View metadata, citation and similar papers at core.ac.uk brought to you by CORE
provided by Elsevier - Publisher Connector
10.1111/j.1469-0691.2008.02238.x
Rickettsia raoultii and Anaplasma phagocytophilum in Dermacentor reticulatus ticks collected from Bialowieza Primeval Forest European bison (Bison bonasus bonasus), Poland K. Matsumoto1,2, A. Grzeszczuk3, P. Brouqui1 and D. Raoult1
1Unite´ des Rickettsies, Faculte´ de Me´decine, Marseille, France, 2Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Japan and 3Department of Infectious Diseases, Medical University of Bialystok, Bialystok, Poland
bison and kept at 4 C until use. Species of ticks were identified INTRODUCTION using a morphological key. The Bialowieza Primeval Forest – UNESCO Bio- sphere Reserve – is a well-known endemic focus of DNA extraction from blood and ticks tick-borne diseases: tick-borne encephalitis, Lyme borreliosis and granulocytic anaplasmosis [1]. A DNA was extracted from the blood samples of bison using QIAamp DNA Mini Kit (Qiagen, GmbH, Germany) according broad range of wild animals, including rare mam- to the manufacturer’s manual. The surface of the tick was mals such as the wolf, the lynx, the otter and disinfected by immersion in 70% ethanol for 10 min. After European bison (Bison bonasus bonasus), live there. rinsing two times with sterilised DW, an individual tick was l The herd of about 350–400 free ranging bison is the put in a tube with 200 L of medium and homogenised. DNA was extracted from 20 lL of homogenate using QIA- biggest in the world. Bison may serve as ‘tick amp DNA Mini Kit. The rest of the homogenate was kept at shelter’ in winter for the most prevalent species: 4 C. Ixodes ricinus and Dermacentor reticulatus. Recently, more than 50% of European bison were found to be infected with Anaplasma phagocytophilum [2]. PCR, sequencing and phylogenetic analysis Spotted fever group (SFG) rickettsiae are oblig- DNA samples from bison blood were tested by nested-PCR atory intracellular Gram-negative bacteria that using primers ge3a and ge10r, and ge9f and ge2, which belong to the genus Rickettsia. They are associated amplify 546 bp of the 16SrRNA gene of A. phagocytophilum [4]. Tick samples from A. phagocytophilum positive bison were with arthropods, mainly ticks, which act as vectors tested by PCR for A. phagocytophilum using primers P44F and ⁄ or reservoirs. In recent years, new species or (GAG-TTT-GCT-AAG-GCC-GTG) and P44R (ACA-GCT-TTG- subspecies of rickettsiae have been identified as GCG-TTG-TCG), which amplify approximately 300 bp of the emerging agents of human tick-borne diseases [3]. P44 gene of A. phagocytophilum and SFG Rickettsia spp. using The objectives of this study were (i) to evaluate primers 190-70 and 190-701, which amplify about 632 bp of the ompA gene of SFG rickettsiae. PCR products were sequenced the bison as a tick shelter for winter, (ii) to and analysed by BLAST search. demonstrate the presence of A. phagocytophilum and rickettsiae in ticks collected on bison, and (iii) to isolate the microorganisms. Shell vial culture The homogenate samples of ticks positive for A. phagocytophi- lum and Rickettsia spp. were used for shell vial culture using MATERIALS AND METHODS HL-60 and L929, respectively. Bison and ticks
Ticks were collected from free ranging European bison (B. RESULTS bonasus bonasus) culled in the Bialowieza Primeval Forest, north-eastern Poland, in March 2005. Blood was taken from A total of 132 ticks were collected from five bison and all of them were identified as D. reticulatus males. They were found on bison’s ears and the Corresponding author and reprint requests: Kotaro Matsumo- number of live ticks infesting one animal varied to Department of Applied Veterinary Medicine, Obihiro from 6 to 66 (mean ± SD, 26.4 ± 26.1). No Ixodes University of Agriculture and Veterinary Medicine, Inada- ricinus ticks, usually parasitising in groins, were cho, Obihiro 080-8555, Japan found. Blood samples were collected from five Email: [email protected] culled bison. One of five DNA samples from bison No conflicts of interest declared.