Isolation of anAnaplasmasp. Organism from White-Tailed Deer by Tick Cell Culture

Abstract

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...

Tick cell lines: tools for tick and tick-borne disease research

“…Subsequently, a previously uncultivable Anaplasma sp. was isolated into ISE6 cells from the blood of white-tailed deer [33], and the same cell line was used for the first isolation of the aetiological agent of southern tick-associated rash illness in humans, Borrelia lonestari, by co-cultivation with tissues of the vector tick Amblyomma americanum [34]. Similarly, the R. appendiculatus cell line RAE25 was used to isolate a Rickettsia sp.…”

Experimental Infection of White-Tailed Deer with Anaplasma phagocytophilum , Etiologic Agent of Human Granulocytic Anaplasmosis

“…of WTD (33,44). All deer were determined to be culture negative for Trypanosoma cervi, a protozoan known to interfere with tissue culture of deer blood (35,36,44). Two deer (WTD132 and WTD133) were diagnosed with Theileria cervi infection by observation of intraerythrocytic protozoans in whole-blood smears.…”

“…The deer were determined to be seronegative by indirect fluorescence antibody (IFA) test for A. phagocytophilum and Ehrlichia chaffeensis (8,17) and PCR negative for A. phagocytophilum, E. chaffeensis, Ehrlichia ewingii, and an undescribed Anaplasma sp. of WTD (33,44). All deer were determined to be culture negative for Trypanosoma cervi, a protozoan known to interfere with tissue culture of deer blood (35,36,44).…”

“…Reisolation attempts of A. phagocytophilum from mouse and deer blood were performed as previously described (44,45) except that resuspended ISE6 stock cell monolayers and washed WTD buffy coat cells were mixed, pelleted at 720 ϫ g for 20 min, and then allowed to stand at room temperature for 30 min before the pellet was resuspended in "ehrlichia medium" (46) and divided into two flasks. Duplicate flasks were monitored and maintained separately using different sets of reagents as a precaution against contamination; antibiotics were not used at any time.…”

Sequence Analysis of the msp4 Gene of Anaplasma phagocytophilum Strains

“…Alternatively, the sensitivity of the msp4 PCR was below the detection level for these samples (five copies of msp4 per nanogram of DNA). Nevertheless, although A. phagocytophilum-like organisms isolated from WTD may be closely related to A. phagocytophilum (33), they could prove to be a separate species after further analysis. The A. phagocytophilum variant 1 (sample AP-V1) coexists with and is closely related to the human strains of A. phagocytophilum but has never been associated with human infection (9).…”