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.

Fig. 1

A. phagocytophilum (strain ApMuc01c)-infected IRE/CTVM20 cell culture showing one heavily infected tick cell containing several endosomes with individual bacteria (centre), and two uninfected cells. Scale bar represents 10 μm.

Fig. 2

Ultrastructure of A. phagocytophilum (strain ApMuc01c) in IRE/CTVM20 cells examined by transmission electron microscopy. (A) Vacuole (black arrow) containing single bacteria (N: nucleus). (B) A. phagocytophilum inclusion in a tick cell showing electron-dense forms; note the double polar dots possibly representing a condensed nucleoside (black arrows). (C) Highly electron-dense organisms (black arrow) in a vacuole. (D) Electron-dense (black arrows) and reticulate forms (white arrow), which were visible in two vacuoles of an infected cell; some individual bacteria are surrounded by a double membrane. (E) and (F) Vacuoles containing electron-lucent reticulate organisms (white arrows); note the electron-dense pinpoint structures in some bacteria. Scale bars in all pictures represent 2 μm.