Research note Immunological inter-strain crossreactivity correlated to 18S rDNA sequence types in Acanthamoeba spp. Julia Walochnik, Andreas Obwaller, Horst Aspo Èck * Department of Medical Parasitology, Clinical Institute of Hygiene, University of Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria Received 17 October 2000; received in revised form 6 December 2000; accepted 6 December 2000 Abstract Various species of the genus Acanthamoeba have been described as potential pathogens; however, differentiation of acanthamoebae remains problematic. The genus has been divided into 12 18S rDNA sequence types, most keratitis causing strains exhibiting sequence type T4. We recently isolated a keratitis causing Acanthamoeba strain showing sequence type T6, but being morphologically identical to a T4 strain. The aim of our study was to ®nd out, whether the 18S rDNA sequence based identi®cation correlates to immunological differentiation. The protein and antigen pro®les of the T6 isolate and three reference Acanthamoeba strains were investigated using two sera from Acanthamoeba keratitis patients and one serum from an asymptomatic individual. It was shown, that the T6 strain produces a distinctly different immunological pattern, while patterns within T4 were identical. Af®nity puri®ed antibodies were used to further explore immu- nological cross-reactivity between sequence types. Altogether, the results of our study support the Acanthamoeba 18S rDNA sequence type classi®cation in the investigated strains. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Acanthamoeba; Strain differentiation; Immunology; Sequence type Acanthamoebae are free-living protozoa that feed on bacteria, algae and yeasts. However, various representatives of Acanthamoeba spp. are known as potential pathogens causing two different disease entities, namely the chronic granulomatous amebic encephalitis (GAE) being associated with immunode®ciency and the Acanthamoeba keratitis occurring predominantly in contact lens wearers (Marciano-Cabral et al., 2000; Martinez and Visvesvara, 1997). Eighteen different Acanthamoeba species have been described, of which 15 have been implicated in human infections (Martinez and Visvesvara, 1997), but the differ- entiation of acanthamoebae at the species level remains problematic. Moreover, it has been demonstrated that species identi®cation gives no information on the patho- genicity of an isolate (De Jonckheere, 1980). Pussard and Pons (1977) divided the genus into three morphological groups according to the size and shape of the cysts. The vast majority of strains isolated from human infections are representatives of group II or group III. Several studies have shown that group II and group III are not only morphologi- cally more alike, but are also genetically closer related than either of them is to group I (Badenoch et al., 1995; Moura et al., 1992; Stothard et al., 1998). A number of attempts have been made in order to enable a more precise identi®cation of Acanthamoeba isolates, but a universally accepted system does not yet exist. A very promising method for differentiation and identi®cation of microorganisms is the comparison of the 18S rRNA gene sequences. Twelve Acanthamoeba sequence types accord- ing to 18S rDNA sequence similarities have been estab- lished and it has become apparent that Acanthamoeba keratitis seems to be associated with sequence type T4. However, T4 generally seems to be the most prevalent sequence type. Most other sequence types are to date only represented by very few, sometimes only one single isolate (Stothard et al., 1998). We have recently isolated a keratitis causing Acantha- moeba strain showing sequence type T6, a sequence type not previously associated with keratitis (Walochnik et al., 2000a). Interestingly, this isolate was morphologically iden- tical to a T4 strain of A. hatchetti, morphological group II. T6 is, however, described to be associated with morpholo- gical group III (Stothard et al., 1998). It has been demonstrated that human serum contains anti- bodies against Acanthamoeba (Cerva, 1989; Cursons et al., International Journal for Parasitology 31 (2001) 163±167 0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S0020-7519(00)00166-1 www.parasitology-online.com * Corresponding author. Tel.: 143-1-4277-79430; fax: 143-1-4277- 9794. E-mail address: horst.aspoeck@univie.ac.at (H. Aspo Èck).