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Journal of Clinical Microbiology, April 2002, p. 1470-1474, Vol. 40, No. 4
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.4.1470-1474.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cloning of a Truncated Babesia equi Gene Encoding an 82-Kilodalton Protein and Its Potential Use in an Enzyme-Linked Immunosorbent Assay

Haruyuki Hirata,1 Hiromi Ikadai,2 Naoaki Yokoyama,1 Xuenan Xuan,1 Kozo Fujisaki,1 Naoyoshi Suzuki,1 Takeshi Mikami,1 and Ikuo Igarashi1*

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555,1 Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan2

Received 19 July 2001/ Returned for modification 8 November 2001/ Accepted 4 December 2001

To isolate Babesia equi genes encoding immunodominant proteins, a cDNA expression library prepared from B. equi mRNA was immunoscreened with B. equi-infected horse serum. Eighteen positive cDNA clones were obtained, and the clone that showed the strongest immunoreactivity, designated Be82, was further characterized. The Be82 gene consisted of 1,953 bp and contained a partial open reading frame lacking the 5'-terminal sequence. As shown by Western blot analyses, immune sera from mice intraperitoneally injected with the Be82 gene product recognized the 82- and 52-kDa proteins of B. equi but not those of Babesia caballi. The glutathione S-transferase fusion protein expressed in Escherichia coli that was purified and used as the antigen in the enzyme-linked immunosorbent assay reacted specifically with B. equi-infected horse sera. These results suggest that the Be82 gene product is a potential diagnostic antigen candidate in the detection of B. equi infection in horses that will be useful both in the performance of epidemiological studies and in the granting of quarantine passes.

* Corresponding author. Mailing address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Phone: 81-155-49-5641. Fax: 81-155-49-5643. E-mail: igarcpmi{at}obihiro.ac.jp.

Journal of Clinical Microbiology, April 2002, p. 1470-1474, Vol. 40, No. 4
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.4.1470-1474.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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