The Intradermal Test in the Diagnosis of Schistosomiasis mansoni. IX. Skin Response to a Purified Fraction Isolated from Cercarial Extracts

Abstract

Butanol powder from lyophilized cercariae (Schistosoma mansoni) was extracted either with 0.9 NaCI or 0.1 M borate buffer pH 8.0, precipitated at pH 4.7 and the supernatant (total extract) fractionated by column chromatography (DEAE-Sephadex). The fractions obtained were assayed for protein and polysaccharide and skin tested in patients with active schistosomiasis mansoni. Several fractions were found to elicit a skin response. One of them (fraction D, in which no polysaccharide was detected) was at least five times more active than the total extract and contained only 20% of the absorbing material at 280 mu. The skin test is a useful and practical tool for diagnosis and epidemiological surveys of schistosomiasis (Kagan and Pellegrino, 1961). However, only crude extracts or incompletely purified antigens from cercariae or adult schistosomes have been used for this test. Maximum advantage would be realized with the use of purified and chemically defined antigens. In the course of a study aiming at the purification and characterization of antigenic, enzymatic, and biologically active components extracted from cercariae of S. mansoni, several fractions were obtained. This paper presents the results of skin tests performed with fractions obtained from column chromatography on DEAE-Sephadex. MATERIAL AND METHODS Cercarial extracts from lyophilized cercariae Large numbers of Australorbis glabratus infected with S. mansoni (200 to 500 snails) were isolated in small volumes of water under conditions favoring the emergence of cercariae. The heavy cercarial suspension was sedimented overnight in conical flasks in the cold room at 5 C. The supernatant was carefully aspirated and the packed cercariae washed twice with distilled water, and then transferred to glass ampules and shell frozen at -70 C. Lyophilized cercariae were extracted with dry n-butanol at 0 C for about 20 min with continuous stirring (5 ml of butanol/100 mg of cercariae), and the extract separated by centrifugation. The residual material was reextracted with dry butanol Received for publication 1 March 1965. * This work was supported by grants from the Research Office of the U. S. Army, the WHO, the Rockefeller Foundation, and the Conselho Nacional de Pesquisas. 753 as before and with dry acetone at 15 C. The organic solvents of the precipitated material were evaporated under vacuum over CaC12. The butanol powder was then extracted twice either with 0.9% NaCl or 0.1 M sodium borate, pH 8.0, for periods of 2 hr with continuous stirring at 0 C. Some extracts were precipitated at pH 4.7 with 0.2 M HC1 according to Melcher (1943). The 10,000-g supernatant (total extract) was dialyzed against 0.02 M EDTA buffered at pH 7.0 before being applied to the chromatography column. Fractionation of the extract The fractionation was carried out in an 0.8 by 27-cm column of DEAE Sephadex A-50, medium (Pharmacia Fine Chemicals, Sweden), previously equilibrated with 0.02 M EDTA (Kent, 1963). The elution was performed with an NaCl gradient up to 1 M NaCl, obtained by interposing in the line between the reservoir and the column, a 50-ml suction flask containing a magnetic stirring bar. The gradient was started as soon as the extract entered into the resin. The flow rate was 2 to 3 ml/hr and fractions of approximately 1 ml were collected by means of an automatic fraction collector and read with Beckman DU spectrophotometer at 280 m,i. Protein and polysaccharide determination The protein content of the samples was determined by the phenol-biuret reagent of Lowry et al. (1951). The polysaccharide was assayed by the anthrone reagent (Seifter et al., 1950). A glucose standard was always included and the results expressed as glucose.