Perfusion of adult S. haematobium worms from infected hamsters
Collection of S. haematobium adult worms is much the same principle as that described for collection of adult S. mansoni worms from mice (e.g., perfusion from the portal venous system). The best time for recovery of adult worms is approximately 3 ½ – 4 months after cercarial exposure. This amount of time allows for recovery of fully mature worms and for maximum egg recovery from the tissues.
Hamster do not exhibit the urogenital form of chronic schistosomiasis typically found in humans. Adult worms will mature in the mesenteric veins draining the large intestine. Roughly 30-50% of worms can be collected from large intestine section.
Equipment
Peristaltic perfusion pump (e.g., Masterflex Console Drive/Cole-Palmer Inst. Co.)
Foot-pedal for peristaltic pump (e.g., Treadlite II/Linemaster Switch Corp.)
Vacuum Pump*
Vacuum suction device with a 45-micrometer sieve*
Silicone tubing (e.g., Masterflex 96420-14) fitted with a 21gauge (2”) needle.
Dissecting instruments
Dissecting microscope
Small pan or tray for collecting perfusate
Materials and reagents
Euthanasia cocktail containing heparin (200U/ml)
Perfusion fluid (0.85% NaCl and 0.75% sodium citrate)
70% ethanol
Procedure
- Euthanize the hamster.
- Before dissection, moisten the fur with warm water, to reduce the amount of hair that collects in the perfusate.
- Dissect the skin from the abdominal wall.
- Make an incision in the abdominal wall to expose the viscera.
- Cut through the diaphragm and the hamster’s left rib cage to expose the descending aorta.
- At this point it is advisable to anchor the carcass on a support, or screen, over the collecting vessel, allowing free use of both hands while perfusing.
- Insert the 21-gauge needle (fitted to the end of the silicone tubing) into the Left ventricle.
- Pump a small amount of fluid to verify that the needle is inserted adequately, as evidenced by a swelling of the portal vein.
- Slit a small hole in the portal vein, about 10 mm below its entrance into the liver.
- Pump perfusion fluid through the venous system.
- The perfusate, which will include many of the adult worms, can be collected in the underlying collecting vessel or pan. *Alternatively, one can use a vacuuming (suctioning) apparatus to collect the perfusate.
- Once the mesenteries are cleared of blood, one should examine the mesenteric vasculature (using a dissecting microscope) to locate other worms. In contrast to S. mansoni adult worm collection from mice (or hamsters), roughly 30- 50% of the S. haematobium adult worms remain in the mesenteric veins, even after prolonged perfusion. For a complete collection of worms, therefore, it is imperative to perform this dissection. Most of the worms will be in the mesenteries of the cecum and large intestine; they are rarely found in the mesenteries of the small intestine.
- When adult worms are in the mesenteries, they can be recovered by opening the large intestine (U-sharp fold) with fine forceps and gently removing the fat tissue section through the opening. Most worms can be recovered by this procedure without breaking them.
- To minimize the impact of damage to the worms from dissecting the large intestine, hold the fine forceps and pull out the small fat tissue from the base of the intestine wall. Place the tissue in a petri dish with perfusion fluid or cell culture medium Some of the worms will migrate from the tissue to the fluid overtime.
*Vacuum method
