maintained in axenic culture through 50 transfers over a period of 6 months. The medium used is diphasic and contains a cell-free extract of chick embryo which is essential for growth. Inocula of 50,000 amoebas yield 150,000 to 200,000 organisms in 72 hours of incubation at 35�C. An extract prepared from boiled embryos supports growth equally well.

Intraspecific Variation and Phylogenetic Relationships in the Genus Entamoeba as Revealed by Riboprinting

ABSTRACT. Eighty‐seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty‐four distinct patterns were obtained, most of which corresponded to previously described species. In three species (Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii) intraspecific variation was detected that led to the grouping of isolates into ‘ribodemes’ (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis, which does not encyst, clusters with the third group indicating secondary loss of this ability.

YI-S, a Casein-free Medium for Axenic Cultivation of Entamoeba histolytica, Related Entamoeba, Giardia intestinalis and Trichomonas vaginalis

ABSTRACT. Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen‐dwelling parasitic protozoa, especially Entamoeba, Giardia, and trichomonads. The digest used almost exclusively in the development of these media, Medo‐Peptone (Trypticase® BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo‐Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica. In response to this problem we have developed a casein‐free medium, YI‐S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein‐dependent medium TYI‐S‐33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen‐dwellers.

Entamoeba histolytica and Giardia lamblia: effects of cysteine and oxygen tension on trophozoite attachment to glass and survival in culture media.

Abstract Attachment of Entamoeba histolytica and of Giardia lamblia trophozoites to glass was monitored during the culture cycle. Attachment of each parasite was greatest during the exponential phase of axenic growth. The effects of l -cysteine upon the kinetics of attachment of trophozoites to glass were determined quantitatively. Attachment in complex growth media required cysteine, even under N2, atmosphere. With cysteine, the rates of attachment were greatest for the first 2 hr, then continued more slowly. The numbers of attached trophozoites decreased immediately upon exposure to medium without cysteine. The role of cysteine in protecting trophozoites of both species from the lethal effects of oxygen was assessed using clonal growth in agar or agarose medium to determine viability following exposure to varying oxygen tensions in liquid medium. Cysteine was required for viability of trophozoites. Without cysteine, decreasing the oxygen tension prolonged survival. Under increased oxygen tension, cysteine delayed the onset of exponential killing. Although it has no thiol reducing group, l -cystine similarly protected E. histolytica.

Entamoeba histolytica: Effect of growth conditions and bacterial associates on isoenzyme patterns and virulence

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Freeze-preservation of protozoa

In this presentation an attempt will be made to summarize briefly our present state of knowledge pertaining to the freeze-preservation of protozoa. Most of the accomplishments in this field have been concerned chiefly with the development of preservation techniques. Accordingly, t’his summary will deal primarily with the technological aspects of the subject. In particular, it will cover protective agents and t,heir use, cooling rates employed in freezing, storage temperatures, and methods of thawing frozen samples. The use of freezing techniques for the preservation of living protozoa was first instituted by Coggeshall in 1939.’ Prior investigations into the effects of flreezing on protozoa, which, according to Smith,“” extend back t,o the work of Antony van Leeuwenhoek in the 17th century, dealt primarily with observations on resistance of protozoa to low temperatures (see Luyet and Gehenio,” and Smit,h3” for reviews of early literature). In the pioneering investigations of Coggeshall’ the erythrocytic stages of two malaria parasites, Plasmodium knowlesi and P. inui, were frozen at -76”C, and a.fter storage at this temperature for 70 days were shown to have retained both viability a,nd pathogenicity. Encouraged by this demonstration, other investigators attempted the freeze-preservation of different species of protozoa as well as the preservation of other malaria parasites. As a result of these efforts, some 44 species representing 9 families ha,ve been preserved in this manner. All except one, Tetrahymena pyriformis, are parasitic. A list of species which have been freezepreserved is presented in Table 1. It includes only those species for which a successful storage time of seven or more days has been recorded.

Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents

Abstract The growth responses of Entamoeba histolytica strains HM-1:IMSS and HK-9 to a variety of reducing agents were tested for one subculture in TYI-S-33 medium, prepared with no cysteine or ascorbic acid. Amoebae did not grow in this medium. Addition of l -ascorbic acid, d - or l -cysteine, or l -cystine each permitted the maximum growth observed. Dithiothreitol supported 68% maximum growth of HK-9 amoebae, but only 12% of HM-1. In contrast, growth of both strains was greatly diminished (0–13% growth) with 11 other compounds tested including glutathione, thiomalic acid, thioglycolic acid, and methionine. The growth responses of Giardia lamblia were similarly tested in TYI-S-33, as well as in TP-S-1 media. If l -cysteine was omitted from either medium, trophozoites did not grow, and eventually lysed. In TYI-S-33 medium, the requirement for l -cysteine was specific, whereas in TP-S-1 medium, other sulfhydryl compounds were partially effective and lower concentrations of l -cysteine satisfied the requirement. Ascorbic acid or l -cystine alone was totally ineffective; however, in combination, 30 to 60% of maximum growth was achieved. Once added to either medium, cysteine was rapidly oxidized. Amino acid analysis of the growth media revealed that the broth components of TP-S-1 medium contained 2.8 mM and TYI-S-33 broth 2.1 mM endogenous levels of cysteine (or half-cystine), with an additional 3 mM contributed by 10% serum.