TITLE: REDI-NET FF-1 FILTH FLY FIELD SAMPLING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
To outline steps for properly collecting filth flies from cattle and environmental locations to evaluate the risk of zoonotic disease transmission by the detection of pathogens from vertebrate DNA (vDNA).

[BEFORE_START]
NOTE: Obey all safety procedures in of this SOP.
Never stand in front of the chute until the animal has been completely restrained.
Always be aware of the location of the gate hinge when standing next to the chute as it is opening and closing.
Always make sure that all drop-down bars and fold-out panels are closed before the chute is opened.
Always communicate verbally with the team operating the squeeze chute when you are finished checking the animal so that they know when it is safe to open the chute.
Always be aware of the fact that the animal can still move somewhat inside the chute. Be aware of where your hands and arms are, and be able to remove them from the chute quickly.
If you ever feel uncomfortable checking an animal (animal is too worked up or aggressive in the chute), or you are unable to safely access part of the animal in the chute, skip it.


 


[GUIDELINES]
OBJECTIVE

To document the field processes for collecting filth flies from cattle and environmental locations.

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 protocol for the correct filth fly sampling at CONUS/OCONUS sites to evaluate and predict the risk of zoonotic disease transmission.

RESPONSIBLE PERSON

Principal Investigator, Study Coordinator, Entomology Component Lead, Managers

[STEPS]
SECTION: SAMPLING TEAM
1. This SOP assumes that the animals’ owners/handlers/veterinarians are on site to corral cattle, move them through the chute, and operate the chute. If collecting from cattle, animals should be restrained in a chute or squeeze chute or in a head gate. If animals are only restrained in a head gate, be aware that cattle can kick to the side and keep an appropriate distance from the animal.


SECTION: SAMPLING TEAM
2. Sampling of vDNA 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: CATTLE COLLECTION
3. Label a previously prepared fly carton with the collection date, collection site, mark as "cattle".

SECTION: CATTLE COLLECTION
4. Once the animal(s) is/are restrained, make three, quick forward and reverse sweeps with the net over the back/sides of the animal

SECTION: CATTLE COLLECTION
5. At the end of the third sweep, quickly grab the net to close the opening with your hand.
SECTION: CATTLE COLLECTION
5.1. For cattle in the chute: Stand on step or step stool to sweep over the back of animals.

SECTION: CATTLE COLLECTION
5.2. For cattle restrained in the squeeze chute: Lower the drop-down bars and fold-out panels of the chute to sweep the sides and legs of the animal.

 

SECTION: CATTLE COLLECTION
6. Untie the sleeve of the fly carton (while still holding the net closed) and carefully transfer the flies from the net to the carton by pushing the opening of the net into the sleeve and turning the net inside out.
7. Re-tie the carton sleeve.
8. Record the ear tag or identification number of the animal sampled.
9. Repeat this process until five animals have been sampled. After five animals have been sampled, start a new carton.
10. Continue sampling until at least 20% of the animals have been sampled. Ideally, all animals will be sampled, but occasionally this will not be possible due to time or personnel restraints.

11. Provide each carton with two water-soaked cotton balls in a condiment cup to help sustain flies until they can be returned to the lab.
SECTION: ENVIRONMENTAL COLLECTION
12. 
 

Label a previously prepared fly carton with the collection date, collection site, mark as "environment".
SECTION: ENVIRONMENTAL COLLECTION
13. Identify appropriate collection locations at the site. Common filth fly development and aggregation locations include manure piles (house/stable flies), intact dung pats (horn/face flies), silage (house/stable flies), litter (house/little house flies), barn/stable walls (house/little house flies)	.
SECTION: ENVIRONMENTAL COLLECTION
14. For each collection location-type sampled (e.g. silage, dung pats), use a new fly carton.
SECTION: ENVIRONMENTAL COLLECTION
15. Label the fly carton with the collection location type.

SECTION: ENVIRONMENTAL COLLECTION
16. Make three, quick forward and reverse sweeps with the net over the collection location.
SECTION: ENVIRONMENTAL COLLECTION
17. At the end of the third sweep, quickly grab the net to close the opening with your hand.
SECTION: ENVIRONMENTAL COLLECTION
18. Untie the sleeve of the fly carton (while still holding the net closed) and carefully transfer the flies from the net to the carton by pushing the opening of the net into the sleeve and turning the net inside out.

SECTION: ENVIRONMENTAL COLLECTION
19. Re-tie the carton sleeve.

SECTION: ENVIRONMENTAL COLLECTION
20. Repeat this process until the carton is full, or all flies have been collected.
SECTION: ENVIRONMENTAL COLLECTION
21. Provide each carton with two water-soaked cotton balls in a condiment cup to help sustain flies until they can be returned to the lab.
SECTION: FLY STORAGE
22. If flies do not need to be kept alive for research purposes:
SECTION: FLY STORAGE
22.1. Place fly cartons into a -20 °C freezer for at least two hours, and up to 24 hours to kill flies.

SECTION: FLY STORAGE
22.2. After flies are dead, label a 50mL Falcon tube with the same information that was written on the fly carton.

SECTION: FLY STORAGE
22.3. Remove the mesh covering on the fly carton and transfer the flies to the Falcon tube.

SECTION: FLY STORAGE
22.4. If samples can not be processed on the same day of collection, all samples should be stored at -80 °C until they are identified to species and processed for TNA extraction. 

SECTION: FLY STORAGE
22.5. If -80 °C is not available, filth fly samples should be stored at -20 °C and processed as soon as possible.

SECTION: FLY STORAGE
23. If flies are to kept alive for research purposes:
SECTION: FLY STORAGE
23.1. Maintain flies in a separate room from the main laboratory if possible to limit escapes. Ideally, this should be an insectary space held at ~23 °C, 75% RH, and 16:8 L:D cycle. However, these conditions are not necessary if flies are only being held temporarily as long as the room temperature is appropriate.

SECTION: FLY STORAGE
23.2. For collections from cattle, or for environmental collections that are primarily horn or stable flies, pour 20 mL of cow blood onto a sanitary pad. Place that pad blood side-down on top of the mesh covering of the fly carton. Replace blood pad daily as necessary.

SECTION: FLY STORAGE
23.3. For environmental collections that are primarily house, little house, or face flies, mix 12 g dehydrated milk powder and 12 g table sugar together in a condiment cup and place it inside the fly carton. Also replace the water-soaked cotton balls as necessary.

SECTION: APPENDIX 1. CARTON CONSTRUCTION
24. For mosquitoes, use the X-acto knife to cut a 3.5x2” access hole in the side of the carton. For flies, cut a 5.5" diameter circle.
SECTION: APPENDIX 1. CARTON CONSTRUCTION
25. For mosquitoes, cut the toe off of the sock and cut a 0.5” slit in the cuff. For flies, cut a 30”x24” rectangle of organza or tulle and hot glue the long sides together, leaving the short ends open.
SECTION: APPENDIX 1. CARTON CONSTRUCTION
26. Apply a line of hot glue around the inside of the access hole and adhere the cuff end of the sock (or short side of organza) to the inside of the carton so that it forms a sleeve (Fig. 1).
SECTION: APPENDIX 1. CARTON CONSTRUCTION
27. Apply a small amount of glue at a time (1-2”) and adhere the sock so that the glue does not dry and you can stretch the sock enough to cover the opening.  
 
 
SECTION: APPENDIX 1. CARTON CONSTRUCTION
28. Apply additional glue as needed to secure the sock or organza.
SECTION: APPENDIX 1. CARTON CONSTRUCTION
29. Cut a 12x12” square (or large enough to cover the top of the carton) of tulle screen. Apply a line of hot glue around the outside rim of the carton and adhere the screen over the top to cover it.
SECTION: APPENDIX 1. CARTON CONSTRUCTION
30. Apply a small amount of glue at a time (1-2”) and adhere the screen so that the glue does not dry and you can stretch the fabric enough to cover the carton.
SECTION: APPENDIX 1. CARTON CONSTRUCTION
31. Stretch an elastic band over the top of the screen-covered carton to further secure the screen.
SECTION: APPENDIX 1. CARTON CONSTRUCTION
32. Tie the sleeve before adding insects to the carton (Fig 2.)