Sep 03, 2025
Necropsy and recovery of ascarids of poultry for burden quantification or transplantation
This protocol is a draft, published without a DOI.
- J.B. Collins1
- 1Johns Hopkins University
Protocol Citation: J.B. Collins 2025. Necropsy and recovery of ascarids of poultry for burden quantification or transplantation. protocols.io https://protocols.io/view/necropsy-and-recovery-of-ascarids-of-poultry-for-b-g535by8q7
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: In development
We are still developing and optimizing this protocol
Created: July 23, 2025
Last Modified: November 05, 2025
Protocol Integer ID: 223069
Keywords: recovery of poultry ascarid, poultry ascarid, mechanisms of benzimidazole resistance, recovery of ascarid, benzimidazole resistance, recovery of parasite, poultry for burden quantification, transplantation the necropsy, parasite burden, ascarid, parasite, poultry, transplantations for genetic cross, necropsy, transplantation
Abstract
The necropsy and recovery of poultry ascarids is essential to ongoing work, determined to study the mechanisms of benzimidazole resistance in ascarids. Recovery of parasites is essential for quantification of parasite burdens, as well as for the preparation of controlled transplantations for genetic crosses.
Guidelines
If recovering adult parasites, euthanize animals 30-35 days post-infection. If recovering L4 parasites for transplantation, euthanize animals 19-20 days post-infection.
Materials
50 mL conicals (Grainger #6VMY3 or similar)
10 cm petri dishes (Grainger #48TD81 or similar)
4 L Physiological saline (8.5 grams of NaCl dissolved into 1 L of dH2O)
Size 5 Gel capsules ()
Necropsy kits (Fisher #S07893 or similar)
Dissecting microscope
85 µm Sieve (Fisher Cat: 04-881-27 or similar)
Troubleshooting
Safety warnings
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Ethics statement
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
General
Humanely euthanize the animal per your IACUC-approved protocol—for example, CO2 asphyxiation followed by cervical dislocation.
Position the animal, ventral side up, with feet facing towards you.
Carefully, with scissors, cut through the ribs on both sides of the breastbone. You should be able to lift and bend the rib cage toward the head, exposing the internal organs.
Open the peritoneum (the sheet of smooth tissue that lines the abdominopelvic cavity and surrounds the abdominal organs) to reveal the internal organs. If recovering Ascaridia galli or Ascaridia dissimilis, proceed to step 5. If recovering Heterakis gallinarum, proceed to step 11.
Ascaridia galli/ Ascaridia dissimilis
Find the top of the duodenal loop. The duodenal loop surrounds the pancreas as shown below. Cut, with scissors, where the duodenum begins (red line below) at the gizzard and gently pull the intestine out of the body cavity.
Locate Meckel’s diverticulum, which should appear as a small protrusion towards the posterior end of the intestine. Cut, with scissors, just below the diverticulum and fully remove the small intestine.
Using a small pair of necropsy scissors, carefully cut up the vertical length of the small intestine. Using your fingers, gently scrape the contents from the intestine. Collect the contents in a 50 mL conical.
Add approximately an equal amount of physiological saline and mix well. If transplanting parasites, proceed to Step 18. If recovering adult parasites, proceed to Step 9.
Pour a small amount (a thin enough layer that it spreads across the plate, but does not make piles) of the intestinal contents into a clean 10 cm petri dish and collect any adult/larval ascarids. Repeat until all of the contents have been processed. Record the total number of ascarids recovered. Place collected ascarids from an individual animal into an individual 15 mL conical tube of physiological saline and label with the strain, treatment status, and bird number.
Dispose of the animal carcass and tissue into a biohazard bag. Be aware of maximum weights for biohazard disposal, ensuring that you do not exceed eight allowed in one bag.weight limits.
Heterakis gallinarum
Find the top of the duodenal loop. The duodenal loop surrounds the pancreas as shown below. Gently, tease out the full length of the intestine using your hands to gently pull the intestine from the carcass.
Locate the cecal pouches attached to the posterior end of the intestinal tract. Remove the ceca by cutting at the junction where it joins the intestine (black line)
Using scissors, open the cecal pouches laterally. Using dH20 water, rinse the ceca over an 85 µm sieve, ensuring that all cecal contents are rinsed from the tissue.
Rinse as much debris as possible through the sieve. Using dH20 water, rinse all remaining contents on the sieve into a 50 mL conical tube.
Add approximately an equal amount of physiological saline and mix well. If transplanting parasites, proceed to Step 18. If recovering adult parasites, proceed to Step 16.
Pour a small amount of the recovered contents (a thin enough layer that it spreads across the plate, but does not make piles) into a clean 10 cm petri dish and collect any adult ascarids. Using a dissecting microscope, observe and enumerate H. gallinarum adults present in the sample. Repeat until all of the contents have been processed. Record the total number of ascarids recovered. Place collected ascarids from an individual animal into an individual 15 mL conical tube of physiological saline and label with the strain, treatment status, and bird number.
Dispose of the animal carcass and tissue into a biohazard bag. Be aware of maximum weights for biohazard disposal, ensuring that you do not exceed weight limits.
Transplantation of larval parasites
Resuspend intestinal/cecal contents in physiological saline. Pour a small amount of the recovered contents (a thin enough layer that it spreads across the plate, but does not make piles) into a clean 10 cm petri dish. Viewing under a dissecting microscope, identify and recover any L4 parasites and place into a 10 cm petri dish containing warmed physiological saline. Store larvae in a 37C incubator while processing additional samples.
Using a dissecting microscope, separate male and female larvae into independent petri dishes of physiological saline. Clearly label dishes for parasite isolate and sex. Males are distinguished by the presence of pre-anal suckers.
Remove the top portion of each gel capsule. Using a 200 uL micropipette set to 20 uL, pipette the desired number of males or females of each isolate for the designated cross. Pipette the animals into the bottom portion of the capsule. Repeat for the opposite sex, for a total volume of 20 uL.
Replace the top of filled capsules and store at 37C while preparing additional capsules.
For infection, gently restrain the recipient animal between your non-dominant arm and body. Gently extend the animal's neck upward.
Using your other hand, pick up a capsule, gently open the animal's mouth, and place the capsule as far back in the throat as possible. Allow the animal to swallow the capsule before releasing.
Monitor animals for 15-20 minutes after infection to ensure no adverse events occur.
Acknowledgements
Graphics made in Biorender.com.