Blood Loss during Hookworm Infection, Determined by Erythrocyte Labeling with Radioactive 51 Chromium. I. Infection of Dogs with Normal and with X-Irradiated Ancylostoma caninum

Abstract

Blood loss in the urine and feces were determined, changes in hematology recorded, and the half-life of the circulating erythrocytes calculated during infection with Ancylostoma caninum. Significant changes in the urinary excretion of 5lchromium were not detected at any time during infection. Blood loss in the feces was first detected on the 8th day after inoculation of infective larvae, except in the case of adults dogs (10th or 11th day) and in pups with light infections of x-irradiated worms (10th day). Blood loss in the feces reached a maximum between 12 and 16 days after inoculation with a second peak in the pups of some groups at 23 to 25 days. Pups infected with less than 30 normal adult A. caninum per pound of body weight experienced significantly greater fecal blood loss per worm per day than did pups infected with more than this number of worms (per pound of body weight), although the pups infected with the greater numbers of worms experienced greater total fecal blood loss per day. The hematologic findings reflected the fecal blood loss results during infection with normal A. caninum. Infection with x-irradiated A. caninum, even with relatively large infections (more than 30 worms per pound of body weight), failed to induce adverse hematologic changes in the infected pups in spite of the occurrence of blood loss during such infection. The technique of measuring accurately gastrointestinal hemorrhage by the labeling of erythrocytes with radioactive 51chromium has been described (Gray and Sterling, 1950; Sterling and Gray, 1950; Owen et al., 1954; Foy et al., 1958; Clark et al., 1961). This procedure has been used to measure the blood loss from the host resulting from infection with hookworm in man (Foy et al., 1958; Roche et al., 1957) and in dogs (Clark et al., 1961; Roche and Martinez Torres, 1960). During experiments designed to investigate the immune responses of dogs following vaccination with x-irradiated Ancylostoma caninum larvae (Miller, 1964, 1965a, b, d, e, 1966) the opportunity was taken to employ this technique to estimate the blood loss caused by hookworms in dogs at various stages of the infection. MATERIALS AND METHODS The management of experimental dogs and procedures for culturing and preparing larvae for infection were described (Miller, 1964). For labeling the erythrocytes, 5 ml of blood was taken aseptically using heparin as anticoagulant, and was incubated for 1 hr at room temperature (about 20 C) with approximately 1 me of 51chromium as sodium chromate in isotonic saline (The Radiochemical Centre, United Kingdom Atomic Energy Authority). The erythrocytes were then washed Received for publication 22 December 1965. five times with 10 ml of physiological saline and suspended in saline for intravenous injection of the donor dog. The experimental dogs were maintained in metabolism cages from which total daily urine and fecal outputs could be recovered separately. Each day a small sample of blood (approximately 1 ml) was taken from the cephalic vein of the dogs into tubes with diamino-ethane-tetra-acetic acid (dipotassium salt) as anticoagulant; aliquots of the samples were diluted in aqueous 0.02% sodium hydroxide to make total volumes of 5 ml for counting radioactivity; and at least two preparations were made each day from the blood sample of each dog. Total daily urine output was measured and two 5-ml samples were retained for counting. Total daily fecal output was weighed and was homogenized with an equal weight of water, from which mixture two weighed samples were extracted for counting. The radioactivities of the prepared samples of blood, urine, and feces were counted in a well-type, thallium-activated, sodium iodide crystal, scintillation counter coupled to an automatic dekatron timer unit (Panax Equipment Ltd.). Calculations were performed to give figures for total fecal and urine counts per day and for counts per ml of blood on each day of the experiment. All radioactivity figures were corrected for radioactive decay as determined by the decline in the radioactivity of 5 ml of a standard solution of 51chromium. The total fecal and urine activities (total counts) on each day were divided by the blood activity (counts per ml) that was recorded from the blood sample taken 24 hr before the time of collection of the respective feces and urine. The radioactivity of the blood per ml on each day, after correction by the factor for