Experimental mice were immunized by two stimulating infections, each with two F. hepatica metacercariae. Afterwards, a challenging infection of the same size was given to them as well as to nonimmunized controls. In the experimental mice, there was an earlier migration of the worms from the liver to the common bile duct, and at 40 days after challenge they harbored significantly fewer worms than the controls. Various host measurements (body weight, total and differential leukocyte numbers, spleen weight, hemagglutinating antibody titer, and liver histopathology) indicated a much more rapid response by the experimental animals. On the basis of the nature and timing of lymphocyte infiltration in, and the histopathology of, the livers, it is suggested that delayed (cellular) hypersensitivity may play a prominent role in the earlier migration of worms to the common bile duct. Several attempts have been made to produce acquired immunity to Fasciola hepatica in laboratory animals. Rats immunized with xirradiated metacercariae and then challenged with nontreated metacercariae showed a reduction in worm burdens (Thorpe and Broome, 1962). However, no attempt was made to separate worms of the two infections, hence the results are not considered by some workers to be conclusive (Dawes and Hughes, 1964). When the latter workers gave mice 20 xirradiated metacercariae and challenged them 21 days later with 10 nontreated metacercariae, acquired immunity was not demonstrated. Also, the use of 40 x-irradiated metacercariae and challenge with five to 10 nontreated metacercariae 22 days later yielded no significant reduction in worm burdens when measured at intervals from 8 to 53 days postchallenge (Dawes, 1964). Therefore, when the present study was undertaken, the evidence for the development of acquired immunity by laboratory hosts was inconclusive. Before designing experiments on acquired immunity, it was necessary to make various determinations after an initial infection. In Received for publication 10 June 1966. * A portion of a dissertation submitted to the Faculty of the University of North Carolina in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Parasitology (1966). t This work was supported in part by a Predoctoral Fellowship (1-Fl-GM-29709-01) from the Institute of General Medical Sciences, NIH. ++ Present address: Department of Zoology, University of Oklahoma, Norman, Oklahoma 73069. 21 this study, reported elsewhere (Lang, 1966b), mice were infected with two metacercariae. The worms migrated through the liver and reached the common bile duct 30 to 40 days after infection. The measurement, at intervals, of various host responses suggested the following phases of the infection: (1) incubation period, (2) acute phase, and (3) chronic phase with repair. During the acute phase (18 to 35 days after infection), the mice were seriously debilitated and many died, but most of those that survived this phase were able to recover. The worms showed rapid, progressive growth and matured in the common bile duct. It seemed apparent that a second infection, if given at least 30 days after the first and terminated by 60 days, could be identified on the basis of worm size alone. It was evident from the above study that immunity produced by a stimulating infection(s) can be measured, provided the challenging infection is given at least 40 days later to allow the host to recover from the acute phase of the infection and to allow time for the worms of the stimulating infection(s) to reach the common bile duct. These observations opened the way for the present studies on acquired immunity. Three separate experiments were performed, two with a single stimulating infection, the other with two stimulating infections (Lang, 1966a). The present paper will deal specifically with the latter experiment only, but certain information provided by the other two experiments will be given in the Discussion. This content downloaded from 157.55.39.78 on Sun, 19 Jun 2016 07:04:28 UTC All use subject to http://about.jstor.org/terms 22 THE JOURNAL OF PARASITOLOGY, VOL. 53, NO. 1, FEBRUARY 1967 MATERIALS AND METHODS A strain of Swiss white mice maintained in this Department for many years was used in this study. Techniques for the maintenance of F. hepatica in the laboratory, infection, killing and necropsy of mice, handling of recovered worms, and determination of host responses, including total body weight, and total plus differential leukocyte counts have been described elsewhere (Lang, 19'66b). In addition, spleen weights, serology, and histopathologic examinations were included.