Isolation of Anaplasma phagocytophilum strain Ap-variant 1 in a tick-derived cell line.

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.

Infection of endothelial cells with Anaplasma marginale and A. phagocytophilum.

Anaplasma marginale and A. phagocytophilum are obligate intracellular, tick-borne pathogens that target erythrocytes and neutrophil granulocytes, respectively. Because ticks do not directly tap blood vessels, an intermediate tissue may mediate infection of blood cells. We considered that vascular endothelium interacts with circulating blood cells in vivo, and could be involved in pathogenesis and dissemination of the organisms. We used light and electron microscopy and immune labeling to show that A. phagocytophilum invaded rhesus (RF/6A), human (HMEC-1, MVEC), as well as bovine (BCE C/D-1b) endothelial cell lines, whereas A. marginale infected rhesus and bovine endothelial cells. A. marginale formed large intracellular inclusions that appeared smooth and solid at first, and subsequently coalesced into discrete granules. A. phagocytophilum formed numerous smaller inclusions in each cell. Within 1-3 weeks, the monolayers were destroyed, and lysed cultures were diluted onto fresh monolayers. Electron microscopy demonstrated uneven distribution of A. marginale inside large inclusions, with reticulated forms grouped more tightly than denser cells, whereas in A. phagocytophilum individual organisms appeared more evenly spaced. Specific polyclonal and monoclonal antibodies both labeled A. marginale and A. phagocytophilum in endothelial cells, and oligonucleotide primers complimentary to either A. marginale or A. phagocytophilum amplified their expected target from these cultures. In conclusion, we demonstrate that relevant microvascular endothelium is susceptible to anaplasmas in vitro and may present a link that could explain development of the immune response and persistent infection.

Isolation and propagation of the Ap-Variant 1 strain of Anaplasma phagocytophilum in a tick cell line.

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.

Isolation of an Anaplasma sp. organism from white-tailed deer by tick cell culture.

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 degrees 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.