Dec 11, 2023

Public workspaceIn vitro excystment of Juvenile Fasciola hepatica

DOI
dx.doi.org/10.17504/protocols.io.14egn212qg5d/v1
  • 1Queen's University Belfast
Paul McCusker
Open access
DOI: dx.doi.org/10.17504/protocols.io.14egn212qg5d/v1
Protocol Citation: Paul McCusker, Rebecca Armstrong, Duncan Wells, Emily Robb, Paul McVeigh, Aaron Maule, Erin McCammick, Nathan Clarke, Erica Gardiner 2023. In vitro excystment of Juvenile Fasciola hepatica. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn212qg5d/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 19, 2023
Last Modified: December 11, 2023
Protocol Integer ID: 79051
Keywords: Fasciola, Metacercariae, Excystment
Funders Acknowledgement:
Developing a 'validation portfolio' to exploit key virulence proteins in Fasciola species for parasite control
Grant ID: BB/H009477/1
LIVER FLUKE MOTOR FUNCTION AND PARASITE CONTROL: EXPLOITING A 'TARGET VALIDATION TOOLBOX' AS A DRUG SCREEN-INTERFACE FOR FLUKICIDE DISCOVERY
Grant ID: BB/K009583/1
Probing in vivo parasite biology in vitro
Grant ID: NC/N001486/1
Exploiting stem cell biology for liver fluke control
Grant ID: BB/T002727/1
Abstract
This protocol describes our excystment protocol for Fasciola hepatica supplied by Ridgeway Research Ltd. F. hepatica metacercariae (also known as mets or cysts) are supplied attached to Visking tubing with both inner and outer walls intact. This protocol builds upon methodology described by McVeigh et al. (2014) and McCusker et al. (2020) and has been tweaked by many lab members over the years. Our thanks to all who have contributed.

Guidelines
Storage of F. hepatica metacercariae
When mets are received from Ridgeway Research we move sheets from original sheets into 50 mL falcon tubes in fresh RO water. They are then stored at Temperature4 °C until required and the 'sheet is use' is moved into a Amount15 mL falcon tube.

Washing watchglasses
Watchglasses are rinsed with Concentration10 % (v/v) bleach before rinsing in RO water and dried out before subsequent use.

Materials
Excystment Salt Solution – 0.9% (Amount450 mg ) and 1.2% NaHCO3 (Amount600 mg ) in Amount50 mL RO Water.
1/20 HCl – Amount125 µL 1 N HCl and Amount2375 µL RO Water
Bleach solution – Amount100 µL ReagentSodium hypochlorite solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #1056142500 withAmount900 µL RO water and vortex

Safety warnings
Attention
Infectious Agent
F. hepatica is a category 2 pathogen. The metacercariae is the infectious stage of the life and if ingested can infect humans. Therefore all plasticware that comes into contact with metacercariae or NEJs is disposed of in Concentration10 % (v/v) bleach.

Outer metacercariae (met) wall removal
Outer metacercariae (met) wall removal
25m
Pipette Amount300 µL 50% chicken serum (CS50) onto large petri dish base and lid, spread with finger.

30s
Fill petri dish base with Amount10 mL RO water.

30s
Gently lift rolled-up met sheet out of tube, place in dish base and unwrap the sheet (ensure you know which way mets are facing).
1m
Add Amount100 µL of RO water to petri dish lid to ensure it stays damp. Lay the met sheet onto the lid with mets facing down. NB: avoid bubble formation.

30s
Use a scalpel to gently pop required number of mets out of outer wall (assume ~70-75% excystment rate). Outer wall is coloured brown whereas 'popped' mets are translucent. NB. only count viable mets (those with a bilobed appearance following popping).
10m
Add Amount10 mL water to petri dish lid before moving sheet back to petri dish base (mets facing up).

1m
Transfer mets from petri dish lid and base to a watchglass using a serum-lined tip.
10m
Solution Preparation
Solution Preparation
5m
Make the following solutions and warm to Temperature37 °C :

5m
Tube 1 – Dissolve Amount20 mg sodium tauroglycocholate inAmount2.5 mL Excystment Salt Solution in a 15 mL falcon tube.

Tube 2 – Dissolve Amount20 mg L-cysteine in Amount2.5 mL 1/20 N HCl in a 15 mL falcon tube.

Bleaching mets
Bleaching mets
1h 10m
Swirl watchglass to gather mets in centre. Remove as much water as possible without drying out mets.
1m
Add Amount1 mL bleach solution to mets and start timer (time varies between sheets/strains - typically ~2 min 30 s)

2m 30s
While bleaching is ongoing add Amount1 mL RO water to a new watchglass and serum line a p100 tip.

As timer approaches 0 swirl watchglass to group mets. As timer hits 0 collect mets in serum-lined p100 tip and move into watchglass with water.
30s
Swirl mets to group, remove as much water as possible and add another Amount1 mL RO water to wash

1m
Repeat step 12 x5.
5m
After final wash move mets to fresh watchglass using a serum lined p100 tip in Amount50 µL .

30s
Mix tubes 1 and 2 from step 8 together (some effervesce should be visible). Briefly vortex and ensure that the resulting solution is not cloudy. NB. if cloudy, do not add to worms and remake.
1m
Pour mixed solutions onto mets and place clean watchglass on top to reduce evaporation.
1m
Incubate in a tupperware lunchbox (line base with damp paper towel) for 1 h at Temperature37 °C .

1h
Collection of new excysted juveniles (NEJs)
Collection of new excysted juveniles (NEJs)
2h
Collect NEJs with a p10 and place into pre-warmed RPMI (glass watchglass is best). After collecting excysted worms continue incubation at Temperature37 °C and collect newly excysted NEJs at 20 min intervals until 3 h after addition of excystment solutions.

2h
Protocol references
McCusker, P., Hussain, W., McVeigh, P., McCammick, E., Clarke, N. G., Robb, E., McKay, F. M., Brophy, P. M., Timson, D. J., Mousley, A., Marks, N. J., & Maule, A. G. (2020). RNA interference dynamics in juvenile Fasciola hepatica are altered during in vitro growth and development. International Journal for Parasitology - drugs & drug resistance, 14, 46-55. https://doi.org/10.1016/j.ijpddr.2020.08.004

McVeigh, P., McCammick, E. M., McCusker, P., Morphew, R. M., Mousley, A., Abidi, A., Saifullah, K. M., Muthusamy, R., Gopalakrishnan, R., Spithill, T. W., Dalton, J. P., Brophy, P. M., Marks, N. J., & Maule, A. G. (2014). RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro. PLoS Neglected Tropical Diseases, 8(9), [e3185]. https://doi.org/10.1371/journal.pntd.0003185