An ultraviolet-attenuated egg vaccine for swine ascariasis: parameters affecting the development of protective immunity.

Pigs inoculated with UV-irradiated Ascaris suum eggs developed immunity against challenge exposure with A suum infective eggs. Trials were conducted to determine the optimal conditions necessary to induce high levels of protective immunity and minimize liver lesions related to larval migration. Protective immunity was defined as the percentage of reduction in the recovery of larvae from the lungs of inoculated pigs, compared with controls, 7 days after challenge exposure with 5 X 10(3) or 10(4) infective eggs. Initial experiments showed that protective immunity was higher in pigs inoculated orally with UV-irradiated eggs than in those inoculated intraperitoneally. The highest level of protection (94%) was obtained by immunization with 5 oral inoculations of 10(4) eggs that had been UV-irradiated at total energy doses (ET) of 150, 100, 75, 75, and 75 mu W-min/cm2, respectively. A level of 83% protection was induced with as few as 2 oral inoculations of eggs irradiated at 150 and 75 ET, respectively. However, protection was reduced when pigs received eggs that were irradiated at and above 400 ET. The minimum number of irradiated eggs required to induce significant (P less than 0.05) protection was determined by feeding pigs 10(4), 10(3), or 10(2) eggs irradiated on 3 consecutive weeks at 150, 100, and 75 ET; the levels of protection induced were 94%, 89%, and 83%, respectively. Pigs inoculated with equal numbers of UV-irradiated eggs or normal eggs developed a similar level of protective immunity after challenge exposure. However, worms developing from the inocula were detected in the intestines of some of those pigs that had received normal eggs, but not in those pigs given UV-irradiated eggs.

Development of immune responsiveness to Ascaris suum antigens in pigs vaccinated with ultraviolet-attenuated eggs.

Pigs fed Ascaris suum eggs attenuated by short wave ultraviolet-radiation developed resistance to challenge infection. Per os inoculation of pigs on three successive weeks with 10,000 eggs irradiated to total exposures of 150,100 and 75 microW-min/cm2, respectively, resulted in an 88% reduction in the number of larvae recovered from the lungs 7 days after challenge with 10,000 infective eggs. Peripheral blood lymphocytes (PBL) from vaccinated pigs were specifically stimulated in vitro to incorporate tritiated thymidine by egg hatching fluid (Ea) and by excretory-secretory products obtained from cultures and from cultures of third-stage larvae developing to fourth-stage (L3-4). PBL were also specifically stimulated by living L2. Ea and L2 stimulated pig PBL significantly at 7 days after the first inoculation; responses to L2-3 and L3-4 developed 7 days after a second inoculation. The antigen-responsive cells in the PBL population were non-immunoglobulin-bearing lymphocytes. Antibodies to Ea and L2-3 were not detected in the serum of vaccinated pigs, and only 3 of 7 pigs had low concentrations of serum antibodies to L3-4.

Immunization of pigs against experimental Ascaris suum infection by feeding ultraviolet-attenuated eggs.

Pigs fed Ascaris suum eggs attenuated by short-wave ultraviolet radiation developed a resistance to challenge infections that varied according to the protocols used. Two doses of eggs irradiated at total exposures (ET) of 150 muW-min/cm2 resulted in a reduction of adult worm burden in 3 groups challenged with 50, 100, or 500 eggs by 45, 35, and 49%, respectively. However, these results were not statistically significant (P greater than 0.05). Three other groups were fed eggs as follows: Group 1, 3 doses irridated at ET's of 150, 150 and 150; Group 2, 3 doses irradiated at ET's of 150, 150, 100; and Group 3, 3 doses irradiated at ET's of 150, 100, and 75. All of these regimens provided significant protection: Group 1 (P less than 0.02), Group 2 (P less than 0.05), Group 3 (P less than 0.001). An analysis of the lengths of worms recovered showed that in controls fed varying doses of eggs, worm sizes were inversely related to the number of eggs in the challenge dose. Measurements of worms from vaccinated animals showed that 23 of 116 adult worms recovered were probably survivors of one or more of the vaccinating doses. A ratio of 18 female to 5 male worms indicated that males are more susceptible to ultraviolet radiation than females.

Scanning electron microscopy of the lip denticiles of Ascaris suum adults of known ages.

Purebred Hampshire pigs, farrowed and maintained under conditions precluding extraneous helminth infection, were exposed to a single dose of 10,000 Ascaris suum infective eggs. The pigs were killed at intervals of 28, 41, 55, 86, 115, 145, 175, and 206 days after infection. At necropsy, no gross lesions were found in the lungs or livers of infected pigs. The worms were recovered from the small intestine, identified, counted, and fixed. The heads were excised, critical point dried, mounted en face, and examined by scanning electron microscopy. Worms 28 to 115 days old had unworn denticles that were triangular when viewed laterally but blunt when viewed tangentially. Wearing of the denticles was observed first with 145-day-old worms; wearing increased with age both in numbers of denticles affected and in degree of wear so that by 206 days after inoculation, almost all denticles in the center of the lip were worn. Worn denticles appear truncated when viewed from any angle. The denticles outside the central area were not affected by wear. The size of the denticles varies not only between specimens of the same age, but also on each specimen. However, average denticle size is directly related to the size and, accordingly, to the age of the worm. External to each denticle is a corresponding depression that we have called the denticular groove. One 28-day-old specimen had some extra denticles aligned irregularly along the lip; this irregularity gave the appearance of a double row. The denticles of the two subventral lips are similar to those of the dorsal and are equally affected by wear. There was no detectable difference in denticles of male and female worms. Since wear can now be specifically correlated with age, we conclude that the denticles are functional and become worn through use. Consequently, adult A. suum may be an even more injurious pathogen than heretofore supposed.

Immunization for kidney worm disease (stephanuriasis) of swine I. Somatic antigens.

Nine somatic antigens derived from the excretory gland cells of adult Stephanurus dentatus were evaluated as vaccines for stephanuriasis of swine. Antigens were mixed v/v with Freund's complete adjuvant and administered intramuscularly or subcutaneously. Efficacy was evaluated by counting and comparing lesions and worms in the lymph nodes, livers, and kidney regions of principals and controls necropsied after oral challenge with infective S. dentatus larvae or after exposure to pastures naturally contaminated with such larvae. Against oral challenge, 6 vaccines produced statistically significant reductions in worm burdens of principals; 5 were significant at the 1% level and 1 at the 5% level. Two of these vaccines also reduced liver lesions significantly (P less than 0.05). Results of the natural challenge were inconclusive; small worm burdens were found in both principals and controls. None of the vaccines completely prevented migration to the liver or subsequent development and migration of a few worms to the kidney region. However, since worm burdens were reduced by as much as 92%, the vaccines could be useful in combating the disease, especially if combined with other control measures.