TITLE: T-1 TICK FIELD SAMPLING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
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
 

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.
 

[STEPS]
SECTION: SAMPLING TEAMS
2. 

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.
SECTION: TICK TRANSECT SELECTION
1. Find two suitable sampling locations at least 10-20 m away from the water body representing different ecological systems, if possible.
SECTION: TICK TRANSECT SELECTION
3. 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.
SECTION: TICK TRANSECT SELECTION
4. 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.
SECTION: TICK DRAGGING
5. 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.
SECTION: TICK DRAGGING
6. 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).
SECTION: TICK DRAGGING
7. Drag sampling should be performed by one person.
SECTION: TICK DRAGGING
8. 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).
 
SECTION: TICK DRAGGING
9. Place a blade of grass in the vial before capping (Figure 3).
 
SECTION: TICK DRAGGING
10. After collecting ticks from the drag from each pass, attention should be taken to remove any foreign object on the drags.


SECTION: TICK DRAGGING
11. 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.
SECTION: TICK DRAGGING
12. Continue to the next transect and continue sampling ticks as in steps 1-10.
SECTION: TICK DRAGGING
13. DO NOT mix ticks from different transects.
SECTION: TICK DRAGGING
14. If needed, use a lint roller to remove debris, burrs or the remaining ticks prior to leaving the site.
SECTION: TICK DRAGGING
15. The lint roller paper should be placed in a ziplock bag (labeled with a barcode) for transport back to the laboratory.
SECTION: TICK STORAGE
16. 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.
SECTION: TICK STORAGE
17. 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.
SECTION: TICK STORAGE
18. 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.
SECTION: APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
19. 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). 
SECTION: APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
20. 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.
SECTION: APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
21. 2 eye hook screws for each end of the dowel, ours are size #12.
SECTION: APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
22. 3 large flat washers to weight the back of the flag, 1-3/4” diameter, 1/8” thick.
SECTION: APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
23. 1 length of rope, 9 ft long.
SECTION: APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
24. 1 48” dowel, ¾” in diameter.
SECTION: APPENDIX 1. TICK DRAG CONSTRUCTION -- TICK DRAG SUPPLIES FOR ONE TICK DRAG
25. Cotton thread and sewing machine for stitching flags together.

 
 
 
SECTION: APPENDIX 2. HOW TO REMOVE A TICK ATTACHED TO SKIN
26. Use fine-tipped forceps to grasp the tick as close to the skin's surface as possible.
SECTION: APPENDIX 2. HOW TO REMOVE A TICK ATTACHED TO SKIN
27. 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.
SECTION: APPENDIX 2. HOW TO REMOVE A TICK ATTACHED TO SKIN
28. After removing the tick, thoroughly clean the bite area and your hands with rubbing alcohol, an iodine scrub, or soap and water.
SECTION: APPENDIX 2. HOW TO REMOVE A TICK ATTACHED TO SKIN
29. 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.
SECTION: APPENDIX 2. HOW TO REMOVE A TICK ATTACHED TO SKIN
30. 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.