Collection of lung Schistosoma mansoni schistosomules

 

Author Fred Lewis

Introduction

 

Schistosoma mansoni lung schistosomules can be obtained in greatest numbers from mice ~5-8 days after cercarial penetration of the skin. The lung tissue must be chopped finely enough to allow the schistosomules to migrate out of the capillary beds and into the tissue culture medium.

Equipment

37ºC, 5% CO2 incubator

Dissecting microscope

Materials and reagents

S. mansoni-infected mice, exposed 5-8 days prior to procedure

RPMI 1640 tissue culture medium containing 10 units heparin/ml

Dissecting instruments

Iris scissors

10 ml syringe and 22 gauge needles

50 ml plastic centrifuge tubes

30 mesh stainless steel screen (~500 µm openings)

Procedure

·         Euthanize the 5-8 day exposed mouse.

·         With dissecting scissors, expose the thoracic cavity and perfuse the lungs (using a syringe with 22 gauge needle) by injecting 5-10 ml RPMI medium into the right ventricle of the heart.

·         Remove the lungs and place them in a petri dish with a minimal volume of RPMI medium. Remove excess tissue that may have been removed with the lungs (heart fragments, esophagus, etc.).

·         Place the lungs in a small beaker, with 1-2 ml RPMI 1640 medium. Mince the lungs into fine tissue fragments using iris scissors.

·         Place the suspension into 50 ml centrifuge tubes, bring the volume up to 50 ml with RPMI medium, and incubate for 3 hours at 37ºC.

·         Resuspend the organisms, and pass the suspension through a crude (30 mesh) screen into another 50 ml centrifuge tube.

·         Gently centrifuge the suspension (3 min at 100 x g), and remove most of the supernatant.

·         Resuspend the pellet and pour it into a petri dish. The schistosomules will be about twice the length of the cercarial body, but they are more vermiform in appearance.

Follow-up comments/recommendations

For S. mansoni, the peak time for recovery of schistosomules from the lungs depends on several factors, among which are the mouse strain and immunization status. In a normal mouse, peak recovery time is around 6 days after cercarial skin penetration. Re-incubation of lung fragments in RPMI medium [after the initial 3-hour collection] will increase the yield of schistosomules eventually collected. For maximal yield, re-incubation may be done up to 24 hours.

 

References

Sher, F.A., MacKenzie, P., and Smithers, S.R. 1974. Decreased recovery of invading parasites from the lungs as a parameter of acquired immunity to schistosomiasis in the laboratory mouse. Journal of Infectious Diseases 130: 626-634.

 

Lewis, F.A. and Colley, D.G. 1977. Modification of the lung recovery assay for schistosomula and correlations with worm burdens in mice infected with Schistosoma mansoni. Journal of Parasitology 63: 413-417.

 

Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Frietas, T. C., and Liang, Y-S. 2013. Schistosomiasis, in Current Protocols in Immunology 19.1.1-19.1.57, John Wiley and Sons, Inc., (R. Coico, Ed).  Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142735.im1901s103.