Mar 20, 2024

Public workspaceREDI-NET FF-1 FILTH FLY FIELD SAMPLING

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8b5wl5r/v1
  • 1University of Notre Dame
REDI-NET Consortium
Open access
DOI: dx.doi.org/10.17504/protocols.io.j8nlk8b5wl5r/v1
Protocol Citation: REDI-NET Consortium 2024. REDI-NET FF-1 FILTH FLY FIELD SAMPLING. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8b5wl5r/v1
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: March 15, 2024
Last Modified: March 20, 2024
Protocol Integer ID: 96797
Keywords: Horse fly, Face fly, House fly, Horn fly, Filth fly, cattle, field collection, zoonotic, vDNA
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
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).
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
Materials

ABC
Equipment / MaterialDescriptionMfg / Product #
Fly CartonsSee instructions in Appendix 1Locally sourced
Tulle fabricFor fly cartonLocally sourced
ScissorsFor fly carton constructionLocally sourced
X-acto knifeFor fly carton constructionLocally sourced
Hot glue gunFor fly carton constructionLocally sourced
Hot glue sticksFor fly carton constructionsLocally sourced
Men’s crew sock (size L)For fly cartonLocally sourced
Rubber bandsFor fly cartonLocally sourced
Sweep netsLocally sourced
GPS UnitWGS 84 and precision of 5 decimal degreesLocally sourced
TabletFor data entry and picture of sampling siteLocally sourced
Writing utensilsSharpies, pens, pencilsLocally sourced
Cotton ballsWater-soakedLocally sourced
50 mL Falcon tubesFor storing collected fliesLocally sourced
ForcepsFor collecting flies from netsLocally sourced
Condiment cupFor the water-soaked cotton ballsLocally sourced
SugarFor house/face flies kept aliveLocally sourced
Dehydrated milkFor house/face flies kept aliveLocally sourced
Defibrinated cow bloodFor horn/stable flies kept aliveLocally sourced
Sanitary padsFor horn/stable flies kept aliveLocally sourced

Safety warnings
Attention
CATTLE COLLECTIONS

  • Cattle are restrained in a squeeze chute for all procedures.
  • 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 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.
  • 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.
  • 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.

ENVIRONMENTAL COLLECTIONS

  • Always use caution when entering pastures with cattle. Use the buddy system whenever possible and avoid turning your back on the animals. If bulls are present in the pasture, always be aware of the location of the bulls and know how to exit the pasture quickly if necessary.
  • If entering pastures for collections, don’t forget to close and lock any gates that you open.

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.


Note
Prepare fly cartons prior to field day; see directions in Carton Construction Appendix 1.

SAMPLING TEAM
SAMPLING TEAM
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.


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.
CATTLE COLLECTION
CATTLE COLLECTION
Label a previously prepared fly carton with the collection date, collection site, mark as "cattle".

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

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

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.

Note
Remember to close the panels before the animal is released from the chute and communicate clearly with the chute operator when you are finished working and the animal is safe to be released.

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.
Re-tie the carton sleeve.
Record the ear tag or identification number of the animal sampled.
Repeat this process until five animals have been sampled. After five animals have been sampled, start a new carton.
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.

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.
ENVIRONMENTAL COLLECTION
ENVIRONMENTAL COLLECTION

Note
If entering pastures for collections, don’t forget to close and lock any gates that you open.

Label a previously prepared fly carton with the collection date, collection site, mark as "environment".
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) .
For each collection location-type sampled (e.g. silage, dung pats), use a new fly carton.
Label the fly carton with the collection location type.

Make three, quick forward and reverse sweeps with the net over the collection location.
At the end of the third sweep, quickly grab the net to close the opening with your hand.
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.

Re-tie the carton sleeve.

Repeat this process until the carton is full, or all flies have been collected.
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.
FLY STORAGE
FLY STORAGE
If flies do not need to be kept alive for research purposes:
Place fly cartons into a Temperature-20 °C freezer for at least two hours, and up to 24 hours to kill flies.

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

Remove the mesh covering on the fly carton and transfer the flies to the Falcon tube.

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

If Temperature-80 °C is not available, filth fly samples should be stored at Temperature-20 °C and processed as soon as possible.

If flies are to kept alive for research purposes:
Maintain flies in a separate room from the main laboratory if possible to limit escapes. Ideally, this should be an insectary space held at ~Temperature23 °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.

For collections from cattle, or for environmental collections that are primarily horn or stable flies, pour Amount20 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.

For environmental collections that are primarily house, little house, or face flies, mix Amount12 g dehydrated milk powder and Amount12 g table sugar together in a condiment cup and place it inside the fly carton. Also replace the water-soaked cotton balls as necessary.

APPENDIX 1. CARTON CONSTRUCTION
APPENDIX 1. CARTON CONSTRUCTION
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.
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.
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).
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.
Figure 1. Access hole with sock stretched over it to create a sleeve.
Apply additional glue as needed to secure the sock or organza.
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.
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.
Stretch an elastic band over the top of the screen-covered carton to further secure the screen.
Tie the sleeve before adding insects to the carton (Fig 2.)
Figure. 2. Completed mosquito carton with sock sleeve knotted to prevent escape.
Protocol references