Percutaneous exposure of mice to Schistosoma mansoni

Author: Fred Lewis, PhD

Percutaneous exposure of mice to S. mansonivia the tail offers significant advantages over other means of infection. Skin penetration is the natural route of cercarial infection, andthere is no need to anesthetize the mice,whichare secured by restrainers. An investigator can obtain a relativeestimate of the percentage of cercarial penetration by countingremainingcercariae after the mousetail has been removed, and the mouse returned to its proper cage.

Mouse restrainers (Fischer Scientific, Broome-Style Rodent Restrainer)
12 x 75 mm plastic test tubes (5cc)
Test tube racks to hold 5cc tubes of cercarial suspensions
Mouse exposure apparatus-two “egg crate” ceiling light diffuser panels and spacers
Micropipette (Eppendorf 200 μl+) and tips
Adhesive tape (Johnson and Johnson zonas porous tape)
4” x 4” gauze pads
Kim wipes
Artificial pond water

1. Preparethe animal procedureroom by cutting two pieces of 3’’adhesive tapefor each mouse. Placesmall amounts of Kimwipes inside of the restrainers to prevent urine and fecal contamination of the cercarial suspensions.
2 .Pipette a pre-calculated*number of cercariae into the 12 x 75 mm tubes, then fill the tube with pondwater to within approximately 10 mm of the rim.
3. Place tubes spaced apart in the bottom rack, then place the spacersin the corners and add top rack(see image below).
4. Pick up a mouse by its tail and gently, but quickly,pull it backward into the restrainer (Fig. 1 left) until it is completely inside the restrainer and the tail is entirely through the opening in the bottom.
5. Hold the tail securely with pinchedfingers fromone hand while tightening the lid of the restrainer with the other hand. Be careful not to cramp the mouse in the restrainer but secure it sufficiently that it cannot escape.
6. With the mouse in the restrainer, attach two small pieces of adhesive tape to anchor the tail in place.
7. Wipe the tail clean with a gauze pad moistened with pond water.
8. Insert the tail into the cercarial suspension, resting the restrainer on the top of the rack(Fig. 1right).
9. After 1 hourremove the mouse, release it from the restrainer and return it to its cage.
10. Collect 5 random tubes from the mouse exposure apparatus and place them on a tube rack in the blue transport box.
11. Combine all 5 samples into a gridded watch glass and add a small volume of iodine to fix (kill) and stain the cercariae for visualization. Using a microscope and counter, count the number of cercariae quantify the penetration percentage.

Penetration percentage

Figure 1: Left: Broome-type mouse. Variations of these can be purchased commercially or can be engineered from plastics companies. Restrainers with diameter openings of about 25mm can restrain mice up to 20 gr, whereas larger ones (up to 30mm diameter openings) are needed for larger mice. Right: materials for mouse tail exposure to Schistosomacercariae. Shown are a base and upper level of fluorescent light panels (available in standard hardware stores), separated by spacers and anchored with Velcro strips. The height is adjusted to 75mm, to accommodate 75 x 20mm test tubes (not shown). A single mouse will be restrained in the Broome‐type restrainer, with its tail extending into the test tube.

*Refer to the Counting Cercariae protocol for pre-calculated number of cercariae. We use infection rates of 180-200 cercariae per mouse. This infection rate is ideal for life cycle propogation. Chronic infections (>8wk) requirefewer cercariae (Tucker et al. 2013).

One can count the number of non-penetrating cercariae by emptying the contents of the test tube into a counting dish after the mouse tail has been removed. One can usually obtain a reasonable estimate by counting thecontents from 4-5 randomly chosen tubes. Some cercariae may be intact (with a tail), whereas detached bodies may also be apparent. Cercarial tails will be numerous, but a count of the tails is an unreliable indicator of penetration success, since many of the detached tailsmay be caught up in the hair or tail skin of the mouse. Under the best of conditions, one can expect a penetration rate of at least 95%, or higher. A penetration rate of less than 90% may indicate a problem in the cercarial pool, or from some other source.

1. Lewis, F.A., Stirewalt, M.A., Souza, C.P., and Gazzinelli, G. 1986. Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations. Journal of Parasitology72: 813-829.
2. Stirewalt, M.A. and Bronson, J.F. 1955. Description of a plastic mouse restraining case.Journal of Parasitology41: 328.
3. Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Frietas, T. C., and Liang, Y-S. 2013. Schistosomiasis, inCurrent Protocols in Immunology19.1.1-19.1.57, John Wiley and Sons, Inc., (R. Coico, Ed).Published online November 2013 in Wiley Online Library ( doi: 10.1002/0471142735.im1901s103.NIH NIAID Schistosomiasis Resource Center, July 2021