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The first tissue culture isolates of the unique Anaplasma 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 five mice, confirming that the Minnesota isolates have the same host tropism as Ap-Variant 1 from the northeastern United States. 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 and both electron-dense and electron-lucent forms.

We used tick cell culture to isolate a bacterium previously referred to as the "white-tailed deer (WTD) agent" from two captive fawns inoculated with blood from wild WTD (Odocoileus virginianus). Buffy coat cells were added to ISE6 tick cell cultures and incubated at 34°C, and 8 days later, Anaplasma-like inclusions were demonstrated in Giemsa-stained culture samples. The microbes became established and could be continuously passaged in tick cells. The identity of a culture isolate designated WTD76 was verified as the WTD agent by using specific PCR primers and by DNA sequencing. Comparison with sequences available in GenBank indicated that the isolate was most closely related first to Anaplasma platys and second to Anaplasma phagocytophilum, supporting its placement in the genus Anaplasma. Transmission electron microscopy of this Anaplasma sp. organism in tick cell cultures revealed large inclusions filled with pleomorphic and rod-shaped bacteria. Tick cells infected with the Anaplasma sp. organism were used to successfully infect a naive deer, thereby proving the infectivity of the isolate for deer.The genus Anaplasma comprises obligate intracellular rickettsial pathogens that are biologically transmitted by ixodid ticks (15,20,32,36,54). They target circulating blood cells of wild and domestic animals, as well as of humans, and are of global veterinary and human health importance. Although the type species, Anaplasma marginale, was described early last century (56) and has been intensively studied (33), it was isolated in continuous culture only recently (46). A continuous culture system for this obligate intraerythrocytic parasite became available with the development of cell lines from the black-legged tick, Ixodes scapularis (45), the vector for a range of different pathogens from viruses to protozoa (52,54,55 , abstr. 165, 1995) and Anaplasma phagocytophilum (47, 48), formerly known as Ehrlichia equi, Ehrlichia phagocytophila, Cytoecetes phagocytophila, or the human granulocytic ehrlichiosis agent (17). Tick cell culture has allowed not only primary isolation and continuous in vitro maintenance of these pathogens but also analysis of differential expression of specific antigens in the vector and vertebrate host (28). Other, as yet uncultivable, tick-borne pathogens might be candidates for isolation in ixodid tick cell lines.White-tailed deer (WTD; Odocoileus virginianus) are proven vertebrate reservoirs for Ehrlichia chaffeensis (40,41) and are known to be naturally infected with at least two related zoonotic rickettsiae, Ehrlichia ewingii (60) and A. phagocytophilum (6, 39). Although some wild WTD have been shown to carry antibodies that recognize A. marginale, their role in the epizootiology of bovine anaplasmosis is debated (31,42,44). WTD also are important hosts for Lone Star ticks, Amblyomma americanum, and the black-legged tick, I. scapularis, which are proven vectors of these zoonotic disease agents (2,18,36,54). Examination of wild WTD also has disclosed molecular evidence of another rickettsia...

Ten isolates of the Ap-Variant 1 strain of Anaplasma phagocytophilum were made in the Ixodes scapularis (I. scapularis)-derived cell line, ISE6. Two isolates were obtained from laboratory-infected goats and eight isolates were obtained from field-collected I. scapularis ticks. Each isolate showed 16S rRNA sequences identical to those as previously described for the Ap-Variant 1 strain. These are the first tissue culture isolates of the Ap-Variant 1 strain and will allow for further characterization of the biological and antigenic properties of this strain.