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Journal of Clinical Microbiology, June 1999, p. 1782-1789, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Simultaneous Detection of Bovine Theileria and Babesia Species by Reverse Line Blot Hybridization

J. M. Gubbels,1 A. P. de Vos,1 M. van der Weide,1,dagger J. Viseras,2 L. M. Schouls,3 E. de Vries,1 and F. Jongejan1,*

Department of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht,1 and Research Laboratory for Infectious Diseases, National Institute of Public Health and Environment, 3720 BA Bilthoven,3 The Netherlands, and Department of Parasitology, Faculty of Pharmacology, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain2

Received 11 November 1998/Returned for modification 22 January 1999/Accepted 15 March 1999

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.

* Corresponding author. Mailing address: Department of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands. Phone: (31-30) 2532568. Fax: (31-30) 2540784. E-mail: F.Jongejan{at}vet.uu.nl.

dagger Present address: Innogenetics, 9052 Ghent, Belgium.

Journal of Clinical Microbiology, June 1999, p. 1782-1789, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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