The Mechanism of Immunity of Mice to Nematospiroides dubius

Abstract

The serum of mice immune to Nematospiroides dubius contains antibodies which can be detected by immunoelectrophoresis, excretory pore precipitates on exsheathed third-stage larvae, passive cutaneous anaphylaxis, and tests for skin-sensitizing antibodies. Immune mice are subject to anaphylactic shock on intravenous injection of worm antigens, and the intestines of such mice participate strongly in the reaction. They also respond with cutaneous anaphylaxis to the intradermal injection of worm antigens. Anaphylactic shock induced by a heterologous antigen-antibody combination (horse serum-antihorse serum) causes a significant reduction in the worm burdens of mice undergoing a primary infection with N. dubius. Hence changes associated with anaphylaxis prevent the establishment of a large proportion of larvae. The stomach of immune mice becomes pale and contracts in diameter about a half an hour after the administration of larvae. Changes in physico-chemical conditions in the stomach probably accompany this reaction. It is concluded that a state of immediate hypersensitivity is involved in the immunity of mice to N. dubius. It is suggested that, in immune mice, a fresh intake of larvae initiates an anaphylactic reaction, and that changes associated with this reaction prevent a large proportion of the inciting larvae from becoming established. In a previous paper (Panter, in press), it was shown that mice develop an immunity to Nematospiroides dubius after two or three stimulating infections of 200 larvae. The reduction in worm burdens of immune mice (mice infected for the third or fourth time) occurred largely before the larvae encysted in the small intestine, i.e. during the first 2 days of infection. For this reason, the action of antibodies, rather than cells, was suspected. The present paper describes experiments on the mechanism of immunity of mice to N. dubius. MATERIALS AND METHODS Preparation of worm antigens and mouse serum Worm antigens were prepared from adult N. dubius worms by grinding washed worms in 0.85% NaCl (allowing 4 ml of saline to 1 g of worms), and standing at 4 C for 2 hr. The sediment was removed by centrifugation for 2 min in a Beckman/Spinco microfuge. The supernatant-worm antigens-was used immediately or stored at -15 C in screw-cap bottles. No preservatives were added. The protein content of the preparation was approximately 30 mg/ml. Mouse blood was obtained by cardiac puncture, allowed to stand for 1 hr at room temperature, and the serum removed and spun at 750 g for 10 min. Immunoelectrophoresis The technique of immunoelectrophoresis was adapted from those presented in Crowle (1961) and Wieme (1959). Difco Bacto-agar (final concentration 1%) in barbital buffer pH 8.4, and containing 1:100 merthiolate as a preservative, was Received for publication 22 March 1968. used to pour slides. The worm antigen preparation was applied to holes cut in the agar. The slides were placed in an electrophoresis box, and each was subjected to a current of 21/2 milliamps, with a potential difference of 30 volts across the slide. The troughs cut between the holes were charged with serum from infected and non-infected mice. Precipitin lines were allowed to develop for 48 hr. The lines were stained with amidoblack (Wieme, 1959). Effect of immune serum on larvae in vitro Exsheathed third-stage larvae were obtained from mice infected 16 to 18 hr previously, by incubating the stomach in saline at 37 C for 30 min. The larvae were washed and suspended in a drop of immune serum (from mice infected 3 or 4 times with 200 N. dubius larvae) or normal serum or saline on a slide. The coverslip was sealed, and the worms observed under the microscope, magnification 300 X. Active systemic anaphylaxis In a preliminary study of anaphylactic reactions in immune mice, Evans blue was injected intravenously in an attempt to show areas of increased capillary permeability at sites of infection. Immune mice, at various times after a fourth infection with N. dubius, were warmed beneath a 100-watt lamp, then injected via a lateral tail vein with 0.07 ml of a 1% solution of Evans blue in saline. Thirty minutes later, the mice were killed and the intestines examined. Uninfected mice were injected and killed in the same way. Cutaneous anaphylaxis The method of Ovary (1958, 1964) for active cutaneous anaphylaxis was followed. Volumes of 0.05 ml of 1:20 antigen solution and saline were injected into the skin of the abdomen of immune and normal mice. Immediately after the intrader-