Tick Cell Culture Analysis of Growth Dynamics and Cellular Tropism of Rickettsia buchneri, an Endosymbiont of the Blacklegged Tick, Ixodes scapularis

SIMPLE SUMMARY: The blacklegged tick, Ixodes scapularis, a species of significant medical and veterinary importance, harbors an endosymbiont, Rickettsia buchneri. This bacterium is largely restricted to the ovaries, but all life stages can harbor different numbers or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. We characterized the cells using microscopy. The doubling time of wildtype R. buchneri and a transformant expressing green fluorescent protein was determined to be >7 days when measured by quantitative PCR. Quantification based on fluorescence indicated that 11 days were needed to double the amount of green fluorescent protein. Two rRNA probes were tested using rickettsiae grown in vitro and adapted to localize R. buchneri in different organs of field-collected female I. scapularis ticks. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. The sequestration of rickettsia in ticks and the slow growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont. ABSTRACT: The blacklegged tick, Ixodes scapularis, a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. Rickettsia buchneri most readily grows and is maintained in the cell line IRE11 from the European tick, Ixodes ricinus. The line was characterized by light and electron microscopy and used to analyze the growth dynamics of wildtype and GFPuv-expressing R. buchneri. qPCR indicated that the genome copy doubling time in IRE11 was >7 days. Measurements of fluorescence using a plate reader indicated that the amount of green fluorescent protein doubled every 11 days. Two 23S rRNA probes were tested via RNA FISH on rickettsiae grown in vitro and adapted to evaluate the tissue tropism of R. buchneri in field-collected female I. scapularis. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. Tissue tropism in I. scapularis and in vitro growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.