Persistence of Immunity Following Double Vaccination of Pups with X-Irradiated Ancylostoma caninum Larvae (1965)

A highly significant resistance to the establishment of a challenge of normal Ancylostoma caninum larvae, in groups of dog and bitch pups following double vaccination with x-irradiated larvae at 3 and 4 months of age, persisted in absence of further exposure to hookworm for at least 7 months after completion of the vaccination schedule. Towards the end of the 7-month period, resistance due to age of the host, as exhibited in the previously uninfected challenge control animals, augmented the immunity of the vaccinated animals against challenge worm establishment. The vaccinated animals at all ages were completely immune to the pathogenic effects of the challenge while older control animals were partially (at 8 months age) or completely (at 11 months) protected against these effects by age resistance per se. The control animals infected at 5 months of age were severely affected in terms of adverse clinical and hematologic changes. Vaccination with x-irradiated larvae stimulated an apparently maximal immunity which was not further improved by numerous small infections of normal larvae to the vaccinated dogs and bitches during the period between vaccination and the challenge at 11 months of age. Previous reports (Miller, 1964a, 1965a, c) have shown that dogs of various ages can be immunized against a challenge of normal Ancylostoma caninum larvae by the administration of two doses, preferably by subcutaneous injection (Miller, 1965b), of 40 kr-irradiated larvae. This report describes experiments designed to investigate the persistence of this immunity over various periods of time after vaccination and to examine the immunity of such pups when exposed during the interval between vaccination and challenge to a lowlevel infection with normal larvae. MATERIALS AND METHODS These have been described previously (Miller, 1964b). Four groups of pups, of mixed breeding and without prior exposure to hookworm, were vaccinated at 3 and 4 months of age with 1,000 40 kr-irradiated A. caninum larvae by subcutaneous injection. Four groups of similar pups were kept as challenge controls. On each occasion on which vaccine was administered, two further groups of pups were infected, one group with normal larvae and the other with the irradiated larvae, the necropsy worm burdens of these pups to serve as larval infectivity and radiation-attenuation controls for each batch of vaccine. At the age of 5 months one group of vaccinated pups and one of the groups of challenge control pups were infected by the subcutaneous inoculation of 1,000 normal A. caninum larvae and were killed 22 to 24 days later. Similarly, at 8 months of age two groups (one vaccinated and one control) were infected and killed. On this occasion, Received for publication 30 November 1964. in view of the possibility of age resistance operating in the control animals, a group of 3-month-old pups were also infected to determine the true potential infectivity of the larvae used for the 8-month challenge. At the age of 11 months two of the remaining groups (one vaccinated and one control) were similarly infected and the necropsy worm burdens of another group of 3-month-old pups again served as larval infectivity controls. The two remaining groups (one vaccinated and one control) were also challenged at this time when 11 months of age, but these differed from all other vaccinates and challenge controls in having been exposed between the ages of 6 and 11 months to a low level of infection with normal larvae. This was done in an attempt to simulate field conditions in dog hookworm enzootic areas although published information upon the intensities of such natural infections is completely lacking. These pups received a total of 1,400 larvae by subcutaneous injection over this period, the infections, each of 100 larvae, being spaced at intervals of approximately 10 to 14 days. Hematologic and coprologic examinations were performed throughout the experiment and the dogs observed for clinical signs of ancylostomiasis. Weight gains were not recorded since it has been shown (Miller, 1965c) that during the vaccination period this method of immunization does not interfere with the normal growth of the vaccinated s and that after challenge differential weight gains between vaccinates and controls appear with regularity, at this level of challenge, only in pups aged less than 5 months at the time of challenge. At necropsy the number of hookworms persisting from the vaccine was determined by microscopic examination for fertility (Miller, 1964b) and where appropriate the normal trickle infection worms were discounted on size. Old adult worms (i.e., from trickle infection) are macroscopically distinctly larger than worms derived from the challenge when the dog is killed 22 to 24 days after