Jan 12, 2024
Version 1
L-1 LEECH FIELD SAMPLING V.1
- REDI-NET Consortium1
- 1REDI-NET Consortium
Protocol Citation: REDI-NET Consortium 2024. L-1 LEECH FIELD SAMPLING. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3peog8j/v1Version 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: March 15, 2024
Protocol Integer ID: 86276
Keywords: LEECH TRAPPING, LEECH STORAGE, DATA MANAGEMENT
Funders Acknowledgement:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059
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 and HT9425-23-C-0059. 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 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
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.
- 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.
Materials
EQUIPMENT AND MATERIALS
Note
NOTE: If product number is listed, please ensure use of this or equivalent product.
| A | B | C | |
| Equipment / Material | Description | Mfg / Product # | |
| Leech traps (4 options) |
see Appendix 2 for detailed description for making and baiting the leech traps | See Appendix 2 | |
| Liver | Beef or Pork; for leech baiting 8oz per trap | Locally sourced | |
| Tight sealing tupperware box | For liver transportation | Locally Sourced | |
| Writing utensils | Pen / pencils, marker pens | Locally Sourced | |
| Container | Large volume (500 ml) jar or container for collecting samples; one per sampling location, pre-labeled Example: pickle jar | Locally Sourced | |
| Bright colored tape | For flagging | Uline; S-6089FP | |
| Flagged stakes | For trap retrieval | Locally Sourced | |
| Fine-tipped forceps | For leech removal | Carolina.com; Item # 624734 | |
| Cooler and ice packs | For cooling leeches during transport | Locally Sourced | |
| GPS unit | WGS 84 and precision of 5 decimal degrees | Locally Sourced | |
| Data sheets | REDI-NET DCS L-1 Leech collection form | REDI-NET Data Portal | |
| Tablet | For data entry and picture of sampling site | Locally sourced |
APPENDIX 2. LEECH TRAP CONSTRUCTION
Trap Option 1: Pie Pan Construction
| A | B | C | |
| Equipment / Material | Description | Mfg / Product # | |
| Pie pan (8ââto 9ââ size) | Aluminum; 8ââto 9ââ size | Locally sourced | |
| Mesh wire | To secure liver bait in the traps | Locally sourced | |
| Paper punch/ Metal skewer | To punch hole in the pie pan | Locally sourced | |
| Needle | To sew the mesh wire around the traps | Locally sourced | |
| Scissors | To cut the mesh wire, the thread, and the twine | Locally sourced | |
| Twist tie | To seal the traps | ||
| Thread | To sew the mesh wire around the traps | Locally sourced | |
| Beef | liver To bait the leeches, 8 oz per trap | Locally sourced | |
| Twine/Fishing line | To attach and retrieve the traps (c.5 m per trap) | Locally sourced |
Trap Option 2: Bottle Trap
| A | B | C | |
| Equipment / Material | Description | Mfg / Product # | |
| Plastic soda bottle (1.5L) | Clean, plastic; 1.5 L size | Locally sourced | |
| Stapler and staples | Stainless steel staples; to close the trap | Locally sourced | |
| Paper punch | To puncture the bottle | Locally sourced | |
| Icepick/Metal skewer | To punch hole in the bottle | Locally sourced | |
| Scissors | To cut the bottle | Locally sourced | |
| Beef liver | To bait the leeches, 8 oz per trap | Locally sourced | |
| Medium size rock | To weight down the traps | Locally sourced | |
| Fishing line | To attach and retrieve the traps (c. 5m per trap) | Locally sourced |
Trap Option 3: PVC Pipe Trap
| A | B | C | |
| Equipment / Material | Description | Mfg / Product # | |
| Sewer drain plug | 4 inch | Home Depot, 052063400112 | |
| Sewer drain adaptor | 4 inch | Home Depot, 052063444116 | |
| Drain pipe drop-in grate PVC | 4 inch, round, qty: 2 | Home Depot, 052063950112 | |
| Sewer drain Tee | 4 inch | Home Depot, 052063404752 | |
| Rope | 50 feet | Home Depot, 030699706525 | |
| Eyebolt | 3/8 inch by 7 inch | Home Depot, 887480036046 | |
| Hex nuts | One for each eyebolt | Home Depot, 887480022445 | |
| Drain pipe | 4 inch by 10 feet (this will make 10-20 traps) | Home Depot, 0000-189-781 |
Trap Option 4: Bioquip Gee Minnow Trap
| A | B | C | |
| Equipment / Material | Description | Mfg / Product # | |
| Gee Minnow Trap | For trapping leeches, made of mesh galvanized wire and steel | BioQuip Products/ 2824 | |
| Rope | 50 feet | Home Depot, 030699706525 | |
| Medium size rock | To weight down the traps | Locally sourced |
Safety warnings
RISKS AND PERSONAL PROTECTION
- 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. Be sure that clothing is sturdy and can withstand long thorns.
SAMPLING TEAMS
SAMPLING TEAMS
Note
NOTE: Suggested sampling frequency at each site is one time per month or, when not logistically possible, at minimum two times per unique season representative of the laboratory setting (e.g., early wet/ late wet; early dry/late dry; rainy/ dry season).
Field sampling of iDNA (leech) samples involves two people. One person serves as the âsamplerâ and the other person serves as a âhelperâ. 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.
SAMPLING SITE SELECTION
SAMPLING SITE SELECTION
Find two suitable sampling locations within the water body representing different ecological systems, if possible.
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.
LEECH TRAPPING
LEECH TRAPPING
Note
NOTE: Leech traps should be constructed and prepared ahead of time, and baited with
fresh liver on site.
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.
Note
Leech trapping should be conducted prior to water, sediment, and tick sampling.
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.
Use nearby rocks to weigh the leech trap when placing it in water to ensure full submersion.
Repeat steps 4 - 6 to place traps at the second sampling location in the water body.
Check traps after 2â3 hours (after completing tick/water and soil collections).
Recover traps and carefully open to expose the liver bait. Using forceps, check each side of the liver for leeches.
Note
NOTE: All leeches should be collected from each trap and should be placed into one large container with water from their original environment by sampling location (see step 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.
Note
DO NOT mix leeches from different sampling locations.
NOTE: Leeches often regurgitate under stress so leeches should not be stored together.
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.
After collecting all leeches from the first sampling location, advance to the second sampling location and repeat steps 9-11.
LEECH STORAGE
LEECH STORAGE
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.
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.
Note
NOTE: If leech regurgitates during handling, soak up any fluid expelled using filter paper and store with leech. If -80 °C storage is unavailable, leeches should be stored individually at -20 °C and processed within 2 weeks.
APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
APPENDIX 1.1 LEECH TRAP CONSTRUCTION -- Trap Option 1: Pie Pan Construction
Gently fold the pie pan in half until the edges meet (Figure 1A).
Note
NOTE: Do NOT fold the center line.
Figure 1A: Leech traps construction.
Push the metal skewer through the edges to create 4 equally spaced holes to thread the ties through (Figure A1B, A1C).
Figure A1: Leech traps construction.
Measure and cut a piece of mesh wire that fits around the folded pie pan.
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).
Figure A2: Leech trap, pie pan design.
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).
Figure A1: Leech traps construction.
Figure A1: Leech traps construction.
Note
NOTE: Steps 1â5 can be done in advance of field trips to save time.
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.
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).
Figure A1: Leech traps construction.
Figure A3: Completed pie pan trap.
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.
Note
NOTE: Where carnivores are present it may be necessary to protect the leech trap by placing it inside a wire frame.
Wait until it sinks and secure the cord of the leech trap to sturdy vegetation, or fashion ground stakes to secure.
APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
APPENDIX 1.2 LEECH TRAP CONSTRUCTION -- Trap Option 2: Bottle Trap
Remove the soda bottle cap as this will not be needed for the trap (Figure A4).
Figure A4: Plastic soda bottle.
Cut the bottle top just above the labeled line as shown in Figure A5.
Figure A5: Cut bottle.
Make holes on the bottom portion and around the bottle using an icepick or scissors as shown in Figure A6.
Figure A6: Holes being made with icepick.
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.
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.
Figure A7: Top of bottle being inserted in bottom part.
Using a stapler, staple the funnel into position.
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.
Figure A8: Tying of bottle with fishing line.
Figure A9: Completed bottle trap.
APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
APPENDIX 1.3 LEECH TRAP CONSTRUCTION -- Trap Option 3: PVC Pipe Trap
Figure A10: PVC pipe trap
Place a sewer grate onto both sides of the âTâ shaped drain.
Cut 6 in of the 10 ft x 4in PVC pipe using a hacksaw.
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.
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).
Drill a hole using a 3/8 in drill bit through the top of the drain plug.
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.
Bait will be placed between these two hex bolts.
The 3/8 hex bolt might not go all the way to the top of the eyebolt and that is ok.
Attach rope to eyebolt loop.
Figure A11: PVC pipe trap construction.
APPENDIX 1.4 LEECH TRAP CONSTRUCTION -- Trap Option 4: Bioquip Gee Minnow Trap
APPENDIX 1.4 LEECH TRAP CONSTRUCTION -- Trap Option 4: Bioquip Gee Minnow Trap
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.
Close the trap using the latch.
Attach the rope to the trap for throwing and retrieving the trap.
Note
NOTE: There is no cap on the number of leeches during sampling - all leeches should be
collected.
Figure A12: Minnow trap.
Protocol references