Perfusion of Schistosoma haematobium from hamsters

 

Authors Yung-san Liang, Mei-Shei Su and Fred Lewis

 

Introduction

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.

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.)

Silicone tubing (e.g., Masterflex 96420-14) fitted with a 21 gauge (2”) needle

Dissecting instruments

Dissecting microscope

Small pan or tray for collecting perfusate

Materials and reagents

Sodium pentobarbital  (200 mg/Kg), containing heparin (200U/ml)

Perfusion fluid (0.85% NaCl and 0.75% sodium citrate)

70% ethanol

Procedure

·         Euthanize the hamster with an intraperitoneal injection (0.5 ml) of sodium pentobarbital.

·         Before dissection, moisten the fur with warm water or 70% ethanol, 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 descending aorta.

·         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 5 mm below its entrance into the liver.

·         With the 21 gauge needle still in place in the aorta, 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 50% of the S. haematobium adult worms still 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 located in the mesenteries, they can be recovered by opening the mesentery vein wall with fine forceps and gently removing them through the opening. Most worms can be recovered by this procedure without breaking them.

 

Follow-up comments/recommendations

Due to the size of the hamster relative to the mouse, collecting S. haematobium adult worms is more time consuming than collecting S. mansoni. In addition, collecting all worms by perfusion alone is not possible, so one should plan to collect additional worms from the mesenteries of the cecum and large intestine.  

 

References

Duvall, R.H. and DeWitt, W.B. 1967. An improved perfusion technique for recovering adult schistosomesfrom laboratory animals. The American Journal of Tropical Medicine and Hygiene 16: 483-486.

 

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.