Comparison of the Immunogenic Efficiencies of Normal and X-Irradiated Ancylostoma caninum Larvae in Dogs

Abstract

An experiment with Ancylostoma caninum in previously uninfected dogs aged 3 to 6 months was designed to compare the immunogenic efficiencies of a double vaccination schedule with 1,000 normal and with 1,000 40-kr-irradiated larvae. Subcutaneous vaccination with 40-kr-irradiated larvae stimulated a more uniform and higher level of immunity than did oral vaccination, when the resistance was measured by hookworm establishment from the challenge inoculation. Subcutaneous vaccination with the irradiated larvae was also more effective than vaccination with normal infective larvae by either oral or subcutaneous administration. Attempted vaccination with normal infective larvae was an extremely hazardous procedure for health and survival of the dog. It seems likely that the arrest of radiation-damaged larvae in some migratory somatic location in the tissues of the dog was responsible for optimum immunogenesis. Previous reports (Miller, 1964, 1965a, b, c), have shown that x-rays can be used as an agent of attenuation for the preparation of a vaccine against Ancylostoma caninum infection of dogs. This vaccine was successful in protecting dogs of various ages against the establishment of a challenge inoculation of normal hookworms, and against the potential pathogenicity and lethality of the challenge infection (Miller, 1965c). Vaccination by subcutaneous inoculation stimulated a more effective immunity than did vaccination by oral administration of the irradiated larvae (Miller, 1965b). The present report describes experiments designed to compare the resistance induced by vaccination with x-irradiated larvae with the resistance stimulated by similar doses of normal infective larvae. MATERIALS AND METHODS The management of experimental dogs and the procedures for culturing, irradiation, and preparing larvae for inoculation have been described (Miller, 1964). The plan of experiment is shown in Table I. Groups of pups of mixed unidentifiable breeding (i.e., mongrel pups purchased from pet dealers in the densely populated areas of Glasgow), and without prior exposure to hookworm, were infected by either subcutaneous (A1 B1 B2) or oral inoculation (A2 Di D2); with either 1,000 normal A. caninum infective larvae (B1 B2 D1 D2) or with 1,000 larvae irradiated at 40 kr (A1 A2). The infections were administered at 3 and 4 months of age and at 5 months the immunity of four of these groups (A1 B1 A, D1) and of two groups of similar but uninfected control dogs (C1 C2) was Received for publication 19 August 1965. challenged with 1,000 normal infective larvae. The groups that received the normal larvae at 3 and 4 months of age were treated with an anthelmintic on the 27th and 28th days after the first and second infection, respectively. The anthelmintic used was thenium p-chlorobenzene sulphonate (Ancaris?, Burroughs Wellcome and Co., London), the treatment schedule consisting of four tablets (total 500 mg thenium base) administered as recommended by the manufacturers. Four of the dogs so treated (B2 D2) did not receive the challenge inoculation at 5 months of age, but were killed with other groups at 6 months to determine the numbers of normal hookworms from the vaccinating doses of normal larvae which had resisted the drug action. The mean number of worms in these groups was discounted at necropsy from the hookworm burdens of dogs that were vaccinated with the normal larvae (B1 Di) to give a figure for the challenge hookworm burdens. The numbers of sterile worms, derived from the x-irradiated vaccine, persisting in the dogs that were vaccinated with the irradiated larvae were determined by microscopic examination (Miller, 1964). These numbers were discounted at necropsy from the total hookworm burdens of these dogs (A1 A2) to give a figure for the challenge hookworm burdens. The worm burdens, expressed as mean group per cent takes with standard deviations, from the challenge infection in each group of vaccinated dogs and in their respective challenge control group of dogs were compared statistically by Student's "t" test to determine the significance of the differences. Based on necropsy worm burdens from the challenge inoculation, a figure for per cent protection was calculated using the equation (Mean challenge control worm burdens) (Mean vaccinate worm burden) (Mean challenge control worm burden) as a measure of the reduction in challenge hookworm establishment, resulting from prior infections in the vaccinated groups of dogs, as compared with the worm burdens in their respective challenge control groups of dogs.