We acknowledge the technical assistance of Alain Bernier French National Institute of Agricultural Research (INRAE), and Oc&#233;ane Le Bidel (ANSES). The study was supported by the DIM One Health - R&#233;gion &#206;le-de-France (Acronym of the project: NeuroPaTick). The mice were purchased by ANSES. Dr. Jeffrey L. Blair is acknowledged for reviewing the earlier version of the manuscript.

Please note that this protocol can be only applied when all welfare and safety measures are met in the laboratory. This protocol received permission to use mice for tick feeding by the Ethics Committee for Animal ExperimentsComEth Anses/ENVA/UPEC, Permit Numbers E 94 046 08. For the endpoint, the animals were exposed to CO2 for 9 min in two phases of 4 and 5 min each one.
1. Preparation of the capsule
<ol>
	<li>Stick 2 mm thick EVA-foam and the adhesive double sticky foam together (Figure 1A).</li>
	<li>Using a 20 mm diameter leather hole punch, cut a circle from the sticked foam pieces. Then, using a 12 mm diameter hole punch, cut the interior to create the double foam circle (Figure 1B). 
	NOTE: The frame thickness of the capsule should be greater than 3 mm in size to guarantee sufficient surface for the gluing process to the host skin (see below).</li>
	<li>Peel the protective paper strip from the adhesive double sticky foam (Figure 1C) and attach a transparent circular plastic of 20 mm diameter (Figure 1D). 
	NOTE: If feeding larvae, do not remove the protective paper strip from the adhesive foam and directly move to the step 2 in the protocol. Glue the double foam ring, including protective paper strip to the mouse.</li>
	<li>Make a ~ 1 cm slit in the transparent plastic (Figure 1E).</li>
	<li>Create at least 10 small holes with an entomological pin (Figure 1F) to allow excessive moisture evaporation during the experiment. 
	NOTE: The capsule (Figure 1G) has a total height of 4 mm (2 mm EVA-foam together with 2 mm adhesive foam) and can be used to feed nymphs and larvae of all the hard tick species. The capsule size (Figure 1H) of 20 mm outer diameter is suitable for most of the mouse strains but can be modified if necessary.</li>
</ol>
2. Preparation of the mice before tick infestation
NOTE: In this study, 10 - 12 weeks old female experimental mice (strain C57BL/6 and BALB/cByJ) were maintained in standard cages with food and water offered ad libitum (Green line ventilated racks at -20 Pa) 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 experienced technicians for any abnormal skin reactions, health problems or complications.
<ol>
	<li>Anesthetize mouse with isoflurane in the induction chamber. Once anesthetized, place mouse to the manipulation pad and attach to a nose cone for the continuous isoflurane supply (Figure 2A). Monitor the breathing rate and reduce isoflurane level to ensure it is less than 80 breaths per minute. 
	NOTE: Prior to the manipulation, label the individual mouse by tattooing or radio-frequency identification chip if necessary. It is recommended to keep the individual mice in separate cages to avoid capsule damage by biting.</li>
	<li>Shave the anterior part of the mouse from behind the shoulder blades up to the area just behind the ears (Figure 2A). 
	NOTE: The shaved area should be greater than the capsule surface.</li>
	<li>Apply non-irritating latex glue to the entire EVA-foam site of the prepared capsule and wait for 1 min (Figure 2B).</li>
	<li>Glue the capsule to the mouse back by slight 3 min constant pressure with the finger(s) (Figure 2C), especially on the left and right side of the capsule. Slightly lift the capsule to visually check its attachment to the skin. If non-attached regions are found, apply more glue using a spatula and press for another 3 minutes.</li>
</ol>
3. Tick Infestation
<ol>
	<li>For nymph infestation, introduce the individual nymphs into the capsule via the cut made in step 1.4&#160;(Figure 2D). 
	NOTE: For Ixodes tick species a maximum of 20 nymphs is recommended per one capsule.</li>
	<li>Slightly squeeze the capsule from two sides to allow the transparent plastic to bend for easier introduction of individual nymphs using fine dissection forceps (Figure 2D). Push the individual nymphs via the cut inside the capsule. Once inside, turn the forceps in 90&#730; and pull out the forceps to deposit ticks inside the capsule.</li>
	<li>For larvae infestation remove the paper slip from the attached capsule (Figure 2E). Place the syringe, containing larvae (Figure 2F), directly inside the capsule and deposit ticks by pushing the syringe plunger. Gently turn the plunger towards the skin to remove the remaining larvae attached. 
	NOTE: Place larvae into a 1 mL syringe with cut end plugged by piece of cotton prior to the experiment.</li>
	<li>Once the larvae are deposited onto the skin, close the capsule by attaching the transparent plastic (Figure 2G).</li>
	<li>Apply the protective plastic band around the capsule (Figure 2H). 
	NOTE: The protective plastic band greatly improved the durability of the capsule for the entire duration of the experiment (Figure 2I,J). It is possible to attach two capsules to one individual mouse (Figure 2K). In this case, a minimum of 3 mm space between the capsules is required and the shaved area should be increased appropriately.</li>
	<li>Return the mice to the cage.</li>
</ol>
4. Collection of Ticks
<ol>
	<li>Anesthetize the mouse as in step 2.1 above.</li>
	<li>Make a cross shaped cut (Figure 3A) to the plastic with a scalpel. 
	NOTE: This cross shaped cut enables easy collection of engorged ticks or detachment of the feeding ticks if necessary.</li>
	<li>If needed, reclose the capsule by sticking an adhesive plastic patch to the transparent plastic (20 mm diameter, Figure 3B). 
	NOTE: If collection of ticks at multiple time points is desired, the same sticky plastic patch can be used. If the protocol requires, one may also euthanize the mouse, remove the capsule, and collect/detach the ticks (Figure 3C).</li>
</ol>
5. Recovery of the mice
<ol>
	<li>Keep the mice in cage for one additional week.</li>
	<li>Let the capsule detach naturally. 
	NOTE: In this case, it takes about 8-9 days for capsules to fall off. When the capsule is removed, it is important to check for abnormal reactions on the skin of the mice. In case of irritation apply an emollient lotion, although normally no treatment is required. If the ethical protocol allows, the recovered mice (Figure 3D) can be reused for another tick infestation or different experiment(s).</li>
</ol>

<ol>
	<li>Sonenshine, D. E., Roe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Ticks. , (2014).</li><li>Kr&#246;ber, T., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+feeding+assays+for+hard+ticks.">In vitro feeding assays for hard ticks.</a> Trends in Parasitology. 23 (9), 445-449 (2007).</li><li>Bonnet, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transstadial+and+transovarial+persistence+of+Babesia+divergens+DNA+in+Ixodes+ricinus+ticks+fed+on+infected+blood+in+a+new+skin-feeding+technique.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+artificial+infection+of+hard+ticks:+A+tool+for+the+analysis+of+tick-borne+pathogen+transmission.">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>Kohls, G. M., Galtsoff, P. S., Lutz, F. E., Welch, P. S., Needham, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+rearing+methods+with+special+reference+to+the+Rocky+Mountain+wood+tick,+Dermacentor+andersoni.">Tick rearing methods with special reference to the Rocky Mountain wood tick, Dermacentor andersoni.</a> Culture methods for invertebrate animals. , 246-256 (1937).</li><li>Faccini, J. L. H., Chacon, S. C., Labruna, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbits+(Oryctolagus+cuniculus)+as+experimental+hosts+for+Amblyomma+dubitatum.+Neumann+(Acari:+Ixodidae).">Rabbits (Oryctolagus cuniculus) as experimental hosts for Amblyomma dubitatum. Neumann (Acari: Ixodidae).</a> Arquivo Brasileiro de Medicina Veterin&#225;ria e Zootecnia. 58 (6), 1236-1239 (2006).</li><li>Chacon, S. C., Freitas, L. H. T., Barbieri, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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+rabbit.">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 rabbit.</a> Brazilian Journal of Veterinary Parasitology. 13, 6-12 (2004).</li><li>Sonenshine, D. E., Maramorsch, K., Mahmood, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+ticks+in+the+laboratory.">Maintenance of ticks in the laboratory.</a> Maintenance of Human, Animal, and Plant Pathogen Vectors. , 57-82 (1999).</li><li>Levin, M. L., Schumacher, L. B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+for+maintenance+of+multi-host+ixodid+ticks+in+the+laboratory.">Manual for maintenance of multi-host ixodid ticks in the laboratory.</a> Experimental and Applied Acarology. 70 (3), 343-367 (2016).</li><li>Almaz&#225;n, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protective+antigens+for+the+control+of+Ixodes+scapularis+infestations+using+cDNA+expression+library+immunization.">Identification of protective antigens for the control of Ixodes scapularis infestations using cDNA expression library immunization.</a> Vaccine. 21 (13-14), 1492-1501 (2003).</li><li>Banks, C. W., Oliver, J. H., Hopla, C. E., Dotson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+life+cycle+of+Ixodes+woodi.+(Acari:Ixodidae).">Laboratory life cycle of Ixodes woodi. (Acari:Ixodidae).</a> Journal of Medical Entomology. 35, 177-179 (1998).</li><li>Almaz&#225;n, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+three+Ixodes+scapularis+cDNAs+protective+against+tick+infestations.">Characterization of three Ixodes scapularis cDNAs protective against tick infestations.</a> Vaccine. 23 (35), 4403-4416 (2005).</li><li>Levin, M. L., Ross, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+different+isolates+of+Anaplasma+phagocytophilum+by+Ixodes+scapularis+from+a+model+animal.">Acquisition of different isolates of Anaplasma phagocytophilum by Ixodes scapularis from a model animal.</a> Vector Borne Zoonotic Diseases. 4 (1), 53-59 (2004).</li><li>Heinze, D. M., Wikel, S. K., Thangamani, S., Alarcon-Chaidez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+murine+cutaneous+response+during+initial+and+subsequent+infestations+with+Ixodes+scapularis+nymphs.">Transcriptional profiling of the murine cutaneous response during initial and subsequent infestations with Ixodes scapularis nymphs.</a> Parasites &amp; Vectors. 6 (5), 26 (2012).</li><li>Nuss, A. B., Mathew, M. G., Gulia-Nuss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing,+Ixodes+scapularis,+the+Black-legged+Tick:+Feeding+Immature+Stages+on+Mice.">Rearing, Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice.</a> Journal of Visualized Experiments. (123), e55286 (2017).</li><li>Wada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+ablation+of+basophils+in+mice+reveals+their+nonredundant+role+in+acquired+immunity+against+ticks.">Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks.</a> Journal of Clinical Investigation. 120 (8), 2867-2875 (2010).</li><li>Saito, T. B., Walker, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tick+Vector+Transmission+Model+of+Monocytotropic+Ehrlichiosis.">A Tick Vector Transmission Model of Monocytotropic Ehrlichiosis.</a> The Journal of Infectious Diseases. 212 (6), 968-977 (2015).</li><li>Boppana, V. D., Thangamani, S., Alarcon-Chaidez, F. J., Adler, A. J., Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+feeding+by+the+Rocky+Mountain+spotted+fever+vector,+Dermacentor+andersoni,+induces+interleukin-4+expression+by+cognate+antigen+responding+CD4++T+cells.">Blood feeding by the Rocky Mountain spotted fever vector, Dermacentor andersoni, induces interleukin-4 expression by cognate antigen responding CD4+ T cells.</a> Parasites &amp; Vectors. 2 (1), 47 (2009).</li><li>Gargili, A., Thangamani, S., Bente, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+laboratory+animal+hosts+on+the+life+cycle+of+Hyalomma+marginatum+and+implications+for+an+in+vivo+transmission+model+for+Crimean-Congo+hemorrhagic+fever+virus.">Influence of laboratory animal hosts on the life cycle of Hyalomma marginatum and implications for an in vivo transmission model for Crimean-Congo hemorrhagic fever virus.</a> Frontiers in Cell and Infection Microbiology. 20 (3), 39 (2013).</li><li>Almaz&#225;n, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Versatile+Model+of+Hard+Tick+Infestation+on+Laboratory+Rabbits.">A Versatile Model of Hard Tick Infestation on Laboratory Rabbits.</a> Journal of Visualized Experiments. (140), e57994 (2018).</li><li>Zhijun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+and+biological+characteristics+of+Dermacentor+silvarum+Olenev+(Acari:+Ixodidae)+under+field+conditions.">The life cycle and biological characteristics of Dermacentor silvarum Olenev (Acari: Ixodidae) under field conditions.</a> Veterinary Parasitology. 168 (3-4), 323-328 (2010).</li><li>Ahmed, B. M., Taha, K. M., El Hussein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+Hyalomma+anatolicum+Koch,+1844+(Acari:+Ixodidae)+fed+on+rabbits,+sheep+and+goats.">Life cycle of Hyalomma anatolicum Koch, 1844 (Acari: Ixodidae) fed on rabbits, sheep and goats.</a> Veterinary Parasitology. 177 (3-4), 353-358 (2011).</li><li>&#352;irok&#253;, P., Erhart, J., Petr&#382;elkov&#225;, K. J., Kamler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+tortoise+tick+Hyalomma+aegyptium+under+laboratory+conditions.">Life cycle of tortoise tick Hyalomma aegyptium under laboratory conditions.</a> Experimental and Applied Acarology. 54, 277-284 (2011).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+Haemaphysalis+doenitzi+(Acari:+Ixodidae)+under+laboratory+conditions+and+its+phylogeny+based+on+mitochondrial+16S+rDNA.">Life cycle of Haemaphysalis doenitzi (Acari: Ixodidae) under laboratory conditions and its phylogeny based on mitochondrial 16S rDNA.</a> Experimental and Applied Acarology. 56, 143-150 (2012).</li><li>Jin, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+Cycle+of+Dermacentor+everestianus+Hirst,+1926+(Acari:+Ixodidae)+under+Laboratory+Conditions.">Life Cycle of Dermacentor everestianus Hirst, 1926 (Acari: Ixodidae) under Laboratory Conditions.</a> Korean Journal of Parasitology. 55 (2), 193-196 (2017).</li><li>Labruna, M. B., Fugisaki, E. Y., Pinter, A., Duarte, J. M., Szab&#243;, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+and+host+specificity+of+Amblyomma+triste+(Acari:+Ixodidae)+under+laboratory+conditions.">Life cycle and host specificity of Amblyomma triste (Acari: Ixodidae) under laboratory conditions.</a> Experimental and Applied Acarology. 30 (4), 305-316 (2003).</li><li>Breuner, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+the+Asian+longhorned+tick,+Haemaphysalis+longicornis,+to+serve+as+an+experimental+vector+of+the+Lyme+disease+spirochete,+Borrelia+burgdorferi+sensu+stricto.">Failure of the Asian longhorned tick, Haemaphysalis longicornis, to serve as an experimental vector of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto.</a> Ticks Tick Borne Diseases. 11 (1), 101311 (2020).</li></ol>

The authors have nothing to disclose.

The most critical step in the protocol is firm gluing of the capsule to the mouse skin. Therefore, the latex glue should be homogenously applied to the entire EVA-foam surface of the capsule and constant pressure for 3 minutes should be applied, especially to the left and right side of the capsule. We also recommend placement of the capsule as far forward on the back as possible to avoid its removal by the mouse using its rear paws. In our experiments, only the adhesion of the EVA-foam and latex glue to the mouse skin ha...

Ticks are important vectors of several pathogens and represent a serious risk to animal and human health1. Setting up an effective feeding system is crucial when studying their biology, tick-host-pathogen interactions, or establishing effective control measures. Currently, several artificial feeding systems, which avoid the use of live animals are available for ticks2,3,4 and these should be utilized whenever experimental conditions allow. However, in various experimental settings these systems fail to appropriately mimic the specific physiologic features and the use of live animals is necessary to achieve relevant results.
Laboratory mice are commonly used for the study of many biological systems and are routinely utilized as hosts for feeding ticks5,6,7,8,9. The two most common methods of feeding immature ticks on mice include free infestations and the use of confinement chambers attached to the mouse. Free infestations are primarily used for larval stages and engorged ticks can drop to an area where they can be recovered. Confinement chambers are usually composed of acrylic or polypropylene caps which are glued to the mouse&#8217;s back. The first technique is an effective natural system for tick feeding but does not allow close monitoring during the experiment because the individual ticks are dispersed in different parts of the host body. Additionally, engorged ticks that drop to a recovery area can become contaminated with feces and urine10,11,12,13,14 that may severely affect the tick fitness or they can be damaged or eaten by the mouse if there is no separation between the animal and the recovery area15. Chamber-based systems allow the confinement of ticks to a defined area, however, the gluing process is laborious and the caps are often weakly adherent to the glue and thus they often detach during the experiment16,17,18,19. The caps are also stiff, uncomfortable, and lead to skin reactions, which prevent the re-use of the mice and necessitates their euthanasia after the experiment.
In our previous study, we successfully developed an effective system using chambers made of ethylene-vinyl acetate (EVA) foam for feeding ticks on laboratory rabbits20. Herein, we adapted this system to a mouse model and propose a simple and clean method to feed immature hard tick stages in closed capsules made from EVA-foam. Specifically, our system uses elastic EVA-foam capsules glued to the shaved mice back with fast drying (3 min), non-irritating latex glue. This technique allows firm and long-lasting attachment of capsules to the experimental mouse, as well as effective tick infestation/collection during the entire course of the experiment. The flat capsule is made from flexible materials and does not impede manipulation of the mouse for blood collection or other purposes. The system is suitable mainly for the nymphal tick stages, but with slight modification it can be used for feeding larvae as well. The method can be completed by one single experienced person and extensive training is not required.

<table>
	<tbody>
		<tr>
			<td>EVA-foam 2 mm thick, (low density)</td>
			<td>Cosplay Shop</td>
			<td>EVA-45kg (950/450/2 mm)</td>
			<td>It can be ordered also via Amazon (ref. no. B07BLMJDXD)</td>
		</tr>
		<tr>
			<td>Heat Shrink Tubing Electric Wire Wrap Sleeve</td>
			<td>Amazon</td>
			<td>B014GMT1AM</td>
			<td>Different diameters of Heat Shrink Tubing are available via Amazon.</td>
		</tr>
		<tr>
			<td>Mice BALB/cByJ</td>
			<td>Charles River</td>
			<td>Strain code 627</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice C57BL/6</td>
			<td>Charles River</td>
			<td>Strain code 664</td>
			<td></td>
		</tr>
		<tr>
			<td>No-toxic Latex Glue</td>
			<td>Tear mender</td>
			<td>Fabric &#38; Leather Adhesive</td>
			<td>Also available also via Amazon (ref. no. B001RQCTUU)</td>
		</tr>
		<tr>
			<td>Punch Tool Hand Art Tool</td>
			<td>Amazon</td>
			<td>B07QPWNGBF</td>
			<td>Saled by amazon as Leather Working Tools 1-25mm Round Steel Leather Craft Cutter Working for Belt Strap</td>
		</tr>
		<tr>
			<td>PVC Binding Covers Transparent</td>
			<td>Amazon</td>
			<td>B078BNLSNP</td>
			<td>Any transparent PVC sheet of ticknes between 0.150 mm to 0.180 mm is suitable</td>
		</tr>
		<tr>
			<td>Self Adhesive Pad Sponge Double Coated Foam Tape</td>
			<td>Amazon</td>
			<td>B07RHDZ35J</td>
			<td>Saled by amazon as 2 Rolls Double Sided Foam Tape, Super Strong White Mounting Tape Foam</td>
		</tr>
		<tr>
			<td>Transparent seal stickers (20 mm diameter circles)</td>
			<td>Amazon</td>
			<td>B01DAA6X66</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We propose the detailed step-by step method for feeding immature hard tick stages in EVA-foam capsules applied to a mouse&#8217;s back (Figure 2). This non-laborious protocol is suitable for various types of experiments when precise tick monitoring and collection is required. The main advantages of this method are its simplicity, easily accessible cost-effective materials, and short duration. In addition, we succeeded in attaching two capsules to one mouse individual (Fi...

Watch this Scientific Journal Video about A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice at JoVE.com

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

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 ...

a-capsule-based-model-for-immature-hard-tick-stages-infestation-on

Research

JoVE Journal

Biology

6.3K Views.  Université Paris-Est. 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.

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

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Immunology and Infection

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 ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Neuroscience

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 ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to 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 ...

Studying Strongyloides ratti Infection in Laboratory Mice

Experimental Infection of Mice with the Parasitic Nematode Strongyloides ratti

Strongyloides ratti is a parasitic nematode that naturally infects wild rats. However, most laboratory rat and mouse strains are fully susceptible to infection. Immunocompetent BALB/c and C57BL/6 mice terminate S. ratti infections within a month in the context of a canonical type 2 immune response and&#160;remain semi-resistant to a re-infection. The course of infection can be divided into three phases: (a) the tissue migration phase of the infective third-stage larvae during the first two days; (b) the early intestinal phase, including the molting to the adult parasites and embedding in the mucosa of the intestine in days 3 to 6 post-infection with reproduction starting by day 5 to 6 post-infection; (c) the later intestinal phase ending with the complete clearance of the parasites. Experimental infections of mice with S. ratti enable the precise study of host-parasite interactions throughout the whole life cycle at the different sites of infection, as well as immune evasion strategies employed by the parasite. The protocol presented here describes the maintenance of the parasite in Wistar rats, the infection of laboratory mice, and the detection and quantification of S. ratti parasites in the tissue migrating phase and during the intestinal phase.

Experimental Infection of Mice with the Parasitic Nematode Strongyloides ratti ratti Infection

Strongyloides ratti is a parasitic nematode that causes transient infections in laboratory mice, displaying tissue-migrating and intestinal life stages. Here, we present a protocol for the maintenance of the parasite cycle in rats and experimental infection of mice, including parasite quantification in the head, lung, and intestine.

Strongyloides ratti is a parasitic nematode that naturally infects wild rats. However, most laboratory rat and mouse strains are fully susceptible to ...

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.

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 ...

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 ...