This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. We thank Linden T. Hu, Sam R. Telford III, Kenneth Dardick, Carla Williams, Erin Chung, and Christina Brandeburg for their participation in the development of the procedures.

Exposing volunteers to tick bites is an experimental method and must be conducted under a clinical research protocol approved by the relevant regulatory authorities. The clinical studies (NCT01143558, NCT02446626, and NCT05036707) were approved by the respective institutional review boards, conducted under investigational device exemptions granted by the US Food and Drug Administration, and carried out in accordance with Good Clinical Practice guidelines. Additionally, these studies were registered with ClinicalTrials.gov, and written informed consent was obtained from all participants.
1. Containment dressing preparation
<ol>
	<li>To create a measurement template, photocopy the 3&#34; x 3&#34; hydrocolloid containment dressing along the side of the measurement lines (Figure 1A and Table of Materials).</li>
	<li>Cut the photocopied template along the 1&#34; line. Then, cut along the 2&#34; line, creating a 2&#34; circle with a 1&#34; center (Figure 1B).</li>
	<li>Using this template, cut the non-adhesive foam dressing to create a 2&#34; circle with a 1&#34; center (Figure 2A,B, and Table of Materials).</li>
	<li>Using the same template, cut a 2&#34; circle in the center of the 4&#34; x 4&#34; extra-thin hydrocolloid dressing (Figure 2C,D).</li>
	<li>Cut the hydrocolloid layer of the 3&#34; x 3&#34; containment dressing at the 1&#189;&#34; line.</li>
	<li>Pull back the paper liner of the hydrocolloid layer of the containment dressing to expose the adhesive. Place the cut foam dressing within the 2&#34; circle of the adhesive (Figure 3A). This will ensure that the foam dressing extends beyond the opening of the containment dressing. Replace the paper liner onto the hydrocolloid layer of the dressing&#160;(Figure 3B). 
	&#8203;NOTE: These measurements were used for studies involving up to 10 larval I. scapularis ticks. For studies involving up to 30 larval I. scapularis ticks, the template for the foam center was cut between the 1&#34; and 1&#189;&#34; markings to allow for a larger area for placement. The measurements of the containment dressing can be adapted accordingly to the study design.</li>
</ol>
2. Tick placement
<ol>
	<li>Tick storage
	<ol>
		<li>Store the larval I. scapularis ticks in a medical-grade refrigerator with the temperature set to 9-10 &#176;C.</li>
		<li>Store the ticks in a vial covered with a mesh cap to allow adequate airflow. Package the vial of ticks in a sealable plastic bag with a lightly moistened sponge to maintain humidity and prevent desiccation.</li>
	</ol>
	</li>
	<li>Placing the ticks
	<ol>
		<li>Remove the ticks from the refrigerator to allow them to warm to room temperature. The ticks can become active in as little as 15 min outside the refrigerator.</li>
		<li>Position a white sheet around the placement area to easily visualize any inadvertently dropped tick.</li>
		<li>Dampen a 4&#34;x 4&#34; gauze pad with clean water or saline and use it to clean the skin of the placement site. The area should be patted dry or allowed to air dry. 
		NOTE: When selecting the body site for tick placement, consider areas that do not restrict the volunteer&#39;s daily activities and where the integrity of the dressing can be easily monitored by the participant. Do not place ticks in areas where they could be crushed due to direct pressure (e.g., the back). Evaluate the participant&#39;s body hair, and if necessary, carefully trim excessive body hair using scissors at the site of the adhesive, avoiding damage to the skin. Avoid using a razor for hair removal, as it may increase the risk of skin injury and infection.</li>
		<li>Once the skin is dry, remove the adhesive liner from the hydrocolloid side of the prepared dressing and firmly affix it to the skin at the placement site, ensuring that the mesh layer remains open.</li>
		<li>Break the wooden swab (see Table of Materials) in half to expose jagged ends.</li>
		<li>Carefully open the vial of ticks.</li>
		<li>Using the jagged end of the wooden swab, transfer active ticks from the vial and place them onto the skin within the containment dressing (Figure 4A). Have an additional team member monitor the tick placement site to ensure the ticks remain inside the containment dressing. Use adhesive tape to trap ticks that escape from the vial during this process. 
		NOTE: Breaking a wooden swab and using the jagged end is effective for tick manipulation and transfer without causing damage to the ticks. A small brush with soft bristles in a light color also works for transferring ticks from the container to the skin site.</li>
		<li>Once the specified number of ticks for the study is placed, remove the adhesive liner from the mesh layer of the containment dressing and close it over the opening.</li>
		<li>After ensuring that the adhesive is securely attached, apply the prepared 4&#34; x 4&#34; extra-thin hydrocolloid dressing over the containment dressing for additional security (Figure 4B). 
		&#8203;NOTE: Additional information regarding the pathogen-free I. scapularis tick colony and the duration of the tick exposure procedures used in the cited studies can be found in the methods sections of the published studies19,22.</li>
	</ol>
	</li>
</ol>
3. Site care
<ol>
	<li>Provide a water barrier cover and 2&#34; hypoallergenic tape (see Table of Materials) to the research participant.</li>
	<li>Provide instructions for caring for the tick containment site.
	<ol>
		<li>Periodically check the dressing and monitor the edges for secure adhesion to the skin. Apply additional tape to reinforce the dressing edges as needed.</li>
		<li>Do not scratch the dressing site, and refrain from opening or removing the dressing during the study period.</li>
		<li>Avoid bathing or soaking in water. Also, avoid hot showers and limit the duration of showering. Before showering, protect the containment dressing with the provided water barrier cover and remove it after completing the shower. Gently pat the dressing dry, and inspect it to ensure it remains secure.</li>
		<li>Avoid activities that may lead to heavy perspiration and compromise the dressing&#39;s integrity (e.g., aerobic exercise, extended hikes).</li>
	</ol>
	</li>
</ol>
4. Tick removal
<ol>
	<li>Collection vials preparation
	<ol>
		<li>Label the tick collection vials with the desired identifiers (e.g., study code, date).</li>
		<li>If preservation of live ticks is necessary, puncture holes in the cap of the vials with a 20 G needle. Use a layer of nylon or polyester mesh along with the cap to seal the vial. Place a small piece of moist sponge in a sealable bag with the vial of ticks to maintain humidity.</li>
	</ol>
	</li>
	<li>Removing the ticks
	<ol>
		<li>Place white sheets around the bandage site and ensure a collection basin is within reach.</li>
		<li>Using alcohol wipes, carefully remove the bandage from the skin.</li>
		<li>Once fully removed, place the bandage in the collection basin and examine it for the presence of detached ticks.</li>
		<li>Assess the placement site for attached ticks. Ticks that have fed to repletion will detach (Figure 5A). For the remaining attached ticks, use fine-tip forceps for removal (Figure 5B).</li>
		<li>Clean the skin with alcohol wipes or soap and water.</li>
	</ol>
	</li>
</ol>

Human Exposure to Larval Ticks for Tickborne Disease Research

<ol>
	<li>Yeh, M. 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=Determining+the+duration+of+Ixodes+scapularis+(Acari:+Ixodidae)+attachment+to+tick-bite+victims.">Determining the duration of Ixodes scapularis (Acari: Ixodidae) attachment to tick-bite victims.</a> J Med Entomol. 32 (6), 853-858 (1995).</li><li>Piesman, J., Mather, T. N., Sinsky, R. J., Spielman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duration+of+tick+attachment+and+Borrelia+burgdorferi+transmission.">Duration of tick attachment and Borrelia burgdorferi transmission.</a> J Clin Microbiol. 25 (3), 557-558 (1987).</li><li>Vora, A., 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=Ticks+elicit+variable+fibrinogenolytic+activities+upon+feeding+on+hosts+with+different+immune+backgrounds.">Ticks elicit variable fibrinogenolytic activities upon feeding on hosts with different immune backgrounds.</a> Sci Rep. 7, 44593 (2017).</li><li>Narasimhan, 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=Immunity+against+Ixodes+scapularis+salivary+proteins+expressed+within+24+hours+of+attachment+thwarts+tick+feeding+and+impairs+Borrelia+transmission.">Immunity against Ixodes scapularis salivary proteins expressed within 24 hours of attachment thwarts tick feeding and impairs Borrelia transmission.</a> PLoS One. 2 (5), 451 (2007).</li><li>Nazario, 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=Prevention+of+Borrelia+burgdorferi+transmission+in+guinea+pigs+by+tick+immunity.">Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity.</a> Am J Trop Med Hyg. 58 (6), 780-785 (1998).</li><li>Krause, P. J., 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=Dermatologic+changes+induced+by+repeated+Ixodes+scapularis+bites+and+implications+for+prevention+of+tick-borne+infection.">Dermatologic changes induced by repeated Ixodes scapularis bites and implications for prevention of tick-borne infection.</a> Vector Borne Zoonotic Dis. 9 (6), 603-610 (2009).</li><li>Anderson, J. M., 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=Ticks,+Ixodes+scapularis,+feed+repeatedly+on+white-footed+mice+despite+strong+inflammatory+response:+an+expanding+paradigm+for+understanding+tick-host+interactions.">Ticks, Ixodes scapularis, feed repeatedly on white-footed mice despite strong inflammatory response: an expanding paradigm for understanding tick-host interactions.</a> Front Immunol. 8, 1784 (2017).</li><li>Rosenberg, R., 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=Vital+signs:+trends+in+reported+vectorborne+disease+cases+-+United+States+and+Territories,+2004-2016.">Vital signs: trends in reported vectorborne disease cases - United States and Territories, 2004-2016.</a> MMWR Morb Mortal Wkly Rep. 67 (17), 496-501 (2018).</li><li>Paules, C. I., Marston, H. D., Bloom, M. E., Fauci, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tickborne+diseases+-+confronting+a+growing+threat.">Tickborne diseases - confronting a growing threat.</a> N Engl J Med. 379 (8), 701-703 (2018).</li><li>Tardy, O., 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=Mechanistic+movement+models+to+predict+geographic+range+expansions+of+ticks+and+tick-borne+pathogens:+Case+studies+with+Ixodes+scapularis+and+Amblyomma+americanum+in+eastern+North+America.">Mechanistic movement models to predict geographic range expansions of ticks and tick-borne pathogens: Case studies with Ixodes scapularis and Amblyomma americanum in eastern North America.</a> Ticks Tick Borne Dis. 14 (4), 102161 (2023).</li><li>Molaei, G., Eisen, L. M., Price, K. J., Eisen, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Range+expansion+of+native+and+invasive+ticks:+a+looming+public+health+threat.">Range expansion of native and invasive ticks: a looming public health threat.</a> J Infect Dis. 226 (3), 370-373 (2022).</li><li>Marques, A. R., Strle, F., Wormser, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Lyme+disease+in+the+United+States+and+Europe.">Comparison of Lyme disease in the United States and Europe.</a> Emerg Infect Dis. 27 (8), 2017-2024 (2021).</li><li>Kugeler, K. J., Schwartz, A. M., Delorey, M. J., Mead, P. S., Hinckley, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+frequency+of+Lyme+disease+diagnoses,+United+States,+2010-2018.">Estimating the frequency of Lyme disease diagnoses, United States, 2010-2018.</a> Emerg Infect Dis. 27 (2), 616-619 (2021).</li><li>Schwartz, A. M., Kugeler, K. J., Nelson, C. A., Marx, G. E., Hinckley, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=use+of+commercial+claims+data+for+evaluating+trends+in+lyme+disease+diagnoses,+United+States,+2010-2018.">use of commercial claims data for evaluating trends in lyme disease diagnoses, United States, 2010-2018.</a> Emerg Infect Dis. 27 (2), 499-507 (2021).</li><li>Hook, S. A., 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=Economic+burden+of+reported+Lyme+disease+in+high-incidence+areas,+United+States.">Economic burden of reported Lyme disease in high-incidence areas, United States.</a> Emerg Infect Dis. 28 (6), 1170-1179 (2022).</li><li>Eisen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+species+infesting+humans+in+the+United+States.">Tick species infesting humans in the United States.</a> Ticks Tick Borne Dis. 13 (6), 102025 (2022).</li><li>Embers, M. 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=Variable+manifestations,+diverse+seroreactivity+and+post-treatment+persistence+in+non-human+primates+exposed+to+Borrelia+burgdorferi+by+tick+feeding.">Variable manifestations, diverse seroreactivity and post-treatment persistence in non-human primates exposed to Borrelia burgdorferi by tick feeding.</a> PLoS One. 12 (12), 0189071 (2017).</li><li>Hodzic, E., Imai, D., Feng, S., Barthold, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resurgence+of+persisting+non-cultivable+Borrelia+burgdorferi+following+antibiotic+treatment+in+mice.">Resurgence of persisting non-cultivable Borrelia burgdorferi following antibiotic treatment in mice.</a> PLoS One. 9 (1), 86907 (2014).</li><li>Marques, A., 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=Xenodiagnosis+to+detect+Borrelia+burgdorferi+infection:+a+first-in-human+study.">Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study.</a> Clin Infect Dis. 58 (7), 937-945 (2014).</li><li>Hodzic, E., Imai, D. M., Escobar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generality+of+post-antimicrobial+treatment+persistence+of+Borrelia+burgdorferi+strains+N40+and+B31+in+genetically+susceptible+and+resistant+mouse+strains.">Generality of post-antimicrobial treatment persistence of Borrelia burgdorferi strains N40 and B31 in genetically susceptible and resistant mouse strains.</a> Infect Immun. 87 (10), e00442 (2019).</li><li>Bockenstedt, L. K., Mao, J., Hodzic, E., Barthold, S. W., Fish, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+attenuated,+noninfectious+spirochetes+in+Borrelia+burgdorferi-infected+mice+after+antibiotic+treatment.">Detection of attenuated, noninfectious spirochetes in Borrelia burgdorferi-infected mice after antibiotic treatment.</a> J Infect Dis. 186 (10), 1430-1437 (2002).</li><li>Turk, S., Williams, C., Marques, A., Pal, U., Buyuktanir, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in Borrelia burgdorferi: Methods in Molecular Biology. , 337-346 (2018).</li><li>Hodzic, E., Feng, S., Holden, K., Freet, K. J., Barthold, S. 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Dr. Adriana Marques has a patent US 8,926,989 B2; and is an unpaid Scientific Advisor to the Global Lyme Alliance and to the American Lyme Disease Foundation. Siu Ping Turk and Aleah Eschman do not have an association that might pose a conflict of interest. The content of this publication does not necessarily reflect the views of or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

While animal studies involving exposures to ticks4,5,6,7,21 have been invaluable in increasing our understanding of host response to tickborne diseases and tick bites, these models have limitations in how well they predict the human host response. This model, which describes the methodology for exposing humans to tick bites in a controlled manner, can be easil...

Hard ticks (Ixodidae: Acari) are obligate blood-feeding ectoparasites that occur worldwide and are capable of transmitting a broad range of pathogens, including bacteria, viruses, and parasites, of major medical and veterinary importance. Ixodid ticks must remain attached to the host for days to complete a blood meal, and they have the capacity to stay attached to the skin while avoiding recognition, preventing local blood coagulation, and facilitating long-term feeding1,2,3. Animal studies have demonstrated that non-permissive hosts acquire resistance to tick bites with repeated tick exposures, which can lead to a decreased ability to transmit a pathogen, while ticks can repeatedly parasitize permissive hosts. Acquired tick resistance is dependent on the nature of the host immune response4,5,6,7.
Tickborne diseases are an increasing threat in the United States (US), with the number of reported cases more than doubling between 2004 and 20168,9. Due to climate change, the geographic ranges of different ticks continue to expand10,11. The leading tickborne diseases in the US include Lyme disease, anaplasmosis, ehrlichiosis, spotted fever rickettsiosis, babesiosis, tularemia, and Powassan virus disease8. Lyme disease, caused by infection with Borrelia burgdorferi sensu lato, is the most common tickborne disease in the US and Europe12. With approximately 476,000 individuals diagnosed with Lyme disease annually in the US, there is both a public health and economic burden to individuals and to society13,14,15.
Ixodes scapularis (the black-legged or deer tick) is the primary vector for Lyme disease, as well as anaplasmosis, babesiosis, Borrelia miyamotoi disease, and Powassan virus disease in the US. Other medically important tick species in the US include Amblyomma americanum (Lone star tick), Dermacentor variabilis (American dog tick), Ixodes pacificus (Western black-legged tick), Dermacentor andersoni (Rocky Mountain wood tick), Ixodes cookei (Groundhog tick), Dermacentor occidentalis (Pacific Coast tick), Rhipicephalus sanguineus (brown dog tick), and Amblyomma maculatum (Gulf Coast tick)16.
Developing a method to expose research volunteers to tick bites supports studies using the natural vector to search for evidence of infection, a procedure known as xenodiagnosis17,18,19,20,21, and to learn more about immunity induced by exposure to ticks, which can contribute to the discovery of an anti-tick vaccine5,6,7. The procedure described here was developed and used in the first human research study using laboratory-reared Ixodes scapularis larva for xenodiagnosis of B. burgdorferi infection after antibiotic therapy (NCT01143558), published in 201419. The system has been successfully used in a phase 2 study investigating whether a positive xenodiagnosis correlated with the persistence of symptoms after antibiotic treatment of Lyme disease (NCT02446626) and in a study exploring the host response to tick bites (NCT05036707).
This procedure protocol describes the process for creating the containment dressing, the tick placement procedure, and the tick removal procedure, as well as the site care needed to maintain the containment dressing. The details regarding the pathogen-free I. scapularis tick colony and tick exposure procedures used for the above-cited studies have been previously described19,22. This methodology offers a flexible research tool that can be adapted to studying different aspects of the human host response to tick bites, the effectiveness of tick prevention medications, as well as Lyme disease and other tickborne illnesses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 G needle</td>
			<td>Any brand</td>
			<td></td>
			<td>For puncturing the vial cap.</td>
		</tr>
		<tr>
			<td>3&#34; x 3&#34; containment dressing</td>
			<td>Monarch Labs Names</td>
			<td>LeFlap</td>
			<td>https://www.monarchlabs.com/ordering</td>
		</tr>
		<tr>
			<td>4&#34; x 4&#34; extra-thin hydrocolloid dressing</td>
			<td>ConvaTec</td>
			<td>DuoDerm</td>
			<td>https://www.convatec.com/products/advanced-wound-care/brand-names/pc-wound-duoderm-granluflex/duoderm-extra-thin-dressing/</td>
		</tr>
		<tr>
			<td>4&#34; x 4&#34; gauze</td>
			<td>Monarch Labs Names</td>
			<td></td>
			<td>For cleaning skin</td>
		</tr>
		<tr>
			<td>Clean water or saline</td>
			<td></td>
			<td></td>
			<td>For cleaning skin</td>
		</tr>
		<tr>
			<td>Moisture barrier (e.g. 7&#34; x 7&#34;)</td>
			<td>AquaGuard</td>
			<td>TIDI</td>
			<td>For showering, ttps://www.tidiproducts.com/product-listing/aquaguard-shower-cover-sheets&#160;</td>
		</tr>
		<tr>
			<td>Non-adhesive foam dressing</td>
			<td>Coloplast</td>
			<td>Biatain</td>
			<td>https://www.coloplast.us/biatain-non-adhesive-en-us.aspx</td>
		</tr>
		<tr>
			<td>Roll of 2&#34; hypoallergenic tape</td>
			<td>Monarch Labs Names</td>
			<td>Durapore</td>
			<td>For reinforcing containment dressing.</td>
		</tr>
		<tr>
			<td>Roll of adhesive tape</td>
			<td></td>
			<td></td>
			<td>For trapping ticks</td>
		</tr>
		<tr>
			<td>Vials for collection (e.g. cryovials)</td>
			<td>Ependorf</td>
			<td>ECC200</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The study demonstrated that the procedure is safe and well-tolerated, with the primary adverse event being mild itching at the site of the bites, observed in 58% of the procedures. There were no serious adverse events related to the procedure when using clean laboratory-reared larval I. scapularis ticks19. In the 43 procedures conducted, the mean percentage of recovery of attached ticks compared to placed ticks was 45% &#177; 27% (SD), with a median percentage of 40% (<strong class="xfig"...

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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.

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 ...

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Immunology and Infection

414 Views.  National Institutes of Health. 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.

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

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

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 ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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 ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

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

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Chapters in this video

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

RNA Interference in Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

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

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

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

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment