Molecular characterization of a non-Babesia divergens organism causing zoonotic babesiosis in Europe

Abstract

In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.

Figure 1

Phylogenetic tree for the complete 18S rRNA gene from selected Babesia spp. The tree was computed by using the quarter puzzling maximum likelihood method of the TREE-PUZZLE program and was oriented by using Theileria annulata as the outgroup. Numbers at the nodes indicate the quartet puzzling support for each internal branch. Scale bar indicates an evolutionary distance of 0.01 nucleotides per position in the sequence. Vertical distances are for clarity only. The GenBank accession numbers for the sequences used in the analysis are as follows: Babesia bigemina A, X59604; B. bovis, L19077; B. caballi, Z15104; B. divergens (Purnell isolate [12]), AY046576; B. gibsoni (genotype Asia 1), AF175300; B. odocoilei (Brushy Creek and Engeling isolates [14]), AY046577; Babesia sp. (isolated from Bos taurus), U09834; EU1 (the etiologic agent of infection in the two cases described here), AY046575; and Theileria annulata, M64243.

Figure 2

Panel of computer-generated electronic images of photomicrographs of Babesia-infected erythrocytes on a Giemsa-stained smear of peripheral blood from the patient who became infected in Austria. The electronic images were edited for uniformity of color, without changing the form or size of the organisms. The image on the far right shows a tetrad (Maltese-cross form). Three glass slides of the actual blood films have been deposited in the Oberösterreichisches Landesmuseum, Biologiezentrum, Linz (i.e., Biology Center of the Upper Austrian Museum, Linz), with the accession number 2002/9. The slides are labeled “Babesia sp. (EU1), patient 001, Austria, Krems Land, July 25, 2000.”