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10.1111/j.1469-0691.2008.02238.x

Rickettsia raoultii and Anaplasma phagocytophilum in reticulatus 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 4C 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 -borne diseases: tick-borne encephalitis, Lyme borreliosis and granulocytic [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: 4C. 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 that using primers ge3a and ge10r, and ge9f and ge2, which belong to the genus . 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.

2009 The Authors Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 286–287 Matsumoto et al. Presence of Dermacentor reticulatus ticks in Bialowieza 287

Rickettsia sp. from ticks on bison may suggest that European Bison could act as 83 R. raoultii Marne ‘tick shelters’ for winter. However, all ticks 56 Rickettsia sp. RpA4 collected in this study were males and no female 100 R. raoultii Khabarovsk 81 Rickettsia sp. DnS14 was found, which may imply that the male ticks 58 Rickettsia sp. DnS28 collected in this study were the survivors of the R. amblyommii last activity peak in the last autumn, or all female 17 R. aeschlimannii ticks that survived winter had already finished R. massiliae 100 97 blood feeding in early spring. 91 R. rhipicephali Our results showed for the first time that 100 R. heilongjiangensis R. japonica D. reticulatus ticks in the Bialowieza area hold 30 R. slovaca R. raoultii. Dermacentor reticulatus was known as a 100 30 R. rickettsii 98 vector of the agent causing Omsk hsemorrhagic R. honei fever, Mediterranean spotted fever, North Asian 100 R. conorii tick , and , and the adult of R. sibirica 42 D. reticulatus feeds on large mammals, including 75 R. africae 68 R. parkeri humans. In north-eastern Poland, SFG rickettsia, R. montanensis which were finally identified as RpA4, were also R. monacensis reported by Stanczak in host-seeking D. reticulatus R. australis ticks [5].

0.02 Though neither A. phagocytophilum nor R. raoultii were successfully isolated in this study, the Fig. 1. Neighbour-joining tree based on the sequence of results of this study confirmed the presence of the ompA gene of Rickettsia spp. (Kimura 2-parameter, 100 bootstrap samples). A. phagocytophilum and R. raoultii in the Bialowieza Primeval Forest ecosystem and increased the list of blood (bison IV) was positive for A. phagocytophi- pathogens identified in the forest. lum. Thirteen ticks from bison IV were evaluated for the presence of A. phagocytophilum and Rickettsia REFERENCES spp. No samples were positive for A. phagocyto- 1. Grzeszczuk A, Stanczak J, Kubica-Biernat B, Racewicz M, philum; however, four samples were positive for Kruminis-Lozowska W, Prokopowicz D. Human anaplas- rickettsiae. All positive samples were applied for mosis in north-eastern Poland: seroprevalence in humans and prevalence in Ixodes ricinus ticks. Ann Agric Environ Med sequencing and all of the obtained sequences % 2004; 11: 99–103. were identical and had 100 similarity to the 2. Grzeszczuk A, Ziarko S, Radziwon PM, Prokopowicz D. ompA gene of Rickettsia raoultii Marne strain Evidence of Anaplasma phagocytophilum infection of Euro- (Rickettsia sp. RpA4) [(587 ⁄ 587 GenBank Acces- pean Bisons in the Bialovieza Primeval Forest, Poland. Med sion number DQ365799) (Fig. 1). The shell vial Weter 2004; 60: 600–601. 3. Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses cultures for A. phagocytophilum and R. raoultii around the world: emerging diseases challenging old con- were unsuccessful. cepts. Clin Microbiol Rev 2005; 18: 719–756. 4. Massung RF, Slater K, Owens JH et al. Nested PCR assay for detection of granulocytic ehrlichiae. J Clin Microbiol 1998; DISCUSSION 36: 1090–1095. 5. Stanczak J. Detection of spotted fever group (SFG) rickett- An average of 26.4 D. reticulatus ticks were siae in Dermacentor reticulatus (Acari: Ixodidae) in Poland. collected from a bison in March in Poland, which Int J Med Microbiol 2006; 296 (Suppl. 40): 144–148.

2009 The Authors Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 286–287