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

Podsumowanie

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.

Streszczenie

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Wprowadzenie

Ticks are obligate hematophagous ectoparasites of vertebrates and are distributed worldwide. In the United States, at least 11 species of ticks are vectors of pathogens of public health importance¹. Ixodes scapularis is responsible for transmission of several pathogens such as the causative agents of Lyme disease (Borrelia burgdorferi) relapsing fever (B. miyamotoi), human granulocytic anaplasmosis (Anaplasma phagocytophilum), and babesiosis (Babesia spp.). Despite the importance of I. scapularis as a disease vector, collecting these arachnids in abundance from the wild for studies in the lab is not always feasible. Therefore, the production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions.

The life cycle of all hard ticks (family Ixodidae), including I. scapularis, consists of the egg and three active stages: larva, nymph, and adult. Each active stage feeds on a vertebrate host. The complex interactions that take place between ticks and their hosts over several days of attachment and feeding are nearly impossible to replicate using artificial feeders, and are unlikely to provide enough numbers of fed ticks for mass rearing^2,3,4. Therefore, live mice and rabbits are used most frequently as hosts for rearing immature (larvae and nymphs), and mature stages (adults) of ticks, respectively. The requirement of multiple hosts for blood feeding during each developmental stage complicates tick rearing, and is time and cost intensive^5,6,7. Most tick rearing protocols require keeping mice in a suspended wire grid floor cage^7,8 or in a cylindrical cage of such dimensions that the animal cannot move freely and groom itself^6,9,10.

These cylindrical cages are later transferred to a shoebox cage with a wire grid. Engorged, detached ticks are then collected from the water underneath. However, this method results in exposing fed ticks to water contaminated with urine and feces that can increase fungal growth and tick mortality⁹. In addition, it increases the possibility of tick escape from the water trough, as well as causing stress to mice. To circumvent these problems, we here demonstrate larval tick feeding on mice within plastic shoebox-type disposable cages. This method allows the normal behavior of mice, increases engorged tick recovery, and decreases tick mortality due to contamination.

Protokół

The protocol (Number-00682) outlined below is approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nevada Reno and follows the guidelines of the University of Nevada, Reno's animal research ethics committee. Briefly, mice were anesthetized with isoflurane and a nose cone was used for maintaining continuous isoflurane levels for 20 min. A vet ointment was used to prevent drying of eyes under anesthesia. Toe pinch was used to ascertain the anesthesia level and breathing rate was measured throughout the procedure. Mice were kept in individual cages and monitored until consciousness was regained. Mice were used only once for tick feeding and were euthanized post tick recovery. Euthanasia was carried out by the Laboratory of Animal Medicine staff personnel. CO₂ and cervical dislocation were used for euthanizing animals.

NOTE: Working on ticks requires use of complete personal protective equipment. For counting immature ticks, wear white lab coats, long nitrile gloves to cover the sleeve opening of the lab coat capped with rubber bands, and closed-toe shoes. For infesting mice with ticks, use a hair net, disposable coveralls, long-sleeved gloves over the sleeves, and foot covers. Use white or light colored coveralls to detect wayward ticks. Periodically check gloves and sleeves for ticks.

1. Preparing Room for Animal Housing

1. Designate a separate room to house mice infested with ticks. Place a sticky mat or double-sided carpet tape outside and inside the door to prevent accidental tick escape.

2. Counting Ticks for Mouse Infestation

1. Put double-sided tape all around the top edges of a 7" x 5" x 14" plastic container and fill with water to approximately 2 cm.
2. Place another small box or Petri dish in the center of the container and fill with water to 1 cm creating a "moat" around this container
3. Store the vial containing larvae or nymphs inside the Petri dish.
4. Use a fine paint brush to remove larvae or nymphs from the vial and count under the microscope. Count 50 larvae or 25 nymphs into separate scintillation vials. Cover the vials immediately with nylon mesh or organdy cloth screen and close with rubber bands.

3. Infesting Mice with Immature Ticks

1. Use a white or light colored work bench and stick double-sided tape around the perimeter of the work area.
2. Anesthetize mouse with isoflurane. Check level of anesthesia by toe pinch. Once anesthetized, transfer the mouse to a heating pad covered with paper towels and attach to a nose cone for continued isoflurane supply.
3. Apply petroleum jelly-based eye ointment to avoid dryness. Note the breathing pattern of the mouse to adjust isoflurane levels (80 - 230 breaths per min is normal., Reduce isoflurane level if breathing rate is less than 80 breaths per min to avoid killing the animal).
4. Take one vial of 50 larvae or 25 nymphs and place ticks under the fur on the head between the ears with a paint brush (Figure 1). Keep mouse under anesthesia for 20 min following placement to give enough time for ticks to attach.
5. Move the mouse to a standard, plastic shoe box-type disposable mouse cage with static lids and white bedding. Provide toys, water, and food ad libitum as per normal mouse care.
6. Store the mouse cage within a larger rat or gerbil cage lined with double-sided sticky tape around top edges. Fill the outer cage with 3 cm of water (Figure 2).

4. Collecting Ticks from Mice

1. Immature ticks detach between days 3 and 6 of feeding. Check the cages and water moat for engorged ticks every day after day 3.
2. Collect detached ticks from the cage between days 4 and 6. Use a paint brush or soft forceps for picking up engorged ticks and store in clean scintillation vials capped with nylon mesh cloth secured with rubber bands.
3. Anesthetize and check mouse on day 7 for any remaining attached ticks.
4. Check the bedding, food trough, and water bottle for engorged ticks on day 7. Euthanize mice following the procedure as described above. Autoclave the disposable cages, bedding, water bottle, and food trough to avoid any escaping unfed ticks.

5. Storage of Fed Ticks

1. Maintain engorged ticks at 90% humidity, 20 °C, and a 12:12 light:dark cycle in a humidity and temperature controlled incubator until molting occurs. This may take approximately 12 to 18 weeks to occur.
2. While high humidity promotes tick survival, it also makes the ticks prone to fungal overgrowth. Check the fed ticks under the microscope at least once a week for mold. If detected, wash survivors in 70% ethanol for 5 min., rinse in water, transfer to a filter paper to dry, and transfer to new, clean vials.

Wyniki

We modified existing tick rearing protocols^6,10 for improved feeding efficiency and reduced stress on the mouse host. The results demonstrate that the standard shoebox style mouse cages are well suited for tick rearing. The white bedding provided a good contrast for easy collection of fed ticks. Most ticks climbed up the walls of the containers after feeding and were easy to collect. In addition, the tight fitted lid of the dispos...

Dyskusje

Critical Steps within the Protocol

It is important to have several levels of safety measures when rearing ticks to avoid accidental escape. Use of sticky tape and a water moat are critical to ensure safety. It is important to keep the anesthetized mouse on a heating pad to keep the body temperature constant. We also found that shaving the mouse does not provide any extra benefit for tick attachment. An individual mouse can be kept in the same cage for one week in a designated "tick room" ...

Materiały

Name	Company	Catalog Number	Comments
Puralube vet ointment	Amazon		Available from any Pet store or online store
Disposable mouse cage	Innovive, San Diego, CA	MV 2	Set of bottom and lid
White Alpha dri bedding	Lab Supply, Fort Worth, TX	ALPHA-Dri™