title: Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20.
abstract: Anaplasma phagocytophilum is an intracellular tick-borne rickettsial pathogen, which causes granulocytic anaplasmosis in various species of livestock and companion animals and also in humans. Previously A. phagocytophilum has been isolated and propagated in cell lines derived from the tick Ixodes scapularis and in the human promyelocytic cell line HL60. In this study we used the Ixodes ricinus-derived cell line IRE/CTVM20 to isolate and propagate two new canine strains of A. phagocytophilum. Blood samples were collected by veterinarians from two dogs, one from Germany and the other from Austria. Suspicion of clinical canine granulocytic anaplasmosis was raised by the treating veterinarians and after confirmation of A. phagocytophilum infection by real-time PCR, buffy coat cells were isolated and co-cultivated with IRE/CTVM20 cells maintained at 28 °C in L15/L15B medium. In the tick cells, rickettsial inclusions were first recognised after 86 days of incubation. Electron microscopic examination of tick cells infected with one of the isolates revealed cytoplasmic vacuoles containing pleomorphic organisms with individual bacteria enveloped by a bilayer membrane. Sequencing of 16S rRNA genes confirmed the isolation of A. phagocytophilum and showed the highest identity to the A. phagocytophilum human HZ strain. The two A. phagocytophilum isolates were passaged several times in IRE/CTVM20 cells and transferred to the I. scapularis cell line ISE6. This confirms for the first time the successful establishment and continuous cultivation of this pathogen in I. ricinus cells as well as infectivity of these canine strains for I. scapularis cells.
authors.affiliation: Institute for Infectious Diseases and Zoonoses, Department for Veterinary Sciences, Faculty for Veterinary Medicine, LMU Munich, Veterinaerstr. 13, 80539 Munich, Germany. Electronic address: v.dyachenko@lmu.de.
authors.forename: Viktor
authors.initials: V
authors.lastname: Dyachenko
authors.affiliation: Institute for Infectious Diseases and Zoonoses, Department for Veterinary Sciences, Faculty for Veterinary Medicine, LMU Munich, Veterinaerstr. 13, 80539 Munich, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Geiger
authors.affiliation: IDEXX Vet Med Lab, Moerikestr. 28/3, 71636 Ludwigsburg, Germany.
authors.forename: Nikola
authors.initials: N
authors.lastname: Pantchev
authors.affiliation: Institute of Veterinary Pathology, Center for Clinical Veterinary Medicine, Faculty for Veterinary Medicine, LMU Munich, Veterinaerstr. 13, 80539 Munich, Germany.
authors.forename: Monir
authors.initials: M
authors.lastname: Majzoub
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK.
authors.forename: Lesley
authors.initials: L
authors.lastname: Bell-Sakyi
authors.affiliation: Institute for Infectious Diseases and Zoonoses, Department for Veterinary Sciences, Faculty for Veterinary Medicine, LMU Munich, Veterinaerstr. 13, 80539 Munich, Germany.
authors.forename: Inke
authors.initials: I
authors.lastname: Krupka
authors.affiliation: Institute for Infectious Diseases and Zoonoses, Department for Veterinary Sciences, Faculty for Veterinary Medicine, LMU Munich, Veterinaerstr. 13, 80539 Munich, Germany.
authors.forename: Reinhard K
authors.initials: RK
authors.lastname: Straubinger
mesh_terms: D041081:Anaplasma phagocytophilum / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D000712:Anaplasmosis / Q000382:microbiology; D000818:Animals; D002460:Cell Line; D004283:Dog Diseases / Q000097:blood / Q000382:microbiology*; D004285:Dogs; D016873:Ehrlichiosis / Q000097:blood / Q000382:microbiology / Q000662:veterinary*; D005260:Female; D018922:HL-60 Cells; D006801:Humans; D018884:Ixodes / Q000382:microbiology*; D008297:Male; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D012336:RNA, Ribosomal, 16S