Acquisition and transmission of the agent of human granulocytic ehrlichiosis by Ixodes scapularis ticks

Abstract

The purpose of the present study was to investigate the transmission of a human isolate of the agent of human granulocytic ehrlichiosis (HGE agent) from infected mice to larval ticks and to examine the population kinetics of the HGE agent in different stages of the tick life cycle. The HGE agent was quantitated by competitive PCR with blood from infected mice and with Ixodes scapularis ticks. The median infectious dose for C3H mice was 10(4) to 10(5) organisms when blood from an infected severe combined immunodeficient mouse was used as an inoculum. Uninfected larval ticks began to acquire infection from infected mice within 24 h of attachment, and the number of HGE agent organisms increased in larval ticks during feeding and after detachment of replete ticks. Molted nymphal ticks, infected as larvae, transmitted infection to mice between 40 and 48 h of attachment. Onset of feeding stimulated replication of the HGE agent within nymphal ticks. These studies suggest that replication of the HGE agent during and after feeding in larvae and during feeding in nymphs is a means by which the HGE agent overcomes inefficiencies in acquisition of infection by ticks and in tick-borne transmission to mammalian hosts.

FIG. 1

Quantitation of HGE agent DNA by competitive PCR with primers specific for a 250-bp 16S rRNA target of the HGE agent and a 549-bp competitive target containing an irrelevant 504-bp internal B. burgdorferi ospA segment. Fivefold decreasing amounts (from 1 pg/μl to 0.04 fg/μl) of competitor (top row, 549 bp) were added to a constant amount of HGE agent target DNA (bottom row, 250 bp). The PCR mixtures were amplified for 40 cycles, and the products were resolved on a 1.6% agarose gel stained with ethidium bromide. When competitor and target band intensities were equivalent, the amount of target DNA was presumed to equal the known amount of competitor DNA.

FIG. 2

The accuracy of the competitive PCR was evaluated by performing a series of competitive PCR assays with a decreasing amount of competitor mixed with a constant volume from each of seven different dilutions of target solution (). In another series of seven assays, for each assay a constant amount of competitor was mixed with a decreasing amount of target (○). The x axis is a log₁₀ scale of the percentage of HGE agent-infected HL-60 cell solution (target solution) in a fivefold serial dilution. The y axis is a log₁₀ scale of the DNA concentration at equivalent competitor and target intensities. For each curve, linear analysis revealed P values of <0.001 and correlation coefficients (ρ) of >0.99.