A Sieving Method for the Collection of Schistosome Eggs from Mouse Intestines

Abstract

The principal lesions in schistosomiasis are caused by host reactions against the eggs of the causative organisms. Increasing interest in the immunology and physiology of schistosome eggs during the last several years necessitates methods for isolation of these eggs in pure form. Such methods generally consist of digestion of tissues from infected animals by either autodigestion or the addition of proteases such as trypsin, homogenization, screening to remove host tissue debris, and collection and concentration by centrifugation and/or sedimentation. The methods published to date have utilized principally liver tissue from infected animals (hamster or mice) because this tissue is relatively easy to work with. Nevertheless, the intestines of infected animals are very rich in eggs and should provide a good source for studies. This report presents details of a method to isolate large numbers of sterile schistosome eggs from the intestines of mice infected with S. mansoni or S. japonicum. Mice infected with Schistosoma mansoni (Puerto Rican strain) or Schistosoma japonicum (Formosan strain) were sacrificed at 7 to 8 wk postinfection by injection with 0.2 ml sodium pentothal and the intestines removed and washed in tap water; the large intestine was cut off and discarded. The small intestines were cut longitudinally with a sharp scalpel and the fecal contents and mucosal layers scraped with a scalpel and discarded; mesenteric tissue and surrounding fatty tissue were also removed surgically. As noted by Coker and Lichtenberg (1956, Proc. Soc. Exp. Biol. Med. 92: 780-782), the more residual fat, the more difficult it is to obtain clean eggs. The intestines were then pooled in 0.2 M NaCl and left overnight at room temperature. The following day, the intestines in batches of ten are suspended in 100 ml of 0.2 M saline and rapidly disrupted in a commercial model Waring Blendor (Model 31BL46). The disruption procedure consists of two consecutive 20sec bursts of homogenization with a short, 10sec period between each burst. The homogenate is immediately poured over a 20-cm diameter U.S. Standard Size Sieve #40 (sieve opening = 420 mgu) and washed with 500 ml saline. After all intestines are disrupted and passed through this filter, the material is passed through a series of 20-cm U.S. Standard sieves stacked as follows: #40 (420 m,i), #80 (177 m,u), #100 (149 mgi), #200 (74 mit), #325 (44 mgx); approximately one liter of saline is used to rinse these sieves, by gravity. The eggs pass through all the filters except the last where they are collected, resuspended in 500 ml saline and once again run through the stack of sieves. The eggs collected on the #325 sieve are washed extensively (some immature eggs, egg shell fragments and small debris pass through this sieve, as well as bacterial contaminants) with sterile saline. The eggs are finally collected by rinsing from the #325 sieve with sterile saline into conical 50-ml centrifuge tubes and centrifuged in a Sorvall GLC-1 centrifuge. By bringing the centrifuge to 2,000 rpm and then immediately shutting it off, the eggs are concentrated while some debris will float to the top from which it can be removed. The methods described above are not novel; similar sieving methods have been reported for mouse liver by Griffith and Beesley (1955, Trans. Roy. Soc. Trop. Med. Hyg. 49: 301), hamster tissues by Ritchie and Berrios-