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Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Soft ticks are medically important ectoparasites of birds and mammals that are found throughout the world. This report describes isolation and partial characterization of two embryonic cell lines, CCE2 and CCE3, from the seabird soft tick Carios capensis (Neumann). Sequencing of the mitochondrial 16S rRNA gene and karyology confirmed the lines were derived from C. capensis. CCE3 cells were diploid with a modal chromosome number of 20. The population doubling time for cell lines CCE2 and 3 in passage 40 was 6-9 d. A rickettsial endosymbiont, RCCE3, was co-isolated along with line CCE3. Nucleotide sequences of polymerase chain reaction (PCR) products generated using primers specific for rickettsial 17-kDa antigen, outer membrane protein (omp) A, ompB, and citrate synthase genes along with phylogenetic analyses demonstrated that RCCE3 is a previously uncultured endosymbiont. The rickettsia was identified as a symbiont of C. capensis, closely related to rickettsiae previously detected by PCR in C. capensis, Ornithodoros moubata (Murray) and Hemaphysalis sulcata CanestriniFanzago, a hard tick. RCCE3 caused a cytopathic effect in C. capensis host cells, and it was transferred to Ixodes scapularis Say cell line ISE6 for maintenance. The rickettsial endosymbiont was eliminated from CCE3 by treatment with oxytetracycline. Cell lines from C. capensis will be useful to researchers investigating interactions between soft ticks and microorganisms, soft tick physiology, and molecular biology. The rickettsia adds to the growing number of Rickettsia species that have been isolated in tick cell culture, and it is available for characterization.

Three new cell lines, IDE8 and IDE12 from embryos of northern specimens of Ixodes scapularis Say and ISE18 from southern specimens of I. scapularis, were compared cytogenetically via conventional karyotyping, C- and G-banding, and nucleolar organizing regions (NORs). The karyotypes were very similar. The standard karyotype in the three cell lines consisted of 28 chromosomes with 26 autosomes and XX (female) or XY (male) sex chromosomes. The X chromosome was the largest, and the Y chromosome the smallest chromosome of the karyotype. Constitutive heterochromatin (C-bands) was almost entirely restricted to the centromeric region. An additional interstitial C-band in chromosome 7 was an important notable characteristic of the three cell lines. In sets showing a similar degree of condensation, individual chromosomes of the three lines had identical G-banding patterns. In addition, there was no difference among the cells in number and position of NORs. There were approximately 100 G-bands per haploid set in chromosomes from cells in metaphase, with three to 18 G-bands in each chromosome arm. After staining with silver nitrate, interstitial NORs were identified in chromosomes 7, 10, and the X chromosome. Male cells had five and female cells had six NORs. These findings support the notion that I. scapularis and I. dammini Spielman et al. are conspecific.

Lyme disease spirochetes, Borrelia burgdorferi, interact with cultured tick cells in ways similar to those reported to occur in the vector Ixodes dammini Spielman, Clifford, Piesman & Corwin. Spirochete adhesion and penetration were examined using a cell line from embryos of Rhipicephalus appendiculatus Neumann that morphologically resembles tick gut cells, RAE25. Cocultivation of B. burgdorferi with these cells permitted prolonged maintenance of infectivity for hamsters. Borrelial adherence to RAE25 cells was time- and density-dependent and increased by 10—15% per h during the first 5.5 h of cocultivation when we used a concentration of 4 × 107 spirochetes/ml. After 6 h, >90% of the cells bound an average of 3*.xml5 spirochetes per cell. Low passage, hamster-infective strains of B. burgdorferi (JMNT and CD16) showed a 2-3-fold higher rate of adhesion to RAE25 cells than the highly passaged, noninfectious strain B31. Inactivation of CD16 or JMNT by heat, starvation, or treatment with puromycin reduced adherence by 40*.xml60%, whereas preheatment with monoclonal antibodies to the outer surface proteins had no effect. Spirochetes adhered to young I. dammini cell lines to a similar degree as they did to RAE25, whereas lines from the ticks Dermacentor variabilis (Say) (RML15) and Boophilus microplus (Canestrini) (BME26) bound 30—60% fewer spirochetes. Electron microscopy revealed epicellular borreliae associated with coated pits and vesicles before endocytosis, and intracellular spirochetes were surrounded by a host cell-derived membrane.

Borrelia burgdorferi, strain 297, was incubated with tick cell lines isolated from Rhipicephalus appendiculatus (Neumann), R. sanguineus (Latreille), Anocentor nitens (Neumann), Dermacentor variabilis (Say), and Boophilus microplus (Canestrini). A tick cell culture medium, L-15B, was modified by the addition of gelatin and N'-acetylglucosamine (L-15B/S) to permit the cocultivation of B. burgdorferi and tick cells. Spirochetes continuously passaged axenically in L-15B/S had longer population doubling times (27.1 ± 4.5 h) than those grown in BSK medium (11.7 ± 2.2 h), which was unsuitable for tick cells. Growth of spirochetes cocultured with five of six lines did not change, but when B. burgdorferi was incubated with R. sanguineus cells, the spirochetes disappeared and could not be detected after 1 wk. Spirochetes bound themselves to tick cells by one end within 30 min after being added to cultures. Not all spirochetes attached to tick cells nor did all cells of a given line bind spirochetes. B. burgdorferi had the greatest affinity for embryonic R. appendiculatus cells and the least for D. variabilis cells. Scanning electron microscopy revealed numerous intertwined, coiled spirochetes attached to foci at the surface of R. appendiculatus cells, apparently causing the formation of round bodies. Fewer spirochetes attached to A. nitens cells. High spirochete concentrations (>107 per ml) elicited cytopathic effects—loss of surface membrane extensions, rounding up, detachment, and lysis. This system should be applicable in the study of tick cell-spirochete interactions and the analysis of spirochete tropism and binding to tick cells.