Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

Transcript

The overall goal of this tick-feeding protocol is to improve the existing standards by increasing the survival of blood-fed ticks, while reducing the stress to the rodent host. In addition to simplifying tick colony rearing, this method provides an easier way of collecting number of blood-fed ticks in a single collection day, which is essential to answer key biological questions in macrobiology fields. The main advantage of this technique is that ticks are not exposed to water containing mouse excreta, thereby reducing tick mortality.

In addition, ticks can be collected on a single day, and this reduces stress to the mouse. To house tick vials for counting, make a container that allows minimal escape. Stick double-sided tape all around the top edges of a plastic container.

Then, fill the container with about two centimeters of water. Next, place another small box or dish centrally in the container, and fill it with about one centimeter of water, so it sinks and stays in position. Store vials of tick larvae or nymphs inside this box or dish.

To count the ticks for an infestation, use a microscope and a fine paintbrush to remove larvae or nymphs from the vial. Transfer 50 larvae or 25 nymphs to a scintillation vial. As soon as a vial is fully loaded, cover it with nylon mesh or organdy cloth screen, and secure the cover with tight rubber bands.

Designate a separate room to house mice infested with ticks. To prevent accidental tick escape, place a sticky mat or double-sided carpet tape outside the room, and place another mat inside the door of the room. First, prepare an area to handle the ticks near the mice.

Use a white or light-colored workbench. Make a border of double-sided tape around the perimeter of the work area on the bench. Now, anesthetize a mouse with isoflurane.

When ready, transfer the mouse to a heating pad covered with paper towels, and attach a nose cone to deliver isoflurane continuously. Check the level of anesthesia using a toe pinch. Adjust the anesthetic based on the breathing pattern.

80 to 230 breaths per minute is normal. Reduce the isoflurane if the breathing is slower. Also, apply a petroleum-jelly-based ophthalmic ointment to the eyes.

Now, take one vial of 50 larvae or 25 nymphs and, using a paintbrush, transfer the ticks to the mouse. Place the ticks under the fur on the head between the ears. After placing the last tick, keep the mouse anesthetized for 20 minutes, so the ticks can attach before the mouse tries to remove them.

After 20 minutes, return the mouse to a new home cage with white bedding for improved tick visibility. Include some toys and provide ad lib access to water and food. Use disposable boxes with tight-fitting lids.

Next, place the home cage within a larger rat or gerbil cage that has double-sided sticky tape around the upper internal edge of the walls. Then, fill the space between the cages with about three centimeters of water. Immature ticks will detach after three to six days of feeding.

From day three and onward, check the cages and the surrounding water for engorged ticks on a daily basis. Collect the detached ticks using a paintbrush or soft forceps, and transfer them to clean scintillation vials capped with nylon mesh cloths secured with rubber bands. On day seven, anesthetize the mouse as before, and check for any remaining attached ticks.

Also, thoroughly check the bedding, the food trough and the water. Then, euthanize the mouse and autoclave the disposable cages, bedding, water bottle and food trough to eliminate any remaining ticks. House the engorged ticks in a controlled incubator.

Within 12 to 18 weeks, the ticks should molt. At least once a week, check the ticks under the microscope for mold. If any mold is detected, wash the surviving ticks with 70%ethanol for five minutes.

Then, rinse the survivors in water and transfer them to a filter paper to dry. Once clean and dry, transfer the ticks to new, clean vials, and continue their life cycle. Most ticks climbed up the walls of the containers after feeding, and were easy to collect.

Ticks rarely escaped the mouse cages. The white bedding provided a good contrast to detect fed ticks. During collections, most ticks detached by day four.

A few waited until day five to release, and by day seven, no mouse was carrying any ticks. About 2/3 of ticks were able to attach and feed to engorgement. The mice all remained healthy and did not show any signs of discomfort.

Once mastered, this method can provide hundreds of blood-fed ticks in a single collection day. While performing this procedure, it's important to remember to wear full PPE to prevent accidental tick attachment.

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Journal (Immunology and Infection)

Journal (Immunology and Infection)

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

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