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J. Clin. Microbiol. doi:10.1128/JCM.00478-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Isolation and Propagation of the Ap-Variant 1 Strain of Anaplasma phagocytophilum in a Tick Cell Line

Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA, 30333; Department of Entomology, University of Minnesota, Saint Paul, MN, USA, 55108; School of Medicine, University of Maryland-Baltimore, Baltimore, MD, USA, 21201; and Armed Forces Research Institute of Medical Sciences, Microbiology, Rajthawee, Bangkok, Thailand

^* To whom correspondence should be addressed. Email: rfm2{at}cdc.gov.

	Abstract

The first tissue culture isolates of the unique A. phagocytophilum strain, Ap-Variant 1, were obtained in the Ixodes scapularis tick-derived cell line, ISE6. Two isolates were from goat blood samples: one from a goat infected with I. scapularis ticks from Rhode Island, and a second from a goat infected by serial passage of blood from the first infected goat. Eight isolates were made directly from I. scapularis ticks collected from white-tailed deer in Minnesota, and represent the first isolations of an Anaplasma species directly from ticks. Each of the 10 isolates had a 16S rRNA gene sequence identical to that previously described for Ap-Variant 1, but differences within the ank gene were found that suggest natural variation. Prevalence of Anaplasma in the Minnesota ticks was 63.9% (23 of 36) ticks tested by PCR were positive. Six of the tick-derived isolates were obtained from a set of 18 PCR-positive ticks for a 33.3% isolation success rate. The conservation of host tropism among the Rhode Island and Minnesota isolates of Ap-Variant 1 was examined by use of experimental infections of mice and a goat. A Minnesota tick-derived isolate (MN-61-2) was used to inoculate naïve animals and this isolate was able to infect a goat but unable to infect each of 5 mice, confirming that the MN isolates have the same host tropism as Ap-Variant 1 from the northeast. Light and electron microscopy of the Ap-Variant 1 isolate MN-61-2 in ISE6 cells showed cytoplasmic inclusions characteristic of A. phagocytophilum with pleomorphic bacteria in membrane-bound vacuoles, with both electron-dense and electron-lucent forms.