Name: a versatile model of hard tick infestation on laboratory rabbits
Uploaded: 2018-10-06T15:00:00
Description: Watch this Scientific Journal Video about A Versatile Model of Hard Tick Infestation on Laboratory Rabbits at JoVE.com

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...

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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 t...

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 border="0" cellpadding="0" cellspacing="0" style="width:1157px;" width="1158">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">New Zealand Rabbits (2.5-3.5 kg)</td>
			<td style="width:197px;">Charles River&#160;</td>
			<td style="width:399px;">Strain Code 571</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Rambouillet sheep</td>
			<td style="width:197px;">Local provider-tick free farm</td>
			<td>Female 6 months old</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EVA foam 5 mm thick&#160;</td>
			<td style="width:197px;">Cosplay Shop</td>
			<td style="width:399px;">EVA-PE451kg (950mm x 450mm)</td>
			<td>10 mm PE45kg foam from the Cosplay Shop may be used for the large adult tick species</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Foam Sheet 9&#34; X 12&#34; 6 mm-White</td>
			<td style="width:197px;">Amazon</td>
			<td style="width:399px;">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 height="21">
			<td height="21" style="height:21px;width:295px;">Full length rabbit jackets&#160;</td>
			<td style="width:197px;">Harvard Apparatus, Inc.&#160;</td>
			<td style="width:399px;">620077- medium, 6270078 - large&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Non-toxic latex glue&#160;</td>
			<td style="width:197px;">Tear mender&#160;</td>
			<td style="width:399px;">Fabric &#38; Leather Adhesive</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tubular cotton orthopedic stockinette</td>
			<td style="width:197px;">BSN Medical</td>
			<td style="width:399px;">9076 (12-15 cm wide)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mosquito mesh&#160;</td>
			<td style="width:197px;">Loisirs Creatifs</td>
			<td style="width:399px;">Very fine filter nylon mesh fabric</td>
			<td>Any mosquito mesh, or curtain material with the mesh size less than 50 microns is suitable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Leukoplast&#160;</td>
			<td style="width:197px;">BSN medical S.A.S</td>
			<td style="width:399px;">LF 72361-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Adhesive hook-and-loop tape</td>
			<td style="width:197px;">AIEX store</td>
			<td style="width:399px;">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 height="21">
			<td height="21" style="height:21px;">Fast drying glue &#160;</td>
			<td>Fixtout&#160;</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&#39;s back, covered by a jacket (Figure&#160;1 and&#160;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 mo...

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 ...

This method is suitable for maintaining hard colonies, studying tick biology or host-vector pathogen interactions or evaluating different control measures of these medically important attributes. The main advantages of these techniques are its simplicity and short duration. and most importantly ability to be modified, according to the experimental needs of the study.Although this method can be used for feeding the different life stages of hard ticks on laboratory rabbits. It can also be adapted to other host systems such as sheep. Begin by cutting an appropriately-sized capsule frame from a sheet of EVA Foam rounding the outer corners to minimize accidental detachment during gluing.Cut four eight millimeter wide strips of self-adhesive hook tape and attach the strips to the sides of the frame. Cut the same size strips from the self-adhesive loop tape and bind them to the hook tape on the frame. Next, cut a piece of pine mosquito mesh big enough to cover the frame and stick the mesh to the self-adhesive loop strips, trimming any overhanging material as necessary.When the capsule is ready, manually restrain the rabbit and shave the experimental regions on the back and sides of the animal. Apply fast-drying non-irritating latex glue to the entire surface of the prepared capsule and after one minute, gently pressed the capsule to the skin. The most critical step of the protocol is the field attachment of the capsule to the skin.Therefore, constant pressure of the capsule onto the rabbit skin especially at the corners for at least three minutes is required. After three minutes, slightly lift the capsule to visually confirm its attachment using a spatula to apply glue to any non-attached parts. When the capsule is secured, apply protective tape to the rear paws to prevent jacket damage and place the front legs through the front jacket openings tightening the neck of the jacket to one finger width between the animal and the jacket.To initiate the tick infestation, first add the ticks into the plunger end of a plastic cotton plug to syringe with a cut-off tip. Next, lift the mesh from one corner of the capsule and place the syringe deep into the capsule through the opening. Insert the plunger all the way into the syringe to infest the animal with the ticks.Then slowly twist the plunger toward the skin to remove any remaining ticks while simultaneously pulling the plunger out of the capsule. When all the ticks have been delivered, refasten the hook and loop tape. Close the jacket by zipping the backside of it and insert the rear legs of the rabbit into the rear elastic enclosures.Then return the rabbit to its cage. At the appropriate experimental time point, transfer the rabbit to the bench and unzip the jacket. Gently restraining the rabbit, open the capsule and use forceps to collect the fully engorged adults and a tick twister to collect the partially fed adults.Engorged larvae and nymphs can be collected by brushing them into the dish. Then re-close the capsule if experimentally appropriate. At the end of the experiment, remove the mosquito mesh completely from the capsule without removing the jacket.Three to four weeks later, gently trim one of the corners to try to remove the capsule. Once the capsule has been removed, remove the jacket and allow the rabbit to recover in its cage. If the protocol and experiments allow, the recovered rabbit can be reused or offered for adoption.This protocol is suitable for various types of experiments including mass rearing of multiple tick groups on the same host. The EVA Foam capsule system has been proven to be highly successful when feeding different developmental stages of various hard tick species. It may also be adapted for other type of laboratory hosts such as sheep.In addition, this versatile method facilitates the creation of a variety of different experimental settings based on the modifiable number, shape and composition of the capsules on the host. The procedure also allows a complete recovery of the rabbit hosts after the experiments. This protocol is not laborious and no intensive training is required to become familiar with the technique.In addition, a minimal amount of materials, steps and stress to the experimental animals is required.

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