Dec 16, 2024
Version 2
REDI-NET T-1 TICK FIELD SAMPLING V.2
- REDI-NET Consortium1
- 1REDI-NET Consortium
- Remote Emerging Disease Intelligence - NETwork (REDI-NET)
Protocol Citation: REDI-NET Consortium 2024. REDI-NET T-1 TICK FIELD SAMPLING. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3435lk5/v2Version created by REDI-NET Consortium
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 09, 2023
Last Modified: December 16, 2024
Protocol Integer ID: 109220
Keywords: TICK FIELD SAMPLING, TICK TRANSECT SELECTION, TICK DRAGGING, TICK STORAGE
Funders Acknowledgements:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059
USAMRAA
Grant ID: HT9425-24-C-0072
Disclaimer
This work is supported by the US Army Medical Research and Development Command under Contract No.W81XWH-21-C-0001, W81XWH-22-C-0093, HT9425-23-C-0059, and HT9425-24-C-0072. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army or Navy position, policy or decision unless so designated by other documentation.
Abstract
This protocol describes tick field sampling.
Guidelines
OBJECTIVE
To clearly document the correct process for effective standardized field collections of ticks, and recommended personal protection measures.
SUMMARY/SCOPE
The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on field tick collections in the vicinity of watering holes by dragging.
RESPONSIBLE PERSON
Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
Note
NOTE: All study procedures must be conducted in compliance with national and local policies for prevention and control of COVID-19 infection.
MAINTENANCE OF EQUIPMENT
BEFORE EACH COLLECTION
- Clean forceps with 70%-ethanol.
- Freeze and clean ice-packs.
- Clean cool-boxes
- Fully charge all equipment (e.g., GPS unit, tablets/phone). Make sure the tablet has enough free-space for field sampling pictures.
AFTER EACH COLLECTION
- Clean all equipment thoroughly between sampling sites, including boots, cooler box (inside and outside), etc.
- Tick drags should be sealed into trash bags for transport and cleaned without detergents (only use tap water and then air-dry) before the next collection.
- Lint roller sheets should be disposed of in the waste bag after all ticks have been removed for testing.
- Store sterile equipment separate from used equipment and samples.
QUALITY CONTROL
This SOP is reviewed by the applicable supervisor annually or as required in order to maintain its relevance.
Materials
EQUIPMENT AND MATERIALS
Note
NOTE: If product number is listed, please ensure use of this or equivalent product.
| A | B | C | D | |
| Equipment / Material | Description | Mfg / Product # | ||
| Tick Drags | (~1m) made out of white brushed cotton denim, flannel, or utility cloth with dowel and rope attached; see Appendix 2 for detailed description and instructions | Locally Sourced | ||
| Writing utensils | Pen / Pencils, Marker Pens | Locally Sourced | ||
| Vials (2 options) |
|
| ||
| Fine-tip forceps | Stainless metal or plastic, for removing ticks from cloth | Carolina.com; # 624734 | ||
| Lint roller | For removing ticks from tick drags | Locally sourced | ||
| Cooler and cool packs | For cooling ticks during transport | Locally Sourced | ||
| Microcentrifuge tubes 1.5 ml, RNAse, pyrogenic free | For individual tick sorting | ThermoFisher, AM12450 equivalent | ||
| Wet towels | For cooling ticks during transport | Locally Sourced | ||
| Ziplock bags | For transporting vials containing ticks | Locally Sourced | ||
| Roll of flagging tape | For transect flagging | Uline; S-6089FP | ||
| Hand counters / tally | For tick counting | Amazon; AMZ011Counter | ||
| Dry Ice | for producing CO2 | Locally Sourced | ||
| Standard Cup for measuring dry ice volume | Techpak plastic 6 oz. | Locally Sourced | ||
| Small Dry Ice Shovel | for scooping the dry ice | Locally Sourced | ||
| Container wth lid | e.g., Cecilia's Muesli container 500g/ volume ~1400 sq. cm | Locally Sourced |
Lint roller.
Safety warnings
RISKS AND PERSONAL PROTECTION
BEFORE COLLECTION
- Know the risks associated with the study site and sampling location and take appropriate personal safety precautions and/or wear appropriate equipment.
- Wear light-colored long pants and a long sleeve shirt. Wear rubber boots or hiking shoes and white socks, and tuck pant legs into socks. This creates a barrier to prevent ticks from contacting skin, and the light color increases the likelihood of finding a tick on clothing and removing it before it can attach and feed. Be sure that clothing is sturdy and can withstand long thorns.
- Spray boots and pants with permethrin (i.e. Permanone®, Sawyer®). This should be done prior to removing the drag from the collection kit, and ideally before arriving at the collection site. Apply DEET to skin only. DO NOT get insect repellant on the tick drag. DO NOT stand near the drag when applying bug spray – it will deter ticks and result in inaccurate estimates.
AFTER COLLECTION
- EVERY 100 meters (5 drags), check yourself for ticks. Any ticks collected from your clothing or body can be placed in a vial and counted in the transect .
- EVERY time you complete a sampling site check yourself for ticks.
- EVERY night check yourself for ticks. Especially where clothing is tight against skin, e.g., waistband, and your hair/head.
- If a tick is found attached, remove using instructions in Appendix 2.
- Optional: After initial tick drag, set up Overground Dry Ice Trap (ODIT) in Appendix 3.
- Optional: After initial tick drag, set up Pitfall Dry Ice Trap (PDIT) in Appendix 4.
TICK TRANSECT SELECTION
TICK TRANSECT SELECTION
Find two suitable sampling locations at least 10-20 m away from the water body representing different ecological systems, if possible.
SAMPLING TEAMS
SAMPLING TEAMS
Field sampling of iDNA (tick) samples involves two people. One person serves as the ‘sampler’ and the other person serves as a ‘helper’. The helper can look up details in these instructions when needed, keep track of samples, handle objects that are contamination risks, serve as a second set of eyes for potential contamination, and ensure safety of the sampler in potentially hazardous field conditions.
TICK TRANSECT SELECTION
TICK TRANSECT SELECTION
Determine two 20m x 20m grid blocks for tick collections. Grid blocks should not be overlapping a location sampled the previous month’s collection trip. Each block-transect is composed of 5 ticks drags/passes.
The two grid-transects should be separated at least by 30 meters at the nearest points, when possible. Both transects should be in areas animals pass through to get to the watering hole.
TICK DRAGGING
TICK DRAGGING
Tick drags should not get very wet when sampling. Wait until the dew has burned off in the morning, and do not sample if it is raining.
For each transect, 5 tick-drags/passes should be performed; each tick-dragging being done in opposite directions (e.g., if the first drag of the first transect is oriented from North to South, the second drag should be performed from South to North, and so on).
Drag sampling should be performed by one person.
After each pass within the transect, turn the drag over and remove any ticks on the drag using tweezers to remove all ticks and place them in the appropriately labeled collection vial. No more than 10 live ticks should be placed in vial. All stages should be collected. Teams of two people can remove ticks from the drags (Figure 2).
Figure 2: Tick removal from the drag.
Place a blade of grass in the vial before capping (Figure 3).
Figure 3: Ticks in labeled vial after drag collection.
After collecting ticks from the drag from each pass, attention should be taken to remove any foreign object on the drags.
Place all labeled snap-cap vials from a completed transect into a Ziploc bag and into a cooler with wet towels in bottom with frozen water bottles, in order to slow them down.
Continue to the next transect and continue sampling ticks as in steps 1-10.
DO NOT mix ticks from different transects.
If needed, use a lint roller to remove debris, burrs or the remaining ticks prior to leaving the site.
The lint roller paper should be placed in a ziplock bag (labeled with a barcode) for transport back to the laboratory.
TICK STORAGE
TICK STORAGE
Keep freshly collected ticks alive, keeping on a 4 °C cold chain (cooler) until return to the laboratory. Ticks are not recommended to be stored at 4 °C for long periods of time.
Once at the laboratory, if samples can not be processed on the same day of collection, all adult ticks (within field vials or on lint roller papers) must be individually sorted into appropriately labeled 1.5mL centrifuge tubes and should be stored at -80 °C . Immature ticks remaining on lint roller papers should also be stored at -80 °C until ready for testing. At -80 °C , ticks can be stored for 2 months or longer.
If -80 °C storage is unavailable, ticks should be stored at -20 °C until processing. Ticks can be stored at -20 °C for up to 2 months.
APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
One piece of white heavy-duty fabric with a soft nap, cut to approximately 1 square meter (39.5” x 39.5”). We used a waterproof white rubber sheeting (utility cloth) from JoAnn fabric and craft store (item #1491315), which is normally used to sew crib liners. White brushed bull denim could also be used (this should have a softer nap than regular bull denim).
Two smaller pieces of the same fabric cut to 39.5” x 4” and 39.5” x 3”. These will be sewn to the top and bottom of the flag to hold the dowel and the washers.
2 eye hook screws for each end of the dowel, ours are size #12.
3 large flat washers to weight the back of the flag, 1-3/4” diameter, 1/8” thick.
1 length of rope, 9 ft long.
1 48” dowel, ¾” in diameter.
Cotton thread and sewing machine for stitching flags together.
Figure 4: Ticks drag construction.
Figure 5: Details of the ticks drag construction.
APPENDIX 2. HOW TO REMOVE A TICK ATTACHED TO SKIN
APPENDIX 2. HOW TO REMOVE A TICK ATTACHED TO SKIN
Use fine-tipped forceps to grasp the tick as close to the skin's surface as possible.
Pull upward with steady, even pressure. Don't twist or jerk the tick; this can cause the mouth-parts to break off and remain in the skin. If this happens, remove the mouth-parts with forceps. If you are unable to remove the mouth easily with clean forceps, leave it alone and let the skin heal.
After removing the tick, thoroughly clean the bite area and your hands with rubbing alcohol, an iodine scrub, or soap and water.
Store the tick individually in ethanol and note the estimated duration of time the tick was attached. If desired, these ticks can be sent with those collected from the sampling areas for pathogen testing.
Seek medical attention if a rash or eschar appears around the site of attachment, and monitor for other symptoms including fever, fatigue, malaise, and any other abnormal symptoms. Be sure to inform the physician that you have been conducting tick research, and that a tick-borne disease is possibly responsible for symptoms.
APPENDIX 3. OVERGROUND DRY ICE TRAP (ODIT) SET UP
APPENDIX 3. OVERGROUND DRY ICE TRAP (ODIT) SET UP
Place the white fabric material (1m x 1m) on the ground and secure fabric with a rock (Fig. 1). The trap should ideally be placed under a shaded area with vegetation and close to animal tracks / visible pathway.
Figure 1
Place the Styrofoam box in the center of the fabric. Fill with 4 scoops of dry ice (560g) (this is important that it is consistent). Place the lid on the ODIT trap and a rock on top to prevent it from blowing away.
Leave the trap in place for 3-12 hours, checking every three hours.
After three hours, go back to the collection site and collect any ticks. Be sure to check around, under (on the ground under the trap), and inside the CO2 trap for ticks.
Complete a final tick drag at the site of 10m x 10m (about 5 big steps as each side of the square). Then, check the drag for ticks and collect as necessary.
After a day/overnight catch, measure 30m and set up the next trap in a similar ecosystem.
APPENDIX 4. PITFALL DRY ICE TRAP (PDIT) SET UP
APPENDIX 4. PITFALL DRY ICE TRAP (PDIT) SET UP
Using a shovel, dig a hole large enough for the container to be flush with the ground; ideally finding an area with some shade.
Fill one cup (6oz) with dry ice (should be weighed) and place upside down in the 1 -1.5L screw cap container.
Place the container in the hole (with the lid loosely screwed on top to ensure dirt is not getting inside of the trap) and fill in with remaining dirt ensuring the top of the container is even with the ground. Remove the lid of the screw cap container after dirt has been added to the hole.
Find three small rocks to place around the trap and one flat rock the size of the container opening.
Place the small rocks around the opening of the container, ensuring the path for ticks to reach the container is not blocked. Then, place a flat rock on top (hovering above the trap) to prevent the CO2 from rapidly escaping and prevent debris or rainfall from entering.
Leave the pitfall trap in place for 3 hours
After three hours, return to the collection site and place ticks around the container or on the rocks around it inside the container and cap it. Place the container inside a cool box containing icepacks.
Complete a final tick drag at the site of 10m x 10m (about 5 big steps as each side of the square). Then, check the drag for ticks and collect as necessary.
Protocol references