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.
Highlights
Anaplasma phagocytophilum is an intracellular rickettsial pathogen, which belongs to the alpha-proteobacteria
Disease caused by A. phagocytophilum in ruminants in Europe has been recognised for over 80 years, and early experiments demonstrated that I. ricinus was the vector (Woldehiwet, 2006)
A European isolate of A. phagocytophilum was only cultivated continuously in vitro for the first time, in the I. scapularis cell line IDE8, a decade ago (Woldehiwet et al, 2002). This delay, compared to the progress made with New World A. phagocytophilum isolates in cells from the North American vector I. scapularis, could be explained by the limited availability of cell lines derived from the European vector I. ricinus
Summary
Anaplasma phagocytophilum is an intracellular rickettsial pathogen, which belongs to the alpha-proteobacteria. Variable pathogenicity for different mammalian hosts as well as genetic variation have been observed in A. phagocytophilum, suggesting a complex ecology of this pathogen (Pusterla et al, 1999, 2001; Foley et al, 2009; Woldehiwet, 2010; Scharf et al, 2011). This pathogen comprises a variety of distinct ecotypes that have evolved a range of strategies to enable their survival (Bown et al, 2009).
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