Infection of Immature Ixodes scapularis (Acari: Ixodidae) by Membrane Feeding

Abstract

A reduction in the use of animals in infectious disease research is desirable for animal welfare as well as for simplification and standardization of experiments. An artificial silicone-based membrane-feeding system was adapted for complete engorgement of adult and nymphal Ixodes scapularis Say (Acari: Ixodidae), and for infecting nymphs with pathogenic, tick-borne bacteria. Six wild-type and genetically transformed strains of four species of bacteria were inoculated into sterile bovine blood and fed to ticks. Pathogens were consistently detected in replete nymphs by polymerase chain reaction. Adult ticks that ingested bacteria as nymphs were evaluated for transstadial transmission. Borrelia burgdorferi and Ehrlichia muris-like agent showed high rates of transstadial transmission to adult ticks, whereas Anaplasma phagocytophilum and Rickettsia monacensis demonstrated low rates of transstadial transmission/maintenance. Artificial membrane feeding can be used to routinely maintain nymphal and adult I. scapularis, and infect nymphs with tick-borne pathogens.

Fig. 1.

Membrane production and tick feeding process. (A) A silicone squeegee is used to remove excess silicone from lens paper substrate. (B) Feeding chambers prior to use. (C) Nymphs in parafilm-sealed chambers with R. monacensis -spiked blood. (D) I. scapularis females engorged after 7 d of feeding.

Fig. 2.

Images of the tick-borne pathogens. (A) A. phagocytophilum HGE1-GFPuv grown in mCherry-expressing RF/6 A cells. Scale bar = 10 μm. (B) B. burgdorferi JMNT cultured in BSK medium, methanol-fixed, and Giemsa-stained. Scale bar = 10 μm. (C) mCherry-expressing EMLA grown in GFP Lifeact-expressing RF/6 A cells. Scale bar = 20 μm. (D) R. monacensis biotin (green) comingled with an mKate-expressing transformant (red) in the gut tissue of a male I. scapularis previously capillary-fed with both strains. Scale bar = 20 µm.

Fig. 3.

Nymphs were allowed to feed for 7 d. On days 2–4, bacteria were inoculated into the blood reservoir with each change of blood. Because nymphs took at least 3 d to engorge, this ensured their exposure to infected blood. Engorged nymphs were sampled for PCR-testing after completion of feeding. Adults were sampled after they had molted and sclerotized 6-8 weeks after completing engorgement as nymphs.

Fig. 4.

Map of pRAM18dSGK[Biotin]. The construct confers resistance to spectinomycin (Spec) and kanamycin (KAN) and expresses GFPuv. The pET vector supports replication in Escherichia coli , and the portion of pRAM18 containing the dnaA and parA genes allows the construct to replicate in rickettsia. The biotin synthesis operon was amplified from R. buchneri pREISMN_2 and inserted into pRAM18dSGK[MCS].

Fig. 5.

The proportion of I. scapularis nymphs that acquired a given pathogen strain is denoted by a gray bar. The proportion of adults that transstadially transmitted infection after acquiring a pathogen as nymphs is denoted by a black bar. The number at the base of each bar indicates the number of ticks tested by PCR at that life stage for each pathogen strain.