BRIEF COMMUNICATIONS AAEM INTRODUCTION Wild canids can play an important role in the transmis- sion and maintenance of metazoan parasites in natural foci [3, 8]. However, little is known about the blood parasites of foxes and other wild living carnivores. In our study, we focused on Anaplasma phagocytophilum infection of red foxes (Vulpes vulpes) in the north-eastern part of Poland. The main vector of A. phagocytophilum in Poland is Ixo- des ricinus tick [1, 13], and the animal reservoirs are small rodents and ruminants [2, 4, 5]. However, other mammals able to serve as animal reservoir are still being found. MATERIALS AND METHODS The diagnostic was made using conventional as well as nested PCR methods. The reactions were conducted on spleen samples from 111 red foxes, obtained from the Sanitary and Epidemiological Station in Ostrołęka, Ma- zovian Province, Poland. DNA was isolated and purified using Genomic Mini AX tissue (A&A Biotechnology), ac- cording to the manufacturer’s protocol. Molecular search- ing for A. phagocytophilum was based on nested PCR am- plification of approximately 640 bp long fragment of 16S rRNA gene [7], and the portion of msp4 gene coding MSP4 major surface protein with a length of 849 bp [2, 7]. PCR amplifications were performed in a total volume of 25 µL of reaction mixture from the MasterTaq DNA polymerase kit (Eppendorf AG, Hamburg, Germany) containing 10.4 μL deionized water, 5 μL 5× TaqMaster PCR Enhancer, 2.5 μL 10× Taq buffer (with 15 mmol/L Mg2+), 1.5 μL 25-mmol/L solution of Mg (OAc)2, 0.1 μL Taq DNA polymerase (5 U/μL), 0.5 μL deoxynucleoside triphosphate (dNTP) mix (10 mmol/L) (Fermentas, Viln- ius, Lithuania), and 1.25 μL of each primer (10 pmol/μL) (Invitrogen, Paisley, Scotland). The PCR assays were car- ried out using primers (EC9,EC12A and SSAP2f, SSAP2r) for the nested PCR amplification of 16S rRNA and primers (MSP4AP5, MSP4AP3) for conventional PCR amplifica- tion of msp4 gene, as published previously [2, 7]. As a tem- plate, 2.5 μL of isolated DNA was used. As a negative con- trol, nuclease free water was added to a PCR mix instead of DNA. Sequenced A. phagocytophilum DNA isolated from A. phagocytophilum – positive deer blood sample was used as a positive control. Amplification was performed using a nested PCR protocol [2, 7]. PCR products were electrophoresed on 1% agarose gel, stained with GoldView Nucleic Acid Stain (Beijing SBS Genetech Co. Ltd.). The fragments amplified, 641 bp in size, were compared with 100 bp DNA molecular weight marker. PCR products were purified by using a QIAquick PCR purification kit (QIAGEN) for further sequencing, ANAPLASMA PHAGOCYTOPHILUM INFECTION OF RED FOXES (VULPES VULPES) Grzegorz Karbowiak 1 , Bronislava Víchová 2 , Viktória Majláthová 2 , Joanna Hapunik 1 , Branislav Peťko 2 1 W. Stefański Institute of Parasitology of the Polish Academy of Sciences, Warsaw, Poland 2 Parasitological Institute of the Slovak Academy of Sciences, Košice, Slovakia Karbowiak G, Víchová B, Majláthová V, Hapunik J, Peťko B: Anaplasma phagocytophilum infection of red foxes (Vulpes vulpes). Ann Agric Environ Med 2007, 14, 71–72. Abstract: The investigation of Anaplasma phagocytophilum infection covered 111 red foxes from the Mazovian Province. Determination was based on PCR amplification of 16S rRNA gene and the portion of msp4 gene coding MSP4 major surface protein. The presence of A. phagocytophilum was found in 2.7% of foxes. Address for correspondence: Dr. Grzegorz Karbowiak, W. Stefański Institute of Parasi- tology of the Polish Academy of Sciences, Twarda 51/55, 00-818 Warsaw, Poland. E-mail: grzgrz@twarda.pan.pl Key words: Anaplasma phagocytophilum, Vulpes vulpes. Received: 7 July 2008 Accepted: 10 October 2009 Ann Agric Environ Med 2009, 16, 71–72