The proper use of personal protective equipment (PPE) is mandatory when working with ticks. PPE requirements vary by institution and country. Full coverage PPE is highly recommended during the entire feeding procedure, and should consist of a hair net, surgical mask, coveralls/white gown, white/light colored gloves, and shoe covers. A depiction of full coverage PPE is shown in Figure 1A. PPE should be white or light-colored to aid in the visualization of ticks. Proper coverage of PPE provides basic protection as a physical barrier, preventing any ticks from crawling inside and attaching to the skin of research or animal facility staff. A hair net is worn to cover the entire crown of the head and extends below the ears as demonstrated in Figure 1B. Adhesive white mats (Fisher Scientific, Cat# 19–181-510) were used as another method of tick-containment and were placed under any container and on any surface where ticks were expected to be handled. The adhesive white mat aids in sticking and visualization in the unlikely event that a tick climbs out, falls, or drops during handling.

Black legged tick larvae and nymphs were purchased from tick rearing facility at Oklahoma State University. The ticks were monitored via PCR assay for Anaplasma phagocytophilum and Borrelia burgdorferi. Single batches of egg clusters from monitored ticks were purchased from Oklahoma State University. Egg clusters were allowed to hatch as larvae before introducing to mice. Prior to feeding the larvae and nymphs, the ticks were stored on a rack inside of a transparent closed chamber (Secador 2.0 vertical desiccator cabinet, SP Bel-Art) that was placed on an adhesive white color mat stored within an incubator (Fisherbrand Isotemp incubator) set to 25°C (Figures 2A,B). Distilled water in an open container was placed on the bottom rack of the chamber to provide and maintain 90% humidity within the chamber (Figures 2A,B). The ticks were stored within a 10x75mm polystyrene test tube (Axiom Medical Supplies M-1035956-3123) with fine nylon mesh (Voile White Decorator fabric) placed between the tube and flange 12 mm polyethylene plug top (Universal Medical Inc. GS-118139C) (Figure 2C). Several holes were poked into the polyethylene plug top with a hypodermic needle to allow for airflow (Figure 2D). Double-sided tape (Scotch) was placed around incubator door openings to further prevent any ticks from escaping (Figure 2E). Two LED light strips were cut to 12 LEDs each, connected to the same power supply, and installed on opposing sides of the internal top of the incubator (Figure 2F). The connected LEDs were run through a programmable timer with a 12:12 light:dark cycle (Figure 2G) (Fisherbrand Traceable Digital Outlet Controller, Cat# 06–662-24).

To study non-native pathogen transmission, ticks must be infected with the pathogens, and for Borrelia burgdorferi, mice need to be infected for the tick nymphs/larvae to feed on them. For our research, we purchased Borrelia burgdorferi (sensu stricto) from BEI resources (NR`-13251, strain B31). Before infecting mice, B. burgdorferi was cultured in Barbour-Stoenner-Kelly (BSK) (Sigma-Aldrich, B8291) medium in 5 mL round-bottom polystyrene tubes (Corning, #352058) in a 37°C incubator (Fisherbrand Isotemp incubator) in the laboratory (Wang et al., 2004). Post-culturing, the bacterial concentration was determined with a Petroff-Hausser counting chamber and a dark-field microscope. Exponentially growing bacterial stock was diluted to 10⁶/ml in BSK broth and 100 μL (10⁵ total Borrelia) was injected via 0.5 cc syringe with 28G needle (0.5 inch) per C3H mouse, intraperitoneally. Ticks were infected with B. burgdorferi by placing and feeding them on B. burgdorferi-infected mice 15 days after injection as described below.

In our protocol, ticks were fed on 6 week old female C3H mice from The Jackson Laboratory, as C3H develops Lyme arthritis (Barthold et al., 1990). All mice were maintained on a regular diurnal lighting cycle (12:12 light:dark) with ad libitum access to food (LabDiet 5P76 IsoPro RMH 3000 Irradiated) and automated reverse osmosis chlorinated water. Room temperature was maintained at 72 +/− 3°F and humidity was 29–50%. Loose soft cellulose (BioFresh Comfort) was used as cage bedding prior to tick-feeding procedures. Prior to tick-feeding procedures, environmental enrichment including nesting material (Bed-r Nest, The Andersons Inc), polycarbonate tunnel (Datesand Limited), and cardboard tunnel (Pakolatus, LLC) were provided. Mice were housed in standard plastic mouse cages (11–1/2″ long x 7–1/2″ wide x 5″ deep) with stainless steel wire flooring during tick feeding. Mice were provided with the same food and water mentioned above during tick feeding. A clear polycarbonate handling tube (Datesand Limited) was added to each cage as enrichment and as a resting place from the wire flooring. Roughly ¼ inch of water was added to the bottom of the mouse cage prior to adding the wire (Figure 3A). Ticks that have fed and detached, drop off the mouse and float in the water below. Mice may attempt to consume fed ticks that climb the walls. The top ½ inch of the interior of the mouse cages were smeared with petroleum jelly, which acts as a second barrier for any ticks that have fed, detached, and climbed the walls of the cage (Figure 3A). Each prepared mouse cage was placed into a larger guinea pig cage (19″ long x 10–1/2″ wide x 8″ deep) that was also prepared with 1 inch of water and the top ½ inch of the interior smeared with petroleum jelly (Figure 3B). The mouse cage inside of the guinea pig cage was then kept on a white sticky mat on a metal shelf.

A 10% bleach solution was prepared fresh daily to sanitize any collected waste liquids and to kill any uncollected ticks and tick-borne pathogens that were present in the wastewater. A 5-gallon jerrican was designated for 10% bleach disposal. Waste liquids that were treated with bleach for 24 h were discarded.

To feed the ticks, mice were anesthetized (100 mg/kg (ketamine) + 10 mg/kg (xylazine), 0.1 mL/100 g intraperitoneally) and placed next to each other in a clean and empty mouse cage with petroleum jelly smeared on the top interior (Figure 4A). A heating pad was placed under the cage for supplemental heat while the mice were anesthetized. Ophthalmic ointment was applied to the eyes of each mouse to prevent drying while anesthetized, as the eyes remained open during anesthetization.

For investigating Borrelia burgdorferi transmission to mice and detection thereof, 3–5 Borrelia-infected nymphs should be placed on naïve mice. For Borrelia acquisition and xenodiagnostic studies, one should put not more than 25–30 nymphs on infected mice (as described below).

During the entire feeding process, the tick-harboring mouse was transferred to a freshly prepared tick feeding cage every morning and evening, with the wastewater having been thoroughly checked for ticks. Wastewater was treated with bleach as described above. During the cage transfer of mice, cages should be kept on a white sticky mat as described above. By Day 3 of tick feeding, 10–50% of ticks drop off after becoming fully engorged. By Day 4, 95–98% of ticks dropped off the mice. Day 5 contained the lowest yield of fully fed ticks, with the subsequent days yielding no additional ticks. Following tick feeding, the mice were euthanized via carbon dioxide and cervical dislocation. Mouse blood and tissues can be harvested at this step to investigate various avenues of tick-host interaction.

The tick nymph was placed into a drop of PBS on a sterile microscope slide (Figure 6A). Using an Olympus SZ61 dissecting microscope to visualize, the legs were each removed by holding the tick with tweezers and cutting off the legs with vannas scissors (World Precision Instrument 501,839). Once the legs were removed, the tick is rendered immobile and easier to dissect. While holding the chelicerae with tweezers, a flat cut was made with vannas scissors on the most posterior portion of the tick (Figure 6B). Two opposing diagonal cuts at roughly 45 degrees and 135 degrees were made connecting with the previous cut, cutting just laterally to the spiracular plates of the trachea (Figure 6B). Using the newly created opening (Figure 6C), the interior of the tick is exposed by peeling the dorsal scutum from the ventral exoskeleton. The scutum was torn away from the ventral exoskeleton, as the ventral side contained all major anchor points for internal tissues (Figure 6D). Care was taken during scutum removal to not disrupt the capitulum and the scutum was discarded (Figure 6D). Both the salivary gland and midgut were located and the tracheal tissue surrounding these organs was removed by gently tearing them away. The midgut was removed first, as it sits on top of the salivary glands (Figure 6E). Using two pairs of tweezers, each portion of the midgut was carefully separated from surrounding tissue to prevent excess tearing, contamination, and leakage. The midgut was placed onto a slide with 1 drop of 1x PBS and fixed with acetone for immunofluorescence assays or into a microcentrifuge tube with 100 μL TRIzol for RNA extraction. Following midgut removal, both salivary glands, which appear as spherical clusters (Figure 6F), were dissected from their respective sides by tearing the anterior lobular salivary duct nearest to the capitulum (Figure 6G), then placed onto a slide with PBS and fixed with acetone for immunofluorescence assays or into a microcentrifuge tube with 100 μL TRIzol for RNA extraction.

Tick guts and salivary glands were washed with 3 drops of PBS after dissection. Post-washing, acetone was added and treated for 10 min to fix and permeabilize the tissues. After fixation, tissues were blocked using PBS-T (0.05% Tween-20 in PBS) supplemented with 5% normal goat serum for 30 min at room-temperature (RT). The blocking buffer was removed, and samples were incubated with primary antibody [Anti-Borrelia (Abcam, ab69219) diluted in fresh blocking buffer overnight at 4°C (primary incubation can also be done for 1 h at RT). Samples were washed three times in PBS-T for 5–10 min each at RT before being incubated with secondary antibody [Goat anti-Rabbit Alexa Fluor® 488 (ab150077) for 1 h at RT. During and following secondary antibody incubation, samples were protected from light by placing them in a foil-wrapped container during incubations. Samples were then washed three times in PBS-T for 5–10 min each at RT. Following washes, the tissues were transferred onto Poly-Lysine coated slides, and a drop of anti-fade mounting media with DAPI was added to the samples before applying a cover slip. Samples were incubated with mounting media for at least 20 min prior to imaging. Positive Borrelia infection results of tick gut tissues are depicted (Figures 7A,B). Western blots of uninfected mammalian cell lysate were compared to cultured Borrelia lysate that were probed with Borrelia-infected mouse serum (Figure 7C) or an anti-Borrelia commercially produced antibody (Figure 7D) and indicated positive Borrelia signals only in the infected mouse.