The authors wish to thank Nicole Hasenkampf and Amanda Tardo for technical support. We also thank Drs. Linden Hu and Adriana Marques for recommendation of the LeFlap containment device, and Dr. Lise Gern for instruction on the capillary feeding method. This work was supported by NIH/NCRR Grant 8 P20 GM103458-09 (MEE) and by the National Center for Research Resources and the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health through grant P51OD011104/P51RR000164.

An experimental outline of tick inoculation and feeding upon animals for Lyme disease research is depicted in Figure 1.
1. Inoculating Nymphal Ixodes Ticks with B. burgdorferi Using Capillary Tube-feeding
When performing manipulations with ticks, white lab coats with elastic sleeves, gloves, and disposable bouffant caps are worn.
<ol>
	<li>Our technique is a modified version of that reported by Broadwater et al. 49. Prepare capillary tubes by heating and pulling Pasteur pipets to breaking thinness using a pipet puller. Using forceps and dissecting scope, break the tips to the optimal diameter (about 0.2 mm). A tube standardized to the tick mouthpart size is used as a sizing guide. A face shield should be worn when preparing the pipets.</li>
	<li>Grow B. burgdorferi to between 2-8 x 107/ml (mid-log phase) in BSK-H medium (Sigma) containing 6% rabbit serum.</li>
	<li>Use nymphal ticks that have been stored at 23 &deg;C for 4-6 weeks post-larval molt. Place ticks onto a small 60 x 15 mm petri dish with double-sided tape on the outer bottom surface of the dish. Place the ticks ventral-side facing up.</li>
	<li>Dip the capillary tube tip into the B. burgdorferi culture tube after mixing. Place the capillary tube over the hypostome of the tick mouth parts using a dissecting scope. Use molding clay to fix the tube in place, as shown in Figure 2A.</li>
	<li>Place the petri dishes with affixed ticks inside a large clear plastic tub for an added level of containment. Wet paper towels are added to provide moisture. Place the ticks in a 37 &deg;C thermal incubator for 30 min-2 hr until defecation is apparent. This indicates that the media containing spirochetes has passed through the tick.</li>
	<li>Rest the ticks for 2-4 weeks at 23 &deg;C to allow adaptation to the tick environment before feeding them on animals.</li>
</ol>
2. Infecting Mice with B. burgdorferi by Tick
<ol>
	<li>Dilute ketamine stock 1:10 in sterile water. Anesthetize each mouse with 100 mg/kg of ketamine by intraperitoneal injection with a tuberculin syringe</li>
	<li>Once the mouse is fully anesthetized, shave the mouse from the ears to the middle back using a fine (Remington smooth &amp; silky) electric trimmer.</li>
	<li>In a white pan with no other objects nearby, transfer the nymphal ticks (that harbor B. burgdorferi) by a moistened paintbrush to the hairless area of the mouse. Alternatively, uninfected ticks can be placed on mice for xenodiagnosis of mice with suspect infection. The use of a clean, white surface for tick placement helps to ensure that any unattached ticks will be readily seen.</li>
	<li>Place the mouse in specialized caging (Allentown Caging, Allentown, PA). The caging consists of a stainless steel grill elevated from the bottom of the cage. The cage top was modified by our in-house machine shop to elevate the water bottle holder enough to allow free movement of the mouse underneath. The pan is filled with about &frac12; inch of water to trap any ticks that fall off mice (Figure 3A). To minimize the risk of hypothermia, reusable heating pads, microwaved prior to use, are placed underneath the cages until mice wake completely from anesthesia. Animals are often ataxic as they recover from anesthesia and rub against food and water trays, so these must be removed. The water level is low enough to prevent limbs of mice from submersion.</li>
	<li>Place the cage inside a tray that has been lined with Tangle trap paste (Contech, Victoria, BC, Canada) and tape to ensure the entrapment of arthropods. Mice are caged singly and observed continuously during the period of anesthesia.</li>
	<li>Within 2 hr, when mice are completely awake from the anesthesia, the food tray and water bottle are replaced to the cage. After 24 hr, the housing enrichment consisting of a plastic hut and nylabone is replaced.</li>
	<li>After 3, 4, and 5 days, check the mouse, cage and cage water for fed ticks. The cage water is sifted through a white metal pan (i.e. "panning for gold"). Rinse fed ticks in clean water and store in plastic jars (Figure 3B). On days 3 and 4, replace water in cage with clean water. On day 5, check not only the cage, but the mouse thoroughly for ticks. Usually by this point all ticks have fed and the mice can be returned to regular caging.</li>
	<li>Place all waste from the mouse cages, including liquids, in biohazard containers for autoclaving and disposal. Keep a log of the number of ticks placed on mice and those recovered at all times.</li>
</ol>
3. Feeding Ticks on Nonhuman Primates for Infection with B. burgdorferi or xenodiagnosis
<ol>
	<li>Prepare the tick containment device: Cut a 1 &frac34; inch diameter circle in the 3 inch x 3 inch LeFlap (flap) using a clean scalpel and the measurement guide. Use the cut-out as a template to cut circles of identical size in the Biatane foam and Duoderm. The foam is used to elevate flap over the surface of the skin and prevent possible crushing of tick. The Duoderm adds another layer of cushioning and overlays the edges of the containment device for added security from tick escape. A diagram of the containment device is depicted in Figure 4.</li>
	<li>Veterinary staff will anesthetize the animal with 5-8 mg/kg Telazol by intramuscular injection.</li>
	<li>Clip the hair of the animal using electric trimmer (Oster) equipped with size 40 blades. All areas that will be covered by the jacket are clipped: back, front, upper arms. Using shaving cream and dual-blade disposable razors, closely shave an area of approximately 25 cm vertical x 20 cm horizontal. Wipe clean with moist paper towels and blow dry with low heat to dry the skin.</li>
	<li>Place the flap on the animal's dorsum, just below the scapula, on either side of the spine. Use a marker to trace the circle in that spot. Prepare the area of skin around the circle by wiping it with SkinPrep. This removes oil in skin that could affect adhesion of glue and containment devices. Leaving about a 1 cm circumference of space around the circle, apply a layer of skin glue (SkinBond) with a width of ~4 cm.</li>
	<li>Remove the adhesive backing from the Biatane foam and affix to the skin in the appropriate spot. Animals are again anesthetized by veterinary staff with 5 mg/kg Telazol. Seal the edges with skin glue and Hypafix tape. Remove the adhesive backing from the flap and affix on top of the Biatane. Place Hypafix tape around the edges of the LeFlap, then tape down the mesh flap of the flap and place the jacket on the animal. Tape and Tangle Trap paste is applied to the floor in a perimeter surrounding the nonhuman primate caging for added security.</li>
	<li>To minimize the effects of chemicals used in Step 3.4 on tick feeding, the ticks are added 24 hr after the containment device is in place. At this point, the security of the device is also inspected and reinforced if needed. Typically, 20 unfed nymphs (4-8 weeks post-larval molt) are added to the skin within the device using a paintbrush.</li>
	<li>Remove the adhesive backing from the mesh of the flap and seal it in place. Lastly, remove the Duoderm backing to expose adhesive, and place on top of the containment device. Add a piece of Hypafix tape across the open mesh circle, and replace the jacket. The completed containment device is shown in Figure 5A.</li>
	<li>After 5 days, anesthetize the animals as above and the jackets are removed. Remove the tape first to inspect tick feeding through the mesh (Figure 5B). Carefully peel the Duoderm away from the flap.</li>
	<li>Pull back the mesh portion at the edges to provide access to the ticks. Fed ticks are frequently found near or under the foam circle (Figure 5C) and are removed and placed in clean water with a paintbrush. Remove the device once all visible fed ticks are collected (Figure 5D).</li>
</ol>
Note: Oftentimes, the containment device can simply be peeled away from the skin. If adhesion is strong and could potentially damage the skin, Unisolve solvent is applied to the area for gentle removal. The skin is wiped with isopropanol and ticks are stored at 23 &deg;C. If used for infection, the ticks can be crushed to confirm the number that contained B. burgdorferi. If used for xenodiagnosis, the ticks are kept for 1-3 weeks prior to analysis of midgut contents.

The authors declare that they have no competing financial interests.

In order to obtain ticks that harbor B. burgdorferi for downstream studies, the ticks can be: (1) fed on infected mice at the larval stage; (2) immersed in B. burgdorferi cultures at either the larval or nymphal stage 48; (3) microinjected with B. burgdorferi 47; or (4) capillary tube-fed B. burgdorferi 49. While each of these methods has its purpose, for ensuring that a large portion of the ticks to be used for infection harbor B. burgdorferi, we favor capillary tube feeding. If inoculation with known quantities of spirochetes is not required, the capillary-feeding methods may have less potential to damage the ticks. This is of importance if they are to be fed on animals. This method may also be preferred if the investigators are testing mutants for transmissibility/infectivity. It is important to recognize that growth in culture can result in plasmid loss 52, so the use of low passage B. burgdorferi is essential. Also, the medium and density of spirochetes is artificial upon introduction, so the ticks should not be used immediately post-capillary feeding. Instead, a period of not less than 2 weeks is allowed for the spirochetes to adapt to the tick microenvironment before use in experiments.
When feeding ticks on mice, it is not necessary to shave the mice beforehand. In a previous study (unpublished) in which we sought to examine the effects of tick saliva on skin, shaving was necessary. In doing so, and counter intuitively, we discovered that the ticks: a) attach readily to hairless skin; b) have a high feeding rate; and c) are easily visible. The feeding rates vary depending on whether larvae or nymphs are used but are consistently above 50% when nymphs are used. As such, shaving mice prior to feeding has become common practice in our laboratory, not only for experiments, but also for propagation of the colony.
Previously, in our Division at the Tulane National Primate Research Center, 34 monkeys have been fed upon by 770 Ixodes nymphs in 5 different studies. Tick feeding rates (# fed/# added to capsules) average 35.2%, ranging between 23.5-52.5%. In infection studies, rates of transmission average 86.5%. In a recent pilot study (unpublished), tick feeding rates varied from 5-75% and no resistance to subsequent feeding was apparent. However, the success rates between attempts 2 and 3 varied significantly, where feeding rates were much higher for the 3rd attempt than for the 2nd. The "attempt 2" ticks were housed at ambient temperature longer than the "attempt 3" ticks. The most important factor we have found that affects tick feeding is the tick age and environment pre-experiment. Those kept at 4 &deg;C post-molt until shortly before use generally feed better. As such, we recommend continuously propagating ticks, storing them accordingly and having two separate lots of ticks available when performing feeding on animals.
In our most recent study (unpublished) we compared feeding ticks on macaques using the hard capsule to feeding with the flap device. Ten monkeys were fed upon once with capsules and twice with LeFlap. In this set of experiments, we observed and average feeding rate of 17% (range 5-25%) with capsules and an average rate of 54.75% (range 35-90%) with the flap. We surmise that the broader surface area for tick feeding and the reduced use of harsh adhesives improves feeding. Use of the flap containment also allows the investigators to either let ticks feed longer or add more ticks, as the tick can be removed without removal of the entire device. Lastly, though the adhesives may cause mild skin irritation in some animals (which could affect or induce cutaneous immune responses) the flap device itself may have limited, if any, discomfort for the animals.

In 2011, Lyme disease was the 6th most common Nationally Notifiable disease in North America (<a href="http://www.cdc.gov/lyme/stats/index.html" target="_blank">http://www.cdc.gov/lyme/stats/index.html</a>). B. burgdorferi is a versatile microbe, both genetically and antigenically (reviewed in 1). Its genetic constitution includes a large (&gt;900 kB) chromosome and up to 21 plasmids (12 linear, 9 circular), with plasmid content varying among isolates. Much is to be learned about this spirochete, as over 90% of the plasmid open reading frames are unrelated to any known bacterial sequences 2,3 . B. burgdorferi presents a wide variety of antigens as potential targets of host immunity. However, an untreated infection often persists. The interaction of spirochetes with the tick milieu and the vertebrate host environment necessitates adaptation by B. burgdorferi throughout the infection process. Several plasmid-encoded genes are known to be differentially expressed in response to changes in temperature, pH, cell density and even stage of the tick life cycle 4-8.
The study of B. burgdorferi adaptation throughout its enzootic cycle, and host responses following infection by the natural route relies on the ability to feed ticks on appropriate animal models. Such studies are met with the technical challenges of generating ticks that harbor B. burgdorferi, and ensuring the efficient transmission and/or feeding of ticks on the model host. In addition, the containment and recovery of infected ticks is essential. Among the models used are mice and nonhuman primates, each of which serves as a valuable tool in Lyme disease research. As with the white-footed mouse, which is a natural reservoir host for B. burgdorferi, the laboratory mouse is a highly susceptible host that supports persistent infection by B. burgdorferi 9. Following infection of disease-susceptible mice, such as the C3H strain, the spirochetes disseminate to multiple tissues, including the skin, bladder, muscles, joints and heart. Inflammatory responses to the infection lead to diseased heart and joint tissue. While the spirochetes persist in this host and remain infectious, inflammatory lesions may become intermittent, not unlike the process in humans. The mouse model has thus provided much information on B. burgdorferi-induced pathology, including arthritis and carditis and host immune responses 10-12. From the perspective of the pathogen, certain genes differentially expressed during mammalian infection have been characterized, as have some necessary for transmission from the tick vector 13-21.
Though several animal species have been used to study Lyme disease 22, rhesus macaques most closely mimic the multi-organ character of human disease 23. Unlike other animal models, the breadth of disease manifestations such as erythema migrans, carditis, arthritis, and neuropathy of the peripheral and central nervous systems are observed in macaques. In mice, the reservoir host for B. burgdorferi, disease varies by mouse strain and age 24, while the early and late-disseminated manifestations are uncommon 9. In addition, other rodents, lagomorphs, and canines all fail to exhibit neurological disease from B. burgdorferi infection 25. Importantly, macaques exhibit signs that are characteristic of all three phases of Lyme borreliosis, namely, early-localized, early-disseminated, and late-stage Lyme disease 26-28. Erythema migrans (EM) is thought to occur in 70-80% of human cases 29, and is also seen in rhesus macaques 28,30 . Following infection, the spirochetes disseminate from the site of inoculation to multiple organs. Spirochetal DNA has been detected in skeletal muscles, heart, bladder, peripheral nerve and plexus, as well as in the central nervous system (cerebrum, brainstem and cerebellum, spinal cord, and dura mater) 31.
Tick feeding on mice has been utilized by us and other research teams for propagation of tick colonies, in reservoir competence studies 32-36 and in studies of B. burgdorferi pathogenesis 37-40. This technique has also been used for xenodiagnosis and testing of vaccine efficacy in mice 41-44. We have fed Ixodes ticks on nonhuman primates for model development 28, a study of vaccine efficacy 45, and for xenodiagnosis in the assessment of persistence post-antibiotic treatment 46. Ticks that harbor B. burgdorferi can be maintained in a natural enzootic cycle by feeding larvae on infected mice and using the nymphs for studies, as the spirochetes are transmitted through the life stages. In this report, we instruct on how to generate ticks infected with wild type or mutant B. burgdorferi, using capillary tube-feeding. This can also be accomplished by microinjection 47 and by immersion 48. The purpose of artificial introduction of B. burgdorferi into ticks can be to study mutant strains whose transmissibility is unknown, to generate a group of ticks with a high infection rate, and to reduce potential for error by maintaining a clean and otherwise uninfected tick colony. In addition, we demonstrate tick feeding on mice and nonhuman primates, so as to assure containment and recovery of replete ticks. The use of tick feeding is essential for future studies of immune responses to B. burgdorferi infection, potential Lyme vaccine efficacy, and xenodiagnosis for detection of occult infections.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>BSK-H</td><td>Sigma</td><td>B-8291</td><td></td></tr><tr><td>Ketamine HCl</td><td></td><td></td><td></td></tr><tr><td>Tangle Trap coating Paste</td><td>Ladd research</td><td>T-131</td><td></td></tr><tr><td>SkinPrep</td><td>Allegro Medical Supplies</td><td>177364</td><td></td></tr><tr><td>LeFlap, 3&quot; x 3&quot;</td><td>Monarch Labs</td><td></td><td></td></tr><tr><td>Hypafix tape</td><td>Allegro Medical Supplies</td><td>191523</td><td></td></tr><tr><td>SkinBond</td><td>Allegro Medical Supplies</td><td>554536</td><td></td></tr><tr><td>UniSolve</td><td>Allegro Medical Supplies</td><td>176640</td><td></td></tr><tr><td>Biatane Foam, adhesive 4&quot;x4&quot;</td><td>Coloplast</td><td>3420</td><td></td></tr><tr><td>DuoDerm CGF Dressing - 4&quot; x 4&quot;, (3/4)&quot; adhesive border </td><td>Convatec</td><td>187971</td><td></td></tr><tr><td>Nonhuman primate jackets with flexible 2&quot; back panels; add drawstrings at top and bottom</td><td>Lomir Biomedical Inc.</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT &lt;/strong&gt;</td></tr><tr><td>Pipet puller</td><td>David Kopf Instruments</td><td>Model 700C</td><td></td></tr><tr><td>Dark field microscope</td><td>Leitz Wetzlar</td><td>Dialux</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>Zoom 2000</td><td></td></tr><tr><td>Mouse caging</td><td>Allentown caging</td><td></td><td></td></tr></tbody></table>

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.

Following the completion of capillary feeding, the ticks are typically rested at 23 &deg;C for 2-3 weeks before they are fed on animals for transmission. Using the capillary-feeding technique, we have found that over 90% of the fed ticks harbor B. burgdorferi. The percentage of positive ticks is determined by washing ticks in peroxide and ethanol, then crushing them in sterile PBS with a microfuge tube-shaped pestle. The midgut contents spilled into the PBS are fixed on slides and stained with an anti-Borrelia species antibody that is FITC-conjugated. Representative tick midgut smears viewed by fluorescent microscopy are depicted in Figure 2B-C.
Mouse infection rates with low passage strain B31 wild type B. burgdorferi are near 100%. A combination of serology and culture of B. burgdorferi from mouse tissues is used to determine if each mouse has become infected. A western blot showing serum antibody responses from mice infected with B. burgdorferi by tick is shown in Figure 3C. This technique has been used to examine the transmissibility and infectivity of B. burgdorferi mutant strains 37-39.
We have used tick feeding on nonhuman primates for infection and for xenodiagnosis. Efforts have been made to improve tick feeding and recovery of fully fed ticks by implementing the flap containment device. The flap product is used for application of medicinal maggots in humans, but we have modified it for tick feeding on primates. In previous studies, we utilized a hard capsule for tick containment 27,28,45,46 and obtained an average feeding rate (# fed ticks/# ticks added to capsules) of 35.2%, ranging between 23.5-52.5%. In infection studies, rates of transmission (# animals infected/# fed upon) averaged 86.5%. In more recent experiments, the feeding rates using LeFlap have been between 50-90%. On rare occasions, using the previous method, ticks have crawled under the capsule and into the adhesive tape, where they desiccate and die. Using the flap the improved feeding and multiple layers of adhesive have kept the ticks contained.
In addition to direct fluorescent staining of tick midgut preparation (Figure 2B-C), more sensitive methods can be used to detect B. burgdorferi within ticks. Molecular detection can, and has been used to detect B. burgdorferi-specific DNA 42,50,51 with either standard or quantitative PCR. Common targets for detection are the flaB 46,50 , OspC 46 and OspA 42,51 genes. The viability of recovered spirochetes has also been examined by culture of midgut preparations and feeding xenodiagnostic ticks on na&iuml;ve mice 42.
<img alt="Figure 1" src="/files/ftp_upload/50617/50617fig1.jpg" /> 
 Figure 1. Feeding Ticks on Animals for Transmission of Borrelia burgdorferi. Overall scheme of the techniques involved in feeding ticks on animals for Lyme disease studies. Ticks are capillary tube-fed cultures of B. burgdorferi and can be fed upon animal models of Lyme disease, such as mice and nonhuman primates (rhesus macaques). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50617/50617fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/50617/50617fig2.jpg" /> 
 Figure 2. The capillary tube-feeding method and results. A) The apparatus used to feed ticks in shown, with a magnified view of the tick and capillary tube on the right. B-C) representative images from the midguts of ticks fed B. burgdorferi. The midgut smears were stained with anti-Borrelia species-FITC polyclonal antibodies (Kirkegaard &amp; Perry Labs) and viewed under fluorescent microscope.
<img alt="Figure 3" src="/files/ftp_upload/50617/50617fig3.jpg" /> 
 Figure 3. Ixodes scapularis tick feeding on laboratory mice. A) The specialized caging for mice when used for tick feeding. The wire floor is elevated above a pan of water to collect ticks. An anesthetized mouse is shown in the image on the right. B) The storage containers used for ticks. C) Representative immunoblots from tick-infected mice. Serum from day 21 post-infection was used to probe blots containing B. burgdorferi lysates and recombinant OspC protein, an immunodominant antigen.
<img alt="Figure 4" src="/files/ftp_upload/50617/50617fig4.jpg" /> 
 Figure 4. Diagram of the tick containment device used to feed ticks on rhesus macaques. The first layer consists of Biatane foam. The flap is placed on top of the foam and the Duoderm is the third layer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50617/50617fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/50617/50617fig5.jpg" /> 
 Figure 5. Ixodes scapularis tick-feeding on rhesus macaques. A) The complete containment device. B,C) Views of ticks feeding through the device, and following removal of the flap. D) The site of tick feeding following complete removal of the device.

Watch this Scientific Journal Video about Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research at JoVE.com

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

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 ...

feeding-ticks-on-animals-for-transmission-xenodiagnosis-lyme-disease

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.1K Views.  Tulane University Health Sciences Center. 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.

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

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

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

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

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

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Genetics

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 ...

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 ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

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 ...

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to humans through the bite of infected Ixodes ticks. The primary etiological agent in North America is Borrelia burgdorferi sensu stricto. As geographic risk regions expand, it is prudent to support robust surveillance programs that can measure tick infection rates, and communicate findings to clinicians, veterinarians, and the general public. The molecular technique of nested polymerase chain reaction (nPCR) has long been used for this purpose, and it remains a central, inexpensive, and robust approach in the detection of Borrelia in both ticks and wildlife. 
This article demonstrates the application of nPCR to tick DNA extracts to identify infected specimens. Two independent B. burgdorferi targets, genes encoding Flagellin B (FlaB) and Outer surface protein A (OspA), have been used extensively with this technique. The protocol involves tick collection, DNA extraction, and then an initial round of PCR to detect each of the two Borrelia-specific loci. Subsequent polymerase chain reaction (PCR) uses the product of the first reaction as a new template to generate smaller, internal amplification fragments. The nested approach improves upon both the specificity and sensitivity of conventional PCR. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to ...

Biology

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 ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

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

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

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

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

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

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

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

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

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

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

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

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

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