We acknowledge the technical assistance of Evelyne Le Naour, French National Institute of Agricultural Research (INRA), Alain Bernier (INRA) and Oc&#233;ane Le Bidel (ANSES). Consuelo Almaz&#225;n was supported by a fellowship from the Laboratory of Excellence, Integrative Biology of Emerging Infectious Diseases (LabEx IBEID), Pasteur Institute. The rabbits and sheep were purchased by ANSES. Part of this work was funded by National Institutes of Health grant RO1AI090062 to Y. Park. Dr. Jeffrey L. Blair is acknowledged for reviewing the manuscript.

NOTE: In this study, rabbits were maintained in standard cages with food and water offered ad libitum at the French Agency for Food, Environmental and Occupational Health &#38; Safety (ANSES) accredited animal facilities in Maisons-Alfort, France. Animals were monitored twice daily by two experienced technicians for any abnormal skin reactions, health problems or complications. The experimental room was secured by framing the interior of the doorway with double-sided tape to avoid accidental escape of ticks. The method works best if two people work as a team, but it is possible to complete single handedly by one experienced person. Although most rabbits are docile and calm, signs of stress may occur during the manipulation. To ensure that a rabbit does not injure itself by struggling, manual restraint may be accomplished by gently holding of the scruff of the neck in one hand while the other hand supports it hind quarters. The six month old, Rambouillet female sheep was kept at the Centre for Biomedical Research (CRBM) facilities at the National Veterinary School of Alfort (ENVA), where water and food were supplied ad libitum, and it was checked twice daily.
NOTE: Our laboratory has received permission to use rabbits and sheep for tick feeding by the Ethics Committee for Animal Experiments ComEth Anses/ENVA/UPEC, Permit Numbers 01741.01 and 11/10/16-5B, respectively. Since we used only pathogen-free ticks in our experiments, all the rabbits used in this study were offered for adoption via the White Rabbit Association, Paris, France.
1. Preparation of the Capsules
<ol>
	<li>Cut the desired size of the capsule from the EVA foam sheet (Figure 1A). Round the outer corners (Figure 1B) of the capsule to minimize accidental detachment when gluing it to the rabbit skin. 
	NOTE: The frame thickness of the capsule should be around 8 mm. Use a 5 mm thick foam sheet for the larvae, nymphs, and small tick adult species such as Ixodes. A 1 cm thickness foam sheet is suitable for large size adult ticks such as Amblyomma sp., Hyalomma sp., etc. The size of the capsule varies based on the experimental requirements. For example, for 20 Ixodes adult couples, 200 nymphs, or 1,000 larvae, we use an inner capsule size of 5 x 5 cm2, 6 x 7 cm2 or 7 x 9 cm2, respectively.</li>
	<li>Cut the 8 mm wide strips of self-adhesive hook tape (see Table of Materials) and stick them to the prepared EVA foam frame (Figure 1C).</li>
	<li>Cut the same size strips from self-adhesive loop tape (see Table of Materials) and bind them to the hook sides attached to the EVA-foam frame (Figure 1D).</li>
	<li>Cut the appropriate size of the fine mosquito mesh (mesh size less than 50 &#181;m) to the size of the EVA foam frame and stick it to the self-adhesive loop (Figure&#160;1E and 1F). Cut the overhangs if necessary. 
	NOTE: This type of capsule can be used to feed nymphs and adults of hard tick species, while a different sealing system of the capsules is needed for larval feeding (Supplemental Figure 1) to prevent accidental escape of the larvae via the fastened hook-and-loop side.</li>
</ol>
2. Preparation of the Rabbit before Tick Infestation
<ol>
	<li>Shave the area of the rabbit back and sides to be used with clippers (Figure 2A).</li>
	<li>Apply non-irritating latex glue to the entire surface of the prepared capsule and wait for 1 min (Figure 2B).</li>
	<li>Glue the capsule by pressing to the skin (especially at the corners) with the fingers for about 3 minutes (Figure&#160;2C and 2D). 
	NOTE: When gluing more than one capsule, make sure to keep at least 5 mm space between them (Figure&#160;2E and 2F). We usually avoid the region of the spine, but it may be used if needed.</li>
	<li>Slightly lift the capsules to visually check their attachment to the skin. If non-attached regions are found, apply the glue using a spatula and press for another 3 minutes.</li>
	<li>Apply protective tape to the rear paws of the rabbit to prevent jacket damage (Figure 2G). 
	NOTE: This step is optional and is mainly to prevent jacket damage, not damage to the tick capsule.</li>
	<li>Put on the rabbit jacket by placing the front legs through the openings and tightening the neck,&#160;making sure rabbit breathing remains comfortable. Do not place the rear legs through the elastic enclosures at this step and leave the zipper open (Figure 2H).</li>
</ol>
3. Tick Infestation
<ol>
	<li>Place the ticks into a plastic syringe (1 or 5 mL depending of the number of the individuals) with the needle-end cut and plugged with cotton (Figure 1G). If a small amount of ticks are to be infested, use forceps. 
	NOTE: For tick colony maintenance allow the engorged tick female(s) to lay eggs with subsequent hatching inside the syringe (5 or 10 mL) covered by the mosquito mesh wrapped by a rubber band12 to avoid laborious manipulation of the larvae at the time they are applied to the host (Figure 1H). Also, fully engorged larvae may be allowed to molt in the syringe (Supplemental Figure 1I; 5 or 10 mL) for direct infestation of the rabbit with nymphs.</li>
	<li>Place the syringe deep into the capsule via the open corner and inoculate the ticks by pushing the syringe plunger. Slowly twist the plunger toward the rabbit skin to remove the remaining ticks attached to the plunger and simultaneously pull it out from the capsule (Figure 2I). 
	NOTE: If some of the individuals crawl out of the capsule, return them using forceps.</li>
	<li>Close the capsule by refastening the hook-and-loop tape.</li>
	<li>Place the rear legs of the rabbit into the rear elastic enclosures of the jacket and zip closed. 
	NOTE: Make that an index finger can fit between the neck of the jacket and the rabbit to ensure comfort and also to avoid chewing on the jacket.</li>
	<li>Return the rabbit to the cage (Figure 2J). 
	NOTE: The time from the infestation to collection of replete ticks vary among different tick species and the developmental stages. For example, for Ixodes scapularis and Ixodes ricinus, the durations of feeding for adults, nymphs, and larvae are 6&#8211;9, 3&#8211;4, and 2&#8211;3 days, respectively. A list of references for 29 different hard tick life cycles under laboratory conditions can be found in Levin and Shumacher (2016)9.</li>
</ol>
4. Collection and Monitoring of Ticks
<ol>
	<li>Take the rabbit from the cage to the bench and unzip the jacket.</li>
	<li>Gently restrain the rabbit with your hands. Open the capsule by unfastening the hook-and-loop tape (Figure&#160;2K and 2L) and collect the ticks by brushing the engorged larvae (Supplemental Figure 1) or nymphs to a plastic dish or using forceps for adults (Figure 2L). If partially fed (not replete) ticks are required, use a tick twister or forceps to detach them. 
	NOTE: If maintaining the tick colonies please see the note in step 3.1. Maintain the engorged ticks in appropriate humid and temperature conditions according to the particular tick species.</li>
	<li>If needed, refasten the hook-and-loop tape to close the capsule.</li>
</ol>
5. Recovery of the Rabbit
<ol>
	<li>Remove the mosquito mesh completely from the capsule and leave the jacket on the rabbit (Figure 2M).</li>
	<li>Wait 3-4 weeks and try to remove the capsule by gently trimming one of the corners (Figure 2N). If the capsule is still firmly attached, repeat this step one week later.</li>
	<li>Remove the jacket and let the rabbit recover in the cage. 
	NOTE: Once the capsule is off, check the skin of the rabbit for abnormal reactions. Although normally no treatment is required, an emollient lotion can be used in case of irritation.</li>
	<li>If the protocol and experiments allow, the recovered rabbit (Figure 2O) can be reused or offered for adoption. 
	NOTE: Rabbits have been shown to acquire tick resistance once exposed to repeated tick infestations13; therefore, reinfestations are not recommended unless the experiment requires.</li>
</ol>

<ol><li>Sonenshine, D. E., Roe, M. Ticks, People and Animals. <a target="_blank" href="https://www.amazon.com/Biology-Ticks-1-Daniel-Sonenshine/dp/019974405X">Biology of Ticks, Vol I</a>. , Oxford University Press. (2014).</li>
<li>Kröber, T., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+feeding+assays+for+hard+ticks%5BTitle%5D)+AND+%22Trends+in+Parasitology%22%5BJournal%5D)">In vitro feeding assays for hard ticks.</a> Trends in Parasitology. 23 (9), 445-449 (2007).</li>
<li>Bonnet, S., Jouglin, M., Malandrin, L., Becker, C., Agoulon, A., L'hostis, M., Chauvin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transstadial+and+transovarial+persistence+of+Babesia+divergens+DNA+in+Ixodes+ricinus.ticks+fed+on+infected+blood+in+a+new+skin-feeding+technique%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus.ticks fed on infected blood in a new skin-feeding technique.</a> Parasitology. 134 (2), 197-207 (2007).</li>
<li>Bonnet, S., Liu, X. <a target="_blank" href="https://hal.archives-ouvertes.fr/hal-01301271">Laboratory artificial infection of hard ticks: A tool for the analysis of tick-borne pathogen transmission.</a> Acarologia. 52 (4), 453-464 (2012).</li>
<li>Khols, G. M. Tick rearing methods with special reference to the Rocky Mountain Wood Tick, Dermacentor andersoni Stiles. Culture methods for invertebrate animals. , Dover Pubs. New York. (1937).</li>
<li>Faccini, J. L. H., Chacon, S. C., Labruna, M. B. <a target="_blank" href="https://www.researchgate.net/publication/250044361_Rabbits_Oryctolagus_cuniculus_as_experimental_hosts_for_Amblyomma_dubitatum_Neumann_Acari_Ixodidae">Rabbits (Oryctolagus cuniculus) as experimental hosts for Amblyomma dubitatum Neumann (Acari: Ixodidae) [Coelhos (Oryctolagus cuniculus) como hospedeiros experimentais de Amblyomma dubitatum Neumann (Acari: Ixodidae).</a> Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 58 (6), 1236-1239 (2006).</li>
<li>Chacon, S. C., Freitas, L. H. T., Barbieri, F. S. Relationship between weight and number of engorged Amblyomma cooperi. Nuttal (sic) and Warburton, 1908 (Acari: Ixodidae) larvae and nymphs and eggs from experimental infestations on domestic rabbits. Brazilian Journal of Veterinary Parasitology. 13, 6-12 (2004).</li>
<li>Sonenshine, D. E. Maintenance of ticks in the laboratory. <a target="_blank" href="https://www.amazon.com/Maintenance-Human-Animal-Pathogen-Vectors/dp/1578080495">Maintenance of Human, Animal, and Plant Pathogen Vectors</a>. , Science Publishers Inc. Enfield. (1999).</li>
<li>Levin, M. L., Schumacher, L. B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Manual+for+maintenance+of+multi-host+ixodid+ticks+in+the+laboratory%5BTitle%5D)+AND+%22Experimental+and+Applied+Acarology%22%5BJournal%5D)">Manual for maintenance of multi-host ixodid ticks in the laboratory.</a> Experimental and Applied Acarology. 70 (3), 343-367 (2016).</li>
<li>Bouchard, K. R., et al. Maintenance and experimental infestation of ticks in the laboratory setting. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBouchard%2C+KR+%22Biology+of+Disease+Vectors%22">Biology of Disease Vectors</a>. , Elsevier. San Diego. (2005).</li>
<li>Jones, L. D., Davies, C. R., Steele, G. M., Nutall, P. A. <a target="_blank" href="https://eurekamag.com/research/001/714/001714602.php">The rearing and maintenance of ixodid and argasid ticks in the laboratory.</a> Animal Technology. 39, 99-106 (1988).</li>
<li>Slovák, M., Labuda, M., Marley, S. E. <a target="_blank" href="https://www.researchgate.net/publication/285818315_Mass_laboratory_rearing_of_Dermacentor_reticulatus_ticks_Acarina_Ixodidae">Mass laboratory rearing of Dermacentor reticulatus ticks (Acarina, Ixodidae).</a> Biologia, Bratislava. 57 (2), 261-266 (2002).</li>
<li>Rechav, Y., Dauth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+resistance+in+rabbits+to+immature+stages+of+the+Ixodid+tick+Rhipicephalus+appendiculatus%5BTitle%5D)+AND+%22Medical+and+Veterinary+Entomology%22%5BJournal%5D)">Development of resistance in rabbits to immature stages of the Ixodid tick Rhipicephalus appendiculatus.</a> Medical and Veterinary Entomology. 1, 177-183 (1987).</li>
<li>Zacarias do Amaral, M. A., Azevedo Prata, M. C., Daemon, E., Furlong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+parameters+of+cattle+ticks+fed+on+rabbits%5BTitle%5D)+AND+%22Brazilian+Journal+of+Veterinary+Parasitology%22%5BJournal%5D)">Biological parameters of cattle ticks fed on rabbits.</a> Brazilian Journal of Veterinary Parasitology. 21 (1), 22-27 (2012).</li>
</ol>

The authors have nothing to disclose.

The most important step in this entire protocol is to glue the capsule firmly to the shaved skin. For this reason, constant pressure for at least 3 minutes is critical, especially at the corners. When inoculating the ticks into the capsule, it is important to apply them deep into the opposite corner from the open one to avoid tick escape during the sealing. When planning the experiments, make sure that all the capsules are covered by the jacket to avoid damage by chewing or scratching. Make sure that the neck region of the jacket is tight enough to prevent chewing but sufficiently loose that the rabbit remains comfortable.
One of the main advantages of the described technique is its simplicity and that modifications in terms of size and number of the capsules can be used. During our experiments, detachment of capsules from skin were not observed. However, occasional damage of the jacket (but not the capsule) by the rabbit may occur.
In rabbits, we have observed that, in the capsule, fully engorged-detached ticks (mainly immature stages such as larvae and nymphs) desiccate faster due to the temperature of the rabbit. For this reason, we suggest to estimate&#160;the duration of the feeding period of particular tick species and stage, to plan engorged tick collection immediately after their detachment. Although our system has been tested for feeding of various hard tick species (Table 1), the rabbit is not a natural host for all hard tick species14. This limitation may be overcome by adapting this system to other animal hosts suitable for a particular hard tick species. Here we reported the use of the EVA foam system adapted to sheep to feed all the developmental stages of I. ricinus (Figure 3). In this particular case, the sheep needs to be shaved and washed with cotton impregnated with 70% ethanol to eliminate the oil present at the surface of the skin. Afterwards the same procedure as the one described for rabbits was followed, but instead of a jacket, a cotton bandage was used around the back as shown in Figure 3.
When developing this system, we paid special attention to minimize the amount of the materials used and steps of the procedure. Compared to other methods, we do not use anesthesia, rabbit collars, ear socks or hobbling of the rear legs5,6,7,8,9,10. In addition, the present protocol is not laborious, and no intensive training is required to become familiar with this technique. The EVA-foam based tick feeding system detailed in this study is expected to be used for a variety of different experiments when studying tick biology, host-vector-pathogen interactions, or evaluating different control measures like acaricides or vaccines. Our future direction will be adapting the EVA foam capsule feeding system to the mouse model to feed immature stages of hard ticks.

The hard ticks (Ixodidae) are well known as slow feeding arthropods and can be attached on a host for several days, or weeks, depending on the species and developmental stage1. These obligatory hematophagous arthropods are vectors of a wide variety of infectious agents, such as bacteria, protozoa, and viruses, and thus present a significant risk to humans and animal health1. When studying tick biology or evaluating new control methods, the establishment of an effective tick feeding system is crucial in order to effectively design the experiments and accomplish the goal(s) of the study. Recently, several artificial tick feeding systems (avoiding the use of live animals) have been developed2,3,4 and they should be used whenever possible. However, these systems have not been able to completely replace tick feeding on live animals, and they are not suitable substitutes for many physiologic conditions required for scientific studies. Therefore, in some cases, the use of experimental animal hosts is crucial to guarantee the relevance of experimental results.
Laboratory New Zealand rabbits have shown to be the most suitable and accessible hosts for several ixodid tick species5,6,7,8,9. Two common strategies of tick feeding on rabbits have been frequently used: a) feeding on rabbit ears covered with cotton cloth or socks6,7, and b) feeding in cotton bags9, nylon bottles10 or neoprene chambers11&#160;glued to the rabbit&#39;s back. The feeding on rabbit&#39;s ears is not an elegant system, because ticks (especially early stages, larvae or nymphs) may crawl and attach deep in the ear canal, which is uncomfortable for the animal and makes the monitoring of tick feeding and/or the recovery of engorged ticks difficult. This system is also limited to only two tick groups on ears fully covered by socks protected by Elizabethan collars,representing a significant discomfort for the animal. Other systems9,10,11 are definitely more advanced and well suited for hard tick colony maintenance. However, they are limited in the number of experimental groups placed on the rabbit as well as in the modifiable sizes/shapes of the feeding chambers. In addition, these protocols often require hobbling the rear rabbit legs to avoid scratching and the use of Elizabethan collars to prevent grooming.
Herein we propose a simple, non-laborious and very effective method to feed multiple groups of hard ticks in closed chambers glued to the rabbit back covered by a jacket, eliminating the need for Elizabethan collars or hobbling during the experiment. Specifically, our system uses elastic capsules made from an ethylene-vinyl acetate (EVA) foam sheet covered by mosquito mesh and glued to the shaved rabbit back with fast solidifying (3 min) non-irritating latex glue. This technique allows the attachment of multiple capsules of desired size or shape, and few weeks after the experiment the rabbits are fully recovered. The system is suitable mainly for the nymphal and adult hard tick stages, but with a little modification it can be used for larvae feeding as well. The EVA foam-based methods for hard tick feeding may be adapted to other types of vertebrate hosts, for example sheep (which is shown as one of the alternatives in this paper).

<table><tbody><tr><td>New Zealand Rabbits (2.5-3.5 kg)</td><td>Charles River&amp;nbsp;</td><td>Strain Code 571</td><td></td></tr><tr><td>Rambouillet sheep</td><td>Local provider-tick free farm</td><td>Female 6 months old</td><td></td></tr><tr><td>EVA foam 5 mm thick&amp;nbsp;</td><td>Cosplay Shop</td><td>EVA-PE451kg (950mm x 450mm)</td><td>10 mm PE45 kg foam from the Cosplay Shop may be used for the large adult tick species</td></tr><tr><td>Foam Sheet 9&quot; X 12&quot; 6 mm-White</td><td>Amazon</td><td>FOAMSHT6-20</td><td>6 mm-EVA foam ca be ordered via Amazon as an alternative to the foam from Cosplay Shop</td></tr><tr><td>Full length rabbit jackets&amp;nbsp;</td><td>Harvard Apparatus, Inc.&amp;nbsp;</td><td>620077- medium, 6270078 - large&amp;nbsp;</td><td></td></tr><tr><td>Non-toxic latex glue&amp;nbsp;</td><td>Tear mender&amp;nbsp;</td><td>Fabric &amp;amp; Leather Adhesive</td><td></td></tr><tr><td>Tubular cotton orthopedic stockinette</td><td>BSN Medical</td><td>9076 (12-15 cm wide)</td><td></td></tr><tr><td>Mosquito mesh&amp;nbsp;</td><td>Loisirs Creatifs</td><td>Very fine filter nylon mesh fabric</td><td>Any mosquito mesh, or curtain material with the mesh size less than 50 &amp;mu;m is suitable.</td></tr><tr><td>Leukoplast&amp;nbsp;</td><td>BSN medical S.A.S</td><td>LF 72361-02</td><td></td></tr><tr><td>Adhesive hook-and-loop tape</td><td>AIEX store</td><td>AIEX 39.37 Feet/12m Hook and Loop Self Adhesive Tape Roll, 20 mm width, white colour</td><td>Fullfiled by Amazon</td></tr><tr><td>Fast drying glue &amp;nbsp;</td><td>Fixtout&amp;nbsp;</td><td>Superglue</td><td></td></tr></tbody></table>

a versatile model of hard tick infestation on laboratory rabbits

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

Here we propose for the first time a detailed step-by step method of hard tick feeding in EVA foam capsules applied to a shaved rabbit's back, covered by a jacket (Figure 1 and Figure 2). This protocol is suitable for various types of experiments when different tick groups on the same host are required and can be also used for mass rearing of hard ticks. The tick feeding success in the laboratory mostly relies on the fitness of the tick individuals and suitability of the rabbit host for the particular tick species, rather than the technique itself. Our system using EVA-foam capsules glued to the rabbit back have been proven to be highly successful when feeding different developmental stages of various hard tick species (Table 1) and may also be adapted for other type of laboratory hosts, such as sheep (Figure 3).
The main advantages of this method are the simplicity, easily accessible materials (Table of Materials) and, most importantly, a comfortable open-close system allowing easy monitoring of the ticks during the feeding. In addition, this versatile method offers the possibility of a variety of different experimental settings based on the modifiable number, shape, and composition of the capsules on the host (Figure 2D-F), meeting the challenges of the particular study. The use of the highly effective, fast-drying, and non-irritating latex glue ensures that the capsule is firmly glued in three minutes and remains attached for at least three weeks. This procedure also allows complete recovery of the rabbit hosts after the experiments (Figure 2O).
<img alt="Assembly and use of a diffusion chamber for passive sampling, involving syringe and filter setup." class="xfigimg" src="/files/ftp_upload/57994/57994fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Preparation of the EVA foam capsule and ticks. (A, B) Cutting the capsule from the EVA foam. (C) Placing the strips of self-adhesive hook tape to the capsule. (D) Binding the loop side strips and peeling the tape from the fasteners. (E, F) Sticking the mosquito mesh to the adhesive strips. (G) Example of adult hard ticks inside the syringe (5 mL) with the cut needle-end covered by cotton. (H) Example of freshly hatching larvae inside the syringe with the cut needle-end covered by the mosquito mesh held in place by a rubber band. <a href="https://www.jove.com/files/ftp_upload/57994/57994fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Rabbit wound healing experiment; frames applied to shaved skin; process, observation sequence." class="xfigimg" src="/files/ftp_upload/57994/57994fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Gluing the capsule to the rabbit, tick infestation/recovering and capsule removal. (A) Shaved rabbit's back. (B) Application of the glue to the prepared EVA foam capsule. (C, D) Attachment of different size capsules to the rabbit. (E, F) Dorsal view of a rabbit showing two or several chambers attached to the back, respectively. (G) The tape is positioned around the rear feet. (H) The jacket is applied starting from the front legs and neck, and the rear part is left open. Opening the corner of the capsule is also shown. (I) Placing the ticks to the capsule via the open corner using the syringe. (J) The jacket is zipped completely and the rabbit is relocated to the cage. (K) Monitoring the ticks during their feeding by opening the capsule. (L) Collecting replete ticks using the forceps. (M) Empty capsule after tick removal. (N) Detachment of the capsule (after 3–4 weeks) from the rabbit. (O) Fully recovered rabbit <a href="https://www.jove.com/files/ftp_upload/57994/57994fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Sheep wound healing experiment; bandages on back, veterinary research methodology, animal model." class="xfigimg" src="/files/ftp_upload/57994/57994fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: EVA foam system adapted to sheep. (A) Attachment of the foam capsules to a shaved and cleaned area in the lateral side of the sheep back. (B) After infestation, the attached capsules are covered with a bandage (orthopedic stockinette), instead of a jacket. <a href="https://www.jove.com/files/ftp_upload/57994/57994fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Tick species</td>
			<td colspan="3">Number of infested ticks/Number of engorged ticks (%)</td>
		</tr>
		<tr>
			<td></td>
			<td>Larvae</td>
			<td>Nymphs</td>
			<td>Females</td>
		</tr>
		<tr>
			<td>Ixodes ricinus</td>
			<td>-</td>
			<td>692/557 (80.49%)</td>
			<td>670/592 (88.35%)</td>
		</tr>
		<tr>
			<td>Ixodes scapularis</td>
			<td>-</td>
			<td>-</td>
			<td>40/34 (85%)</td>
		</tr>
		<tr>
			<td>Dermacentor reticulatus</td>
			<td>3,550/3255 (91.7%)</td>
			<td>900/803 (89.22%)</td>
			<td>323/305 (94.42%)</td>
		</tr>
		<tr>
			<td>Rhipicephalus appendiculatus</td>
			<td>3,300/2822 (85.52%)</td>
			<td>490/421 (85.91%)</td>
			<td>370/362 (97.83%)</td>
		</tr>
		<tr>
			<td>Rhipicephalus pulchelus</td>
			<td>-</td>
			<td>1,920/1831 (95.36%)</td>
			<td>282/257 (91.13%)</td>
		</tr>
		<tr>
			<td>Amblyomma variegatum</td>
			<td>332/225 (67.77%)</td>
			<td>404/308 (76.24%)</td>
			<td>207/146 (70.53%)</td>
		</tr>
		<tr>
			<td>Amblyomma americanum</td>
			<td>-</td>
			<td>140/134 (95.71%)</td>
			<td>31/27 (87.1%)</td>
		</tr>
		<tr>
			<td>Hyalomma excavatum</td>
			<td>1,000*</td>
			<td>510* (58.8%)</td>
			<td>380/313 (82.36%)</td>
		</tr>
	</tbody>
</table>
Table 1: Engorgement rate of different developmental stages of hard tick species feeding on rabbits inside EVA-foam capsules. *Due to the fast molting process of H. excavatum immature stages, the engorged larvae (data not shown) were left to molt into nymphs that subsequently engorged in the same capsule.

Watch this Scientific Journal Video about A Versatile Model of Hard Tick Infestation on Laboratory Rabbits at JoVE.com

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

We have developed a simple and versatile system to feed hard ticks on laboratory rabbits. Our non-laborious protocol uses easily accessible materials and can be adjusted depending on the requirements of the various experimental settings. The method allows comfortable monitoring and/or sampling of ticks during the entire feeding period.

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the ...

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Biology

11.0K Views.  Université Paris-Est. We have developed a simple and versatile system to feed hard ticks on laboratory rabbits. Our non-laborious protocol uses easily accessible materials and can be adjusted depending on the requirements of the various experimental settings. The method allows comfortable monitoring and/or sampling of ticks during the entire feeding period.

Video: A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

Immunology and Infection

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

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

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.

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.

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 ...

Human Exposure to Larval Ticks for Tickborne Disease Research

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick must remain attached to the skin of the host and complete its multi-day feeding process to acquire its blood meal. Exposing animals to ticks is a common practice for studying host responses to tick bites and tickborne diseases. We developed the procedure, conducted the first human research study, and published the findings on exposing human volunteers to uninfected larval Ixodes scapularis ticks. This article describes the methodology used to construct the containment dressing, how to apply and secure the ticks to the host, how to maintain the dressing, and how to remove the ticks from the host. Exposing volunteers to tick bites is an experimental procedure and must be performed under a clinical research protocol approved by the appropriate regulatory authorities. This method allows for translational research to better understand the human response to tick bites and foster the development of diagnostics, prevention, and therapies for tickborne diseases.

Author Spotlight: Controlled Human Exposure Model for Tick Research and Lyme Disease Studies

This article presents the methodology for exposing humans to larval Ixodes scapularis for clinical research. The technique is relatively simple, tolerable by the research volunteers, and can be modified according to experimental needs. Such research involving human subjects must be conducted under clinical study protocols approved by the appropriate regulatory authorities.

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick ...

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Tick Artificial Membrane Feeding for Ixodes scapularis

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ticks during both study and rearing can limit experimental questions or the ability of labs to research ticks and their associated pathogens. An artificial membrane feeding system can reduce these problems and open up new avenues of research that may not have been possible with traditional animal feeding systems. This study describes an artificial membrane feeding system that has been refined for feeding and engorgement success for all Ixodes scapularis life stages. Moreover, the artificial membrane feeding system described in this study can be modified for use with other tick species through simple refinement of the desired membrane thickness. The benefits of an artificial membrane feeding system are counterbalanced by the labor intensiveness of the system, the additional environmental factors that may impact feeding success, and the need to refine the technique for each new species and life stage of ticks.

Tick Artificial Membrane Feeding for Ixodes scapularis

Presented here is a method to blood feed ticks in vitro via an artificial membrane system to allow for partial or full engorgement of a variety of tick life stages.

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ...

Generating Rabbit Model of Bone Infection: A Technique of Inducing Chronic Infection by Injecting Bacteria Directly in Bone Marrow

Source: Zhang, Y et al., <a href="https://www.jove.com/v/57294">Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection.</a> J. Vis. Exp. (2019).
This video describes the technique of surgically inducing bacterial infection in the bone marrow of a rabbit by using bone wax to avoid the leakage of bacteria. This rabbit model can be used to study bone infection treatment and bone regeneration.

Source: Zhang, Y et al., Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection. J. Vis. Exp. ...

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

Summary

Explore More Videos

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

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

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Tick Artificial Membrane Feeding for Ixodes scapularis

Generating Rabbit Model of Bone Infection: A Technique of Inducing Chronic Infection by Injecting Bacteria Directly in Bone Marrow