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Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20.

Dyachenko V, Geiger C, Pantchev N, Majzoub M, Bell-Sakyi L, Krupka I, Straubinger RK - Vet. Microbiol. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Infectious Diseases and Zoonoses, Department for Veterinary Sciences, Faculty for Veterinary Medicine, LMU Munich, Veterinaerstr 13, 80539 Munich, Germany. v.dyachenko@lmu.de

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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.
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fig0010: 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.

Mentions: Electron microscopy was performed on the original culture of ApMuc01c in IRE/CTVM20 cells and on its first subculture. Examination of the infected tick cells revealed cytoplasmic vacuoles of different sizes containing mainly coccoid and pleomorphic organisms with ruffled outer membranes (Fig. 2). Overall the organisms ranged in size from approximately 0.44 μm to 1.60 μm. In most of the inclusions bacteria appeared electron-dense and their size ranged from 0.44 μm to 0.82 μm (median 0.62 μm, n = 30), shown in Fig. 2A and B. However, some of those bacteria appeared highly electron dense (Fig. 2C). Nearly all bacteria were enveloped by a distinct double membrane; rarely one or more additional membranes were seen surrounding the bacteria (Fig. 2D). An intriguing observation was the appearance of electron-dense material inside the coccoid organisms, sometimes as bipolar points or thin crescent-shaped structures (Fig. 2B). Two out of ten infected IRE/CTVM20 cells examined contained some smaller vacuoles with larger (size ranged from 0.5 μm to 1.6 μm, median 0.75 μm, n = 16) pleomorphic reticulate organisms (Fig. 2E and F). These bacteria were easily distinguishable from the electron-dense forms, and sometimes contained small electron-dense pinpoint structures. In some cases these bacteria were arranged close to each other in a single vacuole, while sometimes the reticulate forms were present in vacuoles together with electron dense forms. No intracellular bacteria were seen in preparations of uninfected IRE/CTVM20 cultures.


Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20.

Dyachenko V, Geiger C, Pantchev N, Majzoub M, Bell-Sakyi L, Krupka I, Straubinger RK - Vet. Microbiol. (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757156&req=5

fig0010: 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.
Mentions: Electron microscopy was performed on the original culture of ApMuc01c in IRE/CTVM20 cells and on its first subculture. Examination of the infected tick cells revealed cytoplasmic vacuoles of different sizes containing mainly coccoid and pleomorphic organisms with ruffled outer membranes (Fig. 2). Overall the organisms ranged in size from approximately 0.44 μm to 1.60 μm. In most of the inclusions bacteria appeared electron-dense and their size ranged from 0.44 μm to 0.82 μm (median 0.62 μm, n = 30), shown in Fig. 2A and B. However, some of those bacteria appeared highly electron dense (Fig. 2C). Nearly all bacteria were enveloped by a distinct double membrane; rarely one or more additional membranes were seen surrounding the bacteria (Fig. 2D). An intriguing observation was the appearance of electron-dense material inside the coccoid organisms, sometimes as bipolar points or thin crescent-shaped structures (Fig. 2B). Two out of ten infected IRE/CTVM20 cells examined contained some smaller vacuoles with larger (size ranged from 0.5 μm to 1.6 μm, median 0.75 μm, n = 16) pleomorphic reticulate organisms (Fig. 2E and F). These bacteria were easily distinguishable from the electron-dense forms, and sometimes contained small electron-dense pinpoint structures. In some cases these bacteria were arranged close to each other in a single vacuole, while sometimes the reticulate forms were present in vacuoles together with electron dense forms. No intracellular bacteria were seen in preparations of uninfected IRE/CTVM20 cultures.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Infectious Diseases and Zoonoses, Department for Veterinary Sciences, Faculty for Veterinary Medicine, LMU Munich, Veterinaerstr 13, 80539 Munich, Germany. v.dyachenko@lmu.de

Show MeSH
Related in: MedlinePlus