The Use of Physiological Characterizations in the Classification of Five Species of Acanthamoeba

Abstract

Physiological tests were devised and used to compare 15 strains representing five species of soil and freshwater amoebae assigned to the genus Acanthamoeba. The tests gave an acceptable level of reliability and the pattern of responses was not affected by the differing growth rates of the organisms. There was some degree of overlap between the strains, as shown by the high level of inter-species similarities in relation to those between strains of the same species. Previous classifications of Acanthamoeba, which are based solely on morphological criteria, do not adequately reflect the diversity of these amoebae. I N T R O D U C T I O NSmall amoebae, variously assigned to the genera Acanthamoeba, Hartmannella and Mayorella, occur widely in soil and aquatic habitats and are probably some of the most common protozoa (Page, 1967(Page, , 1976. Some cause certain pathological conditions (Culbertson, 1971). As they are one of the few groups of protozoa which can be isolated and grown in axenic culture they are also being used increasingly in fundamental studies of cell physiology and biochemistry. However, their ecological relationships are poorly understood, due, in part, to the confusion of some of the existing classifications of the group which are based entirely on morphological criteria.At present the most important taxonomic characters are: pseudopodial form; type of amoeboid movement; occurrence of a flagellate stage in the life-cycle; nuclear structure; cyst morphology and method of excystation. The use of such characters can be criticized on several grounds, for example, descriptions of pseudopodial form are often imprecise and cyst morphology can be extremely variable or modified significantly by the environment (Pussard, 1966;Stratford & Griffiths, 1978).The DNA base compositions of representative strains range from 50 to 62 yo GC (Adam & Blewett, 1974) suggesting that existing classifications may not adequately reflect the diversity of this group of organisms. Physiological characters could be used more extensively (Adam, 1964b), and in this present study, methods have been devised to examine further the physiological diversity of 15 strains of amoebae now included in the genus Acanthamoeba. M E T H O D SStrains and their routine maintenance. Fifteen strains representing five species of Acanthamoeba were examined (Table 1). All cultures were axenic and, after receipt, were maintained in PGY medium [0.75 % (w/v) peptone, 0.75 % (w/v) yeast extract, 1.5 % (w/v) glucose; Difco or Oxoid peptones and yeast extracts could be used in this medium with equal effect]. Stock cultures (5 ml) were kept at laboratory temperature in small, screw-capped bottles. These were inoculated with 0.5 ml of a culture which had been maintained similarly and subcultures were made every 4 to 6 weeks.Tests used to characterize cultures of trophozoites. Tests devised for this study are listed in Table 2. PGY

Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp.

“…These include analyses of physiology (Griffiths et al 1978), antigenic differences (Stevens et al 1977), isoenzymes (DeJonckheere 1983Visvesvara et al 1983;Moura et al 1992), restriction-fragmentlength polymorphisms (RFLPs; Bogler et al 1983;Costas et al 1983;McLaughlin et al 1988;Yagita and Endo 1990;Kilvington et al 1991), PCR (Vodkin et al 1992;Lai et al 1994), and hybridization of rDNA-specific probes (Gast and Byers 1995). Several of these studies found considerable diversity between strains within the same species, and the pathogenic phenotype was present across different groups.…”