CHARACTERIZATION OF KINETICS OF ANTI-TRICHINELLA SPIRALIS NEWBORN LARVAE IMMUNITY IN RATS

Ching-Hua Wang

Biology Department, California State University, 5500 University Parkway, San Bernardino, CA 92407, USA

Received 5/11/98 Accepted 5/24/98

3. MATERIALS AND METHODS

3.1. Experimental animals and infection

Adult male or female inbred AO rats weighing 150 to 250 g were used in these experiments and they were bred pathogen-free at the J. A. Baker Institute vivarium. Within one experiment, rats of only one sex were used. T. spiralis was maintained by serial passage in irradiated rats as donors. For infections with muscle larvae, from 500 up to 6,000 infective larvae, isolated by pepsin-hydrochloric acid digestion of infected muscles obtained from the donor rats, were administered orally to each rat. For infections with newborn larvae, 20,000 of the newborn larvae were freshly collected by incubating 6-day-old adult worms obtained from the small intestines of the donor rats for 24 hr in culture medium at 37^oC, separated from the adult worms after incubation, and then injected i.v. to each rat. For challenge infections, newborn larvae of various doses ranging from 10,000 to 100,000 were injected either i.p. or i.v. into each rat.

3.2. Examination of muscle larvae burden

The muscles were ground and digested overnight in 1,500 ml of 1 % pepsin in 1% HCl acid at 37⁰C. The digestant was poured through cheesecloth and passed through a No. 200 mesh sieve to retain the larvae. After rinsing larvae from the screen of the sieve, they were collected and counted under a dissecting microscope.

3.3. Recovery of newborn larvae from peritoneal fluid and portal vein blood

To recover NBL from the peritoneal cavity of the rats, infected or challenged rats were injected i.p. with 10 ml of heparinized 0.85% saline warmed to 37⁰C. The abdomen of the rat was massaged briefly, opened, and the fluid aspirated. The peritoneal fluid was centrifuged at 300 x g, and NBL were resuspended in 5 ml of 0.85% saline for enumeration. To recover NBL from portal vein blood, 1 ml of portal vein blood was taken and added to a test tube containing 1 ml of heparinied medium. After laking the blood with distilled water, the pellet was resuspended in 1 ml of 0.85% saline for counting. Since the number of larvae in 1 ml of blood sample rarely reached 50, the whole sample was examined. The possibility that some larvae might be lost by hypotonic shock in distilled water was investigated by placing NBL in distilled water for as long as 5 hr. No swelling or bursting was observed.

3.4. Determination of cell adherence to newborn larvae and viability of newborn larvae

In normal rats, cell adherence did not occur on more than 96% of larvae recovered from the peritoneal cavity 6 hr after injection. Less than 0.5% of the larvae recovered from the peritoneal cavity had 3 cells on their cuticle and no larva had more than 3 adherent cells. Based on this result, a finding of 4 or more cells adhering to NBL was considered positive adherence. Anything less was considered non-specific binding. To determine the viability of newborn larvae bearing host cells, larval motility was observed. Since live larvae move actively and constantly and dead larvae are straight and non motile, larvae lacking motility for 10 seconds and having a straightened appearance were recorded as dead.

3.5. Quantitation of immune protection against newborn larvae challenge

In the experiments performed here, when no significant difference (p>0.05) was found between muscle larvae burdens of experimental group rats and immunization control rats, this was taken to indicate that the experimental rats were 100% protected from the challenge infection. When the probability value was less than 0.05 between experimental and control groups, the percent immune protection against newborn larvae challenge was calculated as follows:

3.6. Kinetics of cuticular cell adherence estimated in vitro and in vivo

AO rats were randomized into 11 groups of 3-4 rats. On day 1, 8 groups of rats were infected with 2,000 muscle larvae. Then, 7, 9, 10, 12, 13, 16, 18 and 21 days after infection, blood samples of 1 ml were taken from the tail vein of rats in one group per day. Immediately after blood collection, 20,000 newborn larvae were injected into the peritoneal cavity of each rat and then 6 hr later, peritoneal fluid was obtained and examined for newborn larvae. To determine cuticular adherence of blood leukocytes, individual blood samples were added to test tubes containing 0.5 ml of heparinized 0.85% saline and, after addition of 500 newborn larvae, the tubes were incubated at 37⁰C on a hematology mixer (Fisher Scientific Co., U.S.A.) for 6 hr. Each blood sample was then laked with distilled water and examined microscopically for newborn larvae. Rats in 3 control groups were examined on days 7, 14 and 21 with identical experimental procedures.

3.7. Effect of immunization dose on anti-newborn larvae immunity

Rats (16) were randomized into four groups, 1 control and 3 immunized. The three immunized groups were infected respectively with 500, 2,000 and 5,000 T. spiralis muscle larvae on day 1. On day 9, 5 ml of sterile 0.85% saline containing heparin (20 u/ml) was injected into the peritoneal cavity of individual immunized and control rats that had been lightly anesthetized with ether. After a brief abdominal massage the peritoneal fluid was aspirated with a 5 cc syringe under sterile conditions. Each peritoneal fluid sample was centrifuged at 500 xg for 10 min at 4⁰C and the supernatant was kept at room temperature for later usage. The cell pellet was resuspended in 1 ml of the saved peritoneal fluid supernatant and counted in triplicate using a Coulter Counter. The reason for doing this was to include other possible factors, e.g., antibodies (in the fluid or on the cells) in the in vitro incubation experiment so that the results of this experiment would be directly comparable to the in vivo examination done on day 16. When the cell concentration in all 16 test tubes was determined, the one that had the lowest cell concentration, 3.4 x 10⁶ cells/ml (obtained from a rat in the immunized group infected with 5,000 muscle larvae) was selected as the standard. The original peritoneal fluid was used to adjust cell concentration in all other test tubes to 3.4 x 10⁶ cells/ml. When the cell concentration of all 16 samples had been standardized, 2,000 newborn larvae in 0.1 ml of RPMI-1640 were added to each test tube and all were further incubated at 37⁰C in 5% C0₂ in air. After 1, 2, 3 and 5 hr, 50 ul samples were taken from individual test tubes and examined microscopically for newborn larvae, cell adherence and larval killing.

To examine the effect of variation in immunization dose at a later stage of T. spiralis infection, the same rats used above were injected with 25,000 newborn larvae i.p. on day 16. After 6 hr, 10 ml of heparinized 0.85% saline was injected i.p. and peritoneal fluid of these rats was obtained after a midline incision on rats anesthetized with ether. Peritoneal fluid samples were examined for the same parameters described above.

3.8. Statistical analysis

Within experiments, individual groups contained from 4 to 10 rats. Tabular data were recorded as the arithmetic mean + 1 standard deviation, unless otherwise stated. Data were analyzed with analysis of variance and paired means were compared using Student-Newman Keuls

t test. Probability values equal to or less than 0.05 were considered significant.