Ultrastructural Evidence of the Ehrlichial Developmental Cycle in Naturally InfectedIxodes persulcatusTicks in the Course of Coinfection with Rickettsia, Borrelia, and a Flavivirus

Abstract

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.

Tissue Distribution of the Ehrlichia muris-Like Agent in a Tick Vector

“…[15,20–23,26,27,30]. However, the ehrlichial load in some ticks in our study was more extensive than what was previously assumed for Anaplasmataceae other than A .…”

“…marginale and E . muris [21,30]. EML bacteria were observed in multiple organs and tissues including salivary gland (type II and type II acini) (Fig.…”

“…In contrast to previous reports for E . muris infection of Ixodes persulcatus [30], salivary gland cells appeared largely intact.…”

“…ruminantium have been shown in several organs of experimentally infected ticks [28,29]. In addition, there is a single publication containing micrographs of ehrlichial organisms in the salivary glands of wild caught ticks [30]. …”

Mechanisms of Obligatory Intracellular Infection with Anaplasma phagocytophilum

“…The ultrastructures of the DC and RC forms of A. marginale and Ehrlichia muris in tick tissues have been reported (117,186). A. phagocytophilum has been detected in the salivary glands of experimentally infected ticks and in field-derived adult deer ticks as Feulgen-positive inclusions (216), but the bacterial ultrastructure in ticks remains to be studied.…”