Changes in Serum Proteins of Rats Infected with Toxoplasma gondii

Abstract

Rats inoculated with Toxoplasma gondii were bled from the orbital sinus at various intervals postinfection. The most remarkable changes in serum proteins were a rise in -y-globulin and a fall in albumin during the acute stage of the infection. Antibodies measured in the dye test began appearing after 3 days of infection and rose to maximum titers by the 4th and 5th days. Serum protein analysis by starch block electrophoresis revealed the dye test antibodies to reside in the y-globulin fraction. Immunoelectrophoresis indicated major changes in the aand y-globulin fractions. These findings are discussed and compared with results of similar studies in mice. Although there exists many descriptions of the pathologic changes in tissues of man and animals resulting from infection with Toxoplasma gondii, there are only meager reports on the biochemical changes in serum induced by infection with the parasite. Recent observations in our laboratory have revealed remarkable alterations in the electrophoretic and immunoelectrophoretic patterns of the serum proteins of mice infected with various strains of toxoplasma (Remington, in press). Similar studies in rats, which unlike mice do not succumb to infection with toxoplasma, have demonstrated serum protein changes differing from those occurring in mice. MATERIALS AND METHODS Female rats, weighing 150 to 250 g, of the Sprague-Dawley strain were inoculated intraperitoneally with 1 X 106 parasites of the RH strain of toxoplasma obtained from peritoneal fluid of 3-day infected Swiss-Webster mice. To prepare the inocula, peritoneal fluid was centrifuged at 275 g for 15 min, the supernate discarded, and the precipitate washed three times with 0.15 M NaCl. Following the last wash, the precipitate was suspended in 40 ml 0.15 M NaCl and filtered through a Baird-Tatlock P-3 filter to separate white blood cells from organisms. Following centrifugation of the filtrate, the organisms were resuspended in saline and counted in a NeubauerLevy hemocytometer. Appropriate dilutions were Received for publication 8 January 1965. * Presented in part before the First International Congress of Parasitology, Rome, Italy, 21-26 September 1964 (Remington, 1964). Supported by Grants No. AI-04717, No. AI 260 and No. FR-05053 from NIH. t Division of Allergy, Immunology and Infectious Diseases, Palo Alto Medical Research Foundation; Department of Medicine, Stanford University School of Medicine, Palo Alto, California. then made to prepare inocula containing the calculated numbers of parasites desired. Each rat was bled from the orbital sinus prior to and at various time intervals following inoculation of the parasites. To determine if repeated bleedings of amounts necessary for these studies will alter serum protein patterns as noted in infected animals, a separate series of ten rats were bled every 5 days for 30 days as a control. Changes in immunoelectrophoretic and electrophoretic patterns similar to those which occurred in infected animals did not occur in the control series. Variations did occur but were not considered significant. An attempt was made to divide the animals into groups such that, as nearly as possible, a rat would not be bled more often than every 5 days. Samples of blood were allowed to clot at room temperature for 1 hr, centrifuged at 1,000 g for 45 min at 4 C and the sera stored at -20 C. Sera were not used if hemolysis was present. Electrophoresis on paper and immunoelectrophoresis were performed on all serum samples either immediately after separation from the clot or within 10 days after freezing. Sera used for immunoelectrophoresis were not frozen and thawed more than once. Electrophoresis was performed on Whatman 3 mm paper strips using barbital buffer, pH 8.6, in a model-R, series-D Spinco paper electrophoresis cell. Electrophoresis was allowed to continue for 26 hr to obtain satisfactory separation of al-globulins from the albumin fractions. Strips were dried 30 min at 120 C, stained with bromphenol blue, rinsed twice in 5% acetic acid, and fixed in 0.9% sodium acetate in 10% acetic acid. The strips were scanned with a Spinco-model-R analytrol. Each peak (protein fraction) was delimited by marking the lowest point between each peak of the recording trace. The percentage of the total area represented by an individual component was calculated from areas underneath the curves between relevant pairs of delimiting lines using the integrator tracing. Zone electrophoresis in starch was performed as previously described (Merler et al., 1963). Immunoelectrophoresis was performed using the micromethod of Scheidegger (1955) with 1% agar in borate buffer pH 8.6 (, = 0.064). After the precipitin lines developed to maximal intensity