Collection of S. haematobium eggs from hamster guts

 

Authors Fred Lewis, PhD, Sarah Li, Frances Barnes, Mei-Shei Su and Yung-san Liang, PhD

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Introduction

Schistosome eggs are collected for experimental use primarily from either livers or intestinal tissue of infected animal hosts. This procedure describes the isolation of eggs from the large intestine of S. haematobium-infected LVG hamsters. To obtain the cleanest preparation of eggs, the hamster should first be perfused with adult worms lest the final egg preparation contain unwanted adult worm fragments.

Equipment

  • Spray apparatus (Deck sprayer, 2-gallon, pump-type, typically found in hardware stores)
  • Stainless steel sieves of decreasing pore sizes (Newark Wire Cloth Company; mesh openings of
  • 420 µm, 180 µm, 105 µm, and 45 µm)
  • Waring blender, with variable speed control
  • Glass petri dishes (100 mm diameter) with flat bottom
  • Pasteur pipettes
  • Light box
  • 15 mL conical test tubes

Materials and reagents

  • 2% NaCl (2 liters)
  • haematobium infected hamster

Procedure

  1. Dissect the large intestine (the SRC uses the ascending colon to the anus) from the hamster.
  2. Cut the large intestine into small pieces (~15cm) before homogenizing in a Waring blender.
  3. Place the large intestine in cold 1.2% NaCl overnight.
  4. Day 2: homogenize large intestine in a Waring blender for 30 seconds until it has a smooth consistency.
  5. Place the homogenate in the top tier (420 µm) of stainless-steel sieves and allow it to pass through the tier of stacked sieves, from the largest pore size on top to the smallest pore size on the bottom tier while rinsing the tissue continuously on the top sieve with a spray apparatus holding 1.2% NaCl. Agitate the sieve throughout the entire process to ensure that most of the eggs will pass through to the lowest sieve.
  6. For best results, re-homogenize (blender) the homogenate trapped on the top two sieves and pass the homogenate through the sieves again, using the technique described.
  7. Remove the upper three sieves; the fluid remaining in the lowest sieve (45 µm pore size) will contain the eggs.
  8. Pour the suspension into a glass petri dish with about ½ full of 1.2% cold NaCI solution. The egg suspension will contain debris from the large intestine wall and digested food. The debris floating at the top can be removed with a Pasteur pipette.
  9. Add 0.5mL Ampicillin (100mg/mL) into the petri dish with the egg suspension and mix well.
  10. Complete steps 11 and 12 as described below to concentrate more eggs.
  11. Pour the suspension into a glass petri dish and add 1.2% cold NaCl so that it is about ½ full. The egg suspension will contain eggs of several stages of maturation. Cold NaCl will keep most of the eggs from hatching into miracidia; keep eggs on ice in between swirling. To enrich the population for mature eggs, gently swirl the dish over a light box (for better visibility). Mature eggs will concentrate in the center of the vortex. These can be withdrawn with a Pasteur pipette and placed in a 15 mL test tube on ice. Keeping the volume of the egg suspension in the petri dish constant by adding fresh cold 1.2% NaCl as needed, continue to swirl the dish and collect eggs from the center until no more can be seen concentrating in the center. After 3-4 complete cycles, the resulting egg population will be highly enriched in mature eggs.
  12. Centrifuge eggs for 5 minutes at 100 x g. Pour off supernatant and freeze eggs as a dry pellet.

 

Comments

The greatest yield of eggs from hamster liver and large intestine tissue can be obtained from hamsters that were exposed to approximately 350 S. haematobium (Egypt) cercariae, 3 ½ to 4-month post-infection (> 50,000 mature eggs per hamster).

References

  1. 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., Nov 2013 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142735.im1901s103.

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Biomedical Research Institute