The authors would like to thank the Division of Vector-Borne Diseases Animal Resources Branch, specifically Andrea Peterson, Lisa Massoudi, Verna O'Brien, and John Liddell for their care and maintenance of the mice and rabbits. We would also like to thank Amy Ullmann, Theresa Russell, and Barbara J. Johnson for their contributions toward this manuscript. Finally, we would like to acknowledge Alissa Eckert in the Office of the Associate Director for Communication at the CDC for producing the graphic illustrations and Judy Lavelle for directing all the legalities associated with the filming of this manuscript.

1. Hemolymph collection for slide preparation
(Movie 1)
<ol>
	<li>Gently remove actively feeding ticks from an animal and place in 3% topical hydrogen peroxide for 5 minutes and then in 70% ethanol for 10 minutes to surface sterilize.</li>
	<li>With a pap pen draw a circle onto a silane coated microscope slide and place the tick within the pap pen circle.
	<ol>
		<li>Silane coated slides are used for best adherence of the hemolymph to the microscope slide.</li>
	</ol>
	</li>
	<li>View the ti...

<ol>
	<li>Quach, K. A., Boctor, F. N., Elston, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+eating+you?+Hyalomma+ticks.">What's eating you? Hyalomma ticks.</a> Cutis. 87, 165-167 (2011).</li><li>Graham, J., Stockley, K., Goldman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick-borne+illnesses:+a+CME+update.">Tick-borne illnesses: a CME update.</a> Pediatr. Emerg. Care. 27, 141-147 (2011).</li><li>Nuttall, P. A., Paesen, G. C., Lawrie, C. H., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vector-host+interactions+in+disease+transmission.">Vector-host interactions in disease transmission.</a> J. Mol. Microbiol. 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M., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+migration+of+Borrelia+burgdorferi+in+Ixodes+ticks+during+blood+feeding.">Growth and migration of Borrelia burgdorferi in Ixodes ticks during blood feeding.</a> Am. J. Trop. Med. Hyg. 53, 397-404 (1995).</li><li>Horka, H., Cerna-Kyckova, K., Skallova, A., Kopecky, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+saliva+affects+both+proliferation+and+distribution+of+Borrelia+burgdorferi+spirochetes+in+mouse+organs+and+increases+transmission+of+spirochetes+to+ticks.">Tick saliva affects both proliferation and distribution of Borrelia burgdorferi spirochetes in mouse organs and increases transmission of spirochetes to ticks.</a> Int. J. Med. Microbiol. 299, 373-380 (2009).</li><li>Brossard, M., 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=Immunology+of+interactions+between+ticks+and+hosts.">Immunology of interactions between ticks and hosts.</a> Med. Vet. Entomol. 11, 270-276 (1997).</li><li>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=Tick+modulation+of+host+immunity:+an+important+factor+in+pathogen+transmission.">Tick modulation of host immunity: an important factor in pathogen transmission.</a> Int. J. Parasitol. 29 (99), 851-859 (1999).</li><li>Binnington, K. C., Kemp, 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=Role+of+tick+salivary+glands+in+feeding+and+disease+transmission.">Role of tick salivary glands in feeding and disease transmission.</a> Adv. 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We have nothing to disclose.

The collection of tick hemolymph, salivary glands, and saliva is important in the study of tick-borne pathogen transmission, prevalence, dissemination, proliferation, and persistence in both the tick and the host 6,11-13,20,23,29. There are several ways to dissect a tick30,31. However, when collecting salivary glands it is critical to dissect the tick properly so the salivary glands are not ruptured or lost in the tick's remains. Once the salivary glands are removed from the tick they need to be was...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide</td>
 <td>Fisher</td>
 <td>H312-500</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Acros </td>
 <td>61509-5000</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Boston Bioproducts</td>
 <td>BM-2205</td>
 </tr>
 <tr>
 <td>Dumont Fine forceps (3C)</td>
 <td>Fisher</td>
 <td>NC9906085</td>
 </tr>
 <tr>
 <td>Silane treated microscope slides</td>
 <td>Bioworld</td>
 <td>42763007-1</td>
 </tr>
 <tr>
 <td>Pap pen</td>
 <td>Bioworld</td>
 <td>21750008-1</td>
 </tr>
 <tr>
 <td>Super frost plus microscope slides</td>
 <td>Fisher</td>
 <td>12-550-18</td>
 </tr>
 <tr>
 <td>Pilocarpine</td>
 <td>Sigma</td>
 <td>P6503-5G</td>
 </tr>
 <tr>
 <td>Protease inhibitor cocktail</td>
 <td>Sigma</td>
 <td>P2714</td>
 </tr>
 <tr>
 <td>#11 disposable scalpel</td>
 <td>Feather</td>
 <td>2975#11</td>
 </tr>
 <tr>
 <td>Nontoxic modeling clay</td>
 <td>Fisher</td>
 <td>S17307</td>
 </tr>
 <tr>
 <td>Capillary tubes</td>
 <td>Chase scientific Glass, inc </td>
 <td>40A502</td>
 </tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks at JoVE.com

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.

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 ...

saliva-salivary-gland-hemolymph-collection-from-ixodes-scapularis

Research

JoVE Journal

Immunology and Infection

29.2K vistas.  Centers for Disease Control and Prevention. La colección de la hemolinfa garrapata infectada, las glándulas salivales y la saliva es importante para estudiar la forma transmitida por garrapatas patógenos causan enfermedades. En este protocolo se demuestra cómo recoger las glándulas salivales y la hemolinfa de la alimentación Ixodes scapularis Ninfas. También demuestran la toma de saliva de la mujer I. scapularis Adultos.