TITLE: L-1 LEECH FIELD SAMPLING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol describes leech field sampling.

[GUIDELINES]
OBJECTIVE

To document the field processes for collecting hematophagous leeches (Family Hirudinea).

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 the collection of blood-sucking leeches, to be used as sentinels for xenosurveillance of pathogens in wild and domestic animals frequenting the watering holes.


RESPONSIBLE PERSON

Principal Investigator, Study Coordinator, Field Team Lead, Managers
 

MAINTENANCE OF EQUIPMENT

BEFORE EACH COLLECTION

Clean forceps with 70%-ethanol.
Clean and freeze ice-packs
Clean cool-boxes
Save 1cm squared of the bait liver in -80*C - labeled with sites and dates in a cryovial.
Fully charge all equipment (e.g., GPS unit, tablet).
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.
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
1. 
 
Field sampling of iDNA (leech) 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: SAMPLING SITE SELECTION
2. Find two suitable sampling locations within the water body representing different ecological systems, if possible.
SECTION: SAMPLING SITE SELECTION
3. At each sampling location, leech traps should be placed in triplicate at least 10-20 meters apart from the water and sediment sample collection point, and each trap should be distant by 5 meters from each other.
SECTION: LEECH TRAPPING
4. 
 
After identifying sample sites location in the water body, deploy the leech traps. Place a stake in the water body or flag a tree to indicate the sampling site location.
 

SECTION: LEECH TRAPPING
5. Insert a piece of liver into the prepared traps, as instructed in Appendix 1. Leech Traps Construction (see Figures 1 and 2). Attach fishing line to trap and tie the remaining end around the flagged tree or stake to enable trap recovery.
SECTION: LEECH TRAPPING
6. Use nearby rocks to weigh the leech trap when placing it in water to ensure full submersion.
SECTION: LEECH TRAPPING
7. Repeat steps 4 - 6 to place traps at the second sampling location in the water body.
SECTION: LEECH TRAPPING
8. Check traps after 2–3 hours (after completing tick/water and soil collections).
SECTION: LEECH TRAPPING
9. Recover traps and carefully open to expose the liver bait. Using forceps, check each side of the liver for leeches. 
 

SECTION: LEECH TRAPPING
10. Leeches can gently be removed from the liver bait with tweezers and placed directly into the large (example size 500 mL) pre-labeled field transport container with water collected from their original environment. Leeches can be aggregated from the three traps at the same sampling location (1 OR 2) and stored in a single container with water collected from their original environment. 
 

SECTION: LEECH TRAPPING
11. Place all labeled storage containers into a cooler with ice until arriving back to the lab to prevent active swimming during transport; which promotes the consumption of the blood-meal.
SECTION: LEECH TRAPPING
12. After collecting all leeches from the first sampling location, advance to the second sampling location and repeat steps 9-11.
SECTION: LEECH STORAGE
13. Keep freshly collected leeches alive in glass jars which allow air passage (net-lidded or lids with multiple holes) and two-thirds filled with source water and tightly packed (so they will not spill) in a 4 °C cold chain (cooler box), ensuring air movement until return to laboratory. Storage at 4 °C is not recommended for long periods of time.
SECTION: LEECH STORAGE
14. Once at the laboratory, if leech samples can not be processed on the same day of collection, remove from water and freeze individually at -80 °C. Leeches can be stored at -80 °C for up to 1 month.
 

SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
15. Gently fold the pie pan in half until the edges meet (Figure 1A).
 
 
SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
16. Push the metal skewer through the edges to create 4 equally spaced holes to thread the ties through (Figure A1B, A1C).
 
SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
17. Measure and cut a piece of mesh wire that fits around the folded pie pan.
SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
18. Carefully sew the mesh wire around the pie pan ensuring to leave openings for liver placement and to allow leeches into the trap (Figure A2).
 
SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
19. Cut a length of twine 3-4 m long. Tie the twine through the hole on one of the end holes. Thread through and twist tie two other holes shut, leaving one hole open (Figure A1D, A1E, and A1F). 
 
 
 

SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
20. Using rubber gloves, slip approximately 8 oz of liver (add the blood drippings too) into the cavity of the pre-prepared trap and seal the trap by threading and fixing the twist tie in the final hole.
SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
21. Adjust the spaces between the tied parts of the open edge to ensure some space that allows the leeches to enter the trap (Figure A1G and A1H).
 
 
SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
22. Move slightly away from the highest animal traffic area (so as not to damage the trap) and hold the trap in one hand and the end of twine in another, toss the leech trap into the water body, keeping hold of the cord. 
 

SECTION: APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
23. Wait until it sinks and secure the cord of the leech trap to sturdy vegetation, or fashion ground stakes to secure.
SECTION: APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
24. Remove the soda bottle cap as this will not be needed for the trap (Figure A4). 
 
SECTION: APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
25. Cut the bottle top just above the labeled line as shown in Figure A5.
 
SECTION: APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
26. Make holes on the bottom portion and around the bottle using an icepick or scissors as shown in Figure A6. 
 
SECTION: APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
27. Place a piece of beef liver, approximately 8 oz, into the bottom of the bottle along with a medium-size rock. The rock will help sink the trap.
SECTION: APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
28. Invert the top of the soda bottle so it looks like a funnel and insert it into the bottom of the soda bottle as shown in Figure A7.
 
SECTION: APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
29. Using a stapler, staple the funnel into position.
SECTION: APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
30. Punch two holes, one at the top and one at the bottom portion of the bottle, to tie the fishing line for throwing and retrieving the trap.
 
 
SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
31.  
Place a sewer grate onto both sides of the ‘T’ shaped drain.

SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
32. Cut 6 in of the 10 ft x 4in PVC pipe using a hacksaw.
SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
33. Place the 6 inch cut into the lower part of the T shaped drain. Then attach the 4 in sewer drain adapter to the other side.
SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
34. Secure both the adapter and the Tee drain to the 6 in PVC section using nuts and bolts for both sides. (the image below only shows the top attached, the bottom segment will need to be attached with a bolt and nut as well).
SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
35. Drill a hole using a 3/8 in drill bit through the top of the drain plug.
SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
36. Push the 3/8 x 7 in eyebolt through the drain plug, then spin a 3/8 in hex bolt all the way to the top and add a second to the bottom.
SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
37. Bait will be placed between these two hex bolts.
SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
38. The 3/8 hex bolt might not go all the way to the top of the eyebolt and that is ok.
SECTION: APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
39. Attach rope to eyebolt loop.
 
SECTION: APPENDIX 1.4 LEECH TRAP CONSTRUCTION -- Trap Option 4: Bioquip Gee Minnow Trap
40. Place a piece of beef liver, approximately 8 oz, into the bottom of the trap along with a medium-size rock. The rock will help sink the trap.
SECTION: APPENDIX 1.4 LEECH TRAP CONSTRUCTION -- Trap Option 4: Bioquip Gee Minnow Trap
41. Close the trap using the latch.
SECTION: APPENDIX 1.4 LEECH TRAP CONSTRUCTION -- Trap Option 4: Bioquip Gee Minnow Trap
42. Attach the rope to the trap for throwing and retrieving the trap.