Vet. Pathol. 18: 351-357 (1981) Hematology of Experimental Acute Surcocystis bovicanis Infection in Calves. I. Cellular and Serologic Changes R. FAYER and K. W. PRASE Ruminant Parasitic Diseases Laboratory, Animal Parasitology Institute, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Beltsville, Md., and Department of Veterinary Pathology, College of Veterinary Medicine, University of Georgia, Athens, Ga. Abstract. Of four Holstein-Friesian calves infected with 200,000 sporocysts of Sarcocystis bovicanis. three became ill and died on days 3555, and 59 of a 63-day experiment. No control calves became ill or died. Three of the four infected calves developed normocytic normochromic anemia with about a 50% decrease in hemoglobin concentration on days 25 through 35, and had hyperbilirubinemia from day 25 or 26 to death (day 35); this disappeared when the anemia stabilized in surviving calves. Packed cell volume increased slowly after day 35 in the surviving anemic calves. Indirect and direct Coombs tests were negative throughout the experiment in the control and infected calves. A reduction in numbers of neutrophils and lymphocytes paralleled the crisis of anemia in the infected calves. Serum antibody titers to S. bovicanis antigen were increased substantially in all infected calves 35 days or more after inoculation. Hematologic data from animals with sarcocystosis are scant. Virtually all the data have come from three natural outbreaks and four experimental studies in cattle (I, 4, 5, 8,9, 1 I, 121. Anemia was reported in all studies, although data were not always supplied. Other hematologic observations are scattered among the various reports. Because neither a single study nor the composite data from all of them provide an in-depth account of the hematologic changes that accompany sarcocystosis, the present study was undertaken. This study is broad in scope and is divided into three areas presented in two serial reports: the qualitative and quantitative changes in the cellular and serologic components of the blood, and changes in blood chemistry and hemostasis during sarcocystosis [ 131. Experiment 1 Materials and Methods Seven Holstein-Friesian male calves I to 3 days old were obtained from local dairy farms and raised in individual pens in a barn isolated from dogs at the Animal Parasitology Institute. At approximately 3 months of age the calves were assigned to groups. Calves I through 4, in 35 I
352 Fayer and Prase Group 1, were infected with approximately 200,000 Sarcocystis bovicanis sporocysts each, mixed with their daily grain ration. Calves 5 through 8, in Group 2, served as uninfected controls and were fed grain mixed with the aqueous supernatant from an equal number of pelleted sporocysts. The sporocysts were less than 3 months old when used and had been stored in tap water at about 5 C. All calves were observed daily, and changes in feed intake, behavior, and general health were recorded. Beginning on day 0 (the day of infection), body temperature was recorded daily for the 63 days of the experiment. Calves were weighed on day 0 and at eight successive weekly intervals. Blood was taken from the jugular vein on days 0, 7, 14, 21 through 35,42,49, 56, and 63. Blood was collected in four types of vacuum tubes. Tubes contained either no anticoagulant; ethylenediaminetetraacetic acid anticoagulant; 3.8% sodium citrate anticoagulant; or soybean trypsin inhibitor. Within one hour, serum harvested from coagulated blood was frozen and stored at -75OC for later use in an indirect Coombs' test, an indirect hemagglutination test, and a nonspecific hemagglutination test. In the indirect Coombs' test, rabbit anti-bovine globulin (Nutritional Biochemical Corp., Cleveland, Ohio) was inactivated at 56 C for 30 minutes and adsorbed with physiologic saline-washed bovine erythrocytes from a healthy calf to remove nonspecific agglutinins and hemolysins, as follows: Two drops of a 2% suspension of physiologic saline-washed Holstein calf erythrocytes were mixed and incubated with two drops of test serum at 37OC for 15 minutes. The cells were washed three times with physiologic saline, resuspended to 2%, and mixed with two drops of the rabbit anti-bovine globulin. Three tubes were used for each test serum, and the mixtures were incubated at either 4OC, room temperature, or 37 C for 30 minutes. Macroscopic and microscopic examination for agglutination and hemolysis followed the incubation. Serum also was tested for antibody to S. bovicanis on days 0, 35, and 63 (or on the day of death) by the indirect hemagglutination test as described [lo]. Serum from days 0, 21, 25, 29, and 35 was tested at a dilution of 1:16 for nonspecific agglutination of tannic acid-treated bovine erythrocytes (replicated three times) and ovine erythrocytes (tested once) that were prepared similarly to erythrocytes for the indirect hemagglutination test but not sensitized with antigen. Anticoagulated blood was used as follows: 'The packed cell volume was determined by microcapillary-tube assay. Total erythrocyte counts, total leukocyte counts, and hemoglobin concentrations were determined with a Coulter Counter, model ZBI (Coulter Electronics, Hialeah, Fla.). Erythrocyte morphology and staining characteristics were determined from Giemsa-stained blood smears. Leukocyte differential counts also were made from Giemsastained smears; 200 leukocytes were identified, and the absolute number of each type per pl of blood was calculated. We determined the percentage of reticulocytes by mixing equal parts of the anticoagulated blood with new methylene blue, allowing the mixture to incubate at room temperature for 10 minutes, and preparing air-dried blood films from the mixture; the reticulocytes per 1000 erythrocytes were counted. Mean values for infected versus control calves were compared statistically with the Paired T test. Values of p 5 0.05 were considered significant deviations from the null hypothesis. Experiment 2 In light of the possible production of autoantibodies to erythrocytes as a consequence of infection, a direct Coombs' test was made to determine whether bovine immunoglobulins were on the surface of erythrocytes. Five Holstein-Friesian calves were infected with S. bovicanis sporocysts as in experiment 1, and another five were maintained as controls. Blood was collected on days 7, 21, 28, and 35 from the jugular vein of each calf and placed into a vacuum tube containing no anticoagulant. Erythrocytes from clots were washed four times with 0.85% saline and adjusted to a final 3% suspension of cells in saline; 25 p1 of the suspension was added to each well in a microtiter
Hematology of Surcocysris in Calves. 1. 353 plate containing 25 pl of rabbit anti-bovine immunoglobulin. Four microtiter plates were used at each test period. Each plate contained unlabeled anti-bovine IgG,, IgG2, IgM (provided by Dr. A. J. Guidry), or fluorescein-labeled IgM (Miles Laboratories, Elkhart, Ind.) at final concentrations of 1:2, 1:4, 1:8, and 1:16. All plates were incubated at 37OC for I5 minutes, and each well was examined macroscopically and microscopically for agglutination; the fluoresceinlabeled IgM preparations were examined by fluorescence microscopy. The IgGI, IgG2, and IgM plates were incubated overnight at 5 C and then reexamined. Experiment 1 Results Group average daily temperatures for control calves did not exceed 39 C and individual temperatures did not exceed 39.3"C. Group average daily temperatures for infected calves exceeded 39.3"C on days 15, 16, and 26 through 38; the highest group average temperatures were 40.5"C, 40.7"C, and 40.5"C on days 26,27, and 28 respectively. Neither soft feces nor diarrhea was seen from any control calves. All four infected calves had either soft feces or diarrhea: one calf on days 15 through 17 and 24 through 33, another on days 24 through 27, and two others on days 24 through 26. None of the control calves had reduced feed intake. They gained an average of 31.8 kg between day 0 and day 56. All four infected calves had either sharply reduced feed intake or complete anorexia that began on day 28 and lasted for three days in one calf, seven days in another, and nine days in two calves. They gained an average of 6.4 kg between day 0 and day 56. One infected calf had either nasal discharge or hypersalivation for seven consecutive days beginning on day 30. None of the control calves was ill during the study. Only one of the infected calves was alive at the end of the study; the others died on days 35, 55, and 59. Three infected calves developed normocytic normochromic anemia on day 25, and one infected calf had erythron data within the range of values from the control calves (fig. 1). The packed cell volume, erythrocyte count, and hemoglobin concentration decreased by about 50940 in each of the three calves on days 25 through 35. In the two anemic calves that survived beyond day 35, the packed cell volume increased slowly (fig. 1). Hyperbilirubinemia was visible in the sera from each of the three anemic infected calves from day 28 until death of one calf (day 35) and day 49 in the two survivors. The nonanemic infected calf and the controls did not have hyperbilirubinemic serum. Subjective assessment of erythrocyte morphology for all calves in all samples showed immature cells in Giemsa-stained films from one anemic calf on one day only; macrocytic, polychromatophilic, and rarely, basophilic stippled cells were found on day 32. Reticulocyte counts were zero for all control and infected calf samples, with rare exceptions; reticulocytes were seen in nine samples on days 30 through 34 from two control calves and three infected calves (one of which was the nonanemic infected calf). The reticulocyte values in the nine samples ranged from 0.1% to 0.3%.
354 Fayer and Prase I 7 I4 2 1 1 3 0 1 45 49 86 63 DAYS AFTER INFECTION 0 1,,,,,, 0 7 U 2 1 1 3 0 1 6 49 16 Q DAYS AFTER INFECTION Fig. 1: Packed cell volume (PCV), hemoglobin concentration (Hb), erythrocyte count (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemog!obin (MCH). Range for four control calves, shaded areas. Values for four calves infected with Sarcocysris, solid lines for individual calves. Heavy bars below values represent days mean values were significantly different (p 5 0.05) for control and infected calves. Fig. 2 Total leukocyte count (WBC), absolute neutrophil count (NEUTRO), and absolute lymphocyte count (LYMPHO). Range for four control calves, shaded areas. Values for four calves infected with Sarcocysris, solid lines for individual calves. Heavy bars below values represent days mean values were significantly different (p 5 0.05) for control and infected calves. Neutrophil and lymphocyte counts were significantly lower in infected calves than in controls on days 25 through 35 (fig. 2). These counts, however, were never below normal lower limits for cattle [ 141. Serum samples from all calves in experiment 1 from days 0, 21, 25, 29, and 35 were tested for anti-erythrocytic agglutinins or hemolysins by the indirect Coombs test. All 40 samples were negative. Results of the indirect hemagglutination test for calves in experiment 1 (table I) indicate that infected calves had substantially increased serum titers to S. bovicanis antigen 35 days or more after inoculation. Uninoculated control calves had no titer or had very low transient titers. No nonspecific hemagglutination was seen when sera from the calves in experiment 1 were tested against bovine erythrocytes. When ovine erythrocytes were used, serum from all inoculated calves caused hemagglutination beginning on day 2 I, 25, or 29,
Hematology of Sarcocysfis in Calves. I. 355 Table I. Indirect hemagglutination titers to Surcocystis bovicunis in infected and control calves (Exwriment 1) Days after inoculation Infected calves Reciprocal titer Control calves I 2 3' 4 5 6 7 8 0 0 0 0 486 0 0 0 5 4 35 162 486 486 1458 0 0 54 54 63 NS4 4374' 13000 145@ 0 0 0 0 The infected calf that did not become anemic. Last serum taken immediately before death, day 55. ' Last serum taken immediately before death, day 59. NS = no sample, calf died 35 days after infection. and sera from two uninoculated control calves caused hemagglutination on day 2 1 and on days 29 and 35, respectively. Experiment 2 Neither macroscopic nor microscopic agglutination or hemolysis of erythrocytes was seen on day 7, 21, 28, or 35 in the presence of anti-bovine IgGI, IgG2, or IgM. Fluorescence microscopy showed no fluorescence in erythrocytes incubated in fluorescein-labeled anti-bovine IgM. Discussion The experimental induction of anemia by Surcocystis bovicanis infection in calves confirms a previous report [ 1 I] and is consistent with reports of anemia in naturally infected cattle [6, 71. The characterization of anemia as normocytic normochromic, however, contrasts slightly with the previous reports in which one calf was found to have macrocytic hypochromic anemia [6, 71. The onset and development of anemia in experimentally infected calves on days 25 through 35 after ingestion of infective sporocysts overlaps the time of development of second-generation schizonts in vascular endothelial cells throughout the body and multiplication of merozoites in the bloodstream [3, 41. Anemia was not found on days 14 through 16, when firstgeneration schizonts develop in the walls of small arteries [3], and the anemia appears to stabilize and resolve slowly at the time the organism encysts and matures in muscle (on day 35 or later). Our findings support the claim [7] that the anemia of acute bovine sarcocystosis is primarily hemolytic. This contention is supported by the rapidity with which anemia developed and the hyperbilirubinemia that appeared shortly after the onset of anemia and disappeared when the anemia stabilized in surviving calves [ 131. Furthermore, histologic sections of liver and spleen from two anemic calves (previous unpublished trial) that died on days 29 and 3 1, respectively, stained by Perls' test for iron, showed splenic macrophages, Kupffer cells, and hepatocytes with marked iron deposition
356 Fayer and Prase and hemosiderosis, which indicated increased catabolism of hemoglobin. Hemoglobinuria has not been found in infected calves [5], which suggests that hemolysis is by erythrophagocytosis rather than by intravascular lysis. Hemorrhage also may contribute to the anemia. In previous studies, hemorrhage was found at necropsy of naturally infected cattle [2] and of experimentally infected calves [8] on days 26 through 33; it was not of sufficient magnitude, however, to account for the anemia. The possible effects of secondary suppression of erythropoiesis cannot be assessed from the available data. Failure to develop reticulocytosis, a condition classically characteristic of bovine hemolytic anemia [ 141, is not sufficient evidence to invalidate the interpretation of hemolysis. The anemia may have been insufficiently severe or the period of anemia too brief to stimulate detectable or sustained reticulocytosis. Experimentally, massive blood loss is necessary to stimulate reticulocytosis in calves. About a 40% reduction in hemoglobin concentration over 14 days yielded a maximum reticulocyte response of 1.6% in 4-month-old calves [ 11, whereas about a 60% reduction in packed cell volume over 24 hours produced a maximum reticulocyte response of 14% in calves [ 151. Calves in our study experienced about a 50% decrease in hemoglobin concentration over a 10-day period. The mechanism for hemolysis requires further study. The correlation between development of the anemia and the occurrence of blood-borne developmental stages of the parasite suggests either a direct hemolytic effect or an immunohemolytic effect of the organism or its metabolic products. The latter mechanism has been proposed for the hemolytic anemia of bovine trypanosomiasis [ 171. The negative indirect and direct Coombs tests from infected calves during the period of anemic crisis in this study cast some doubt on, but do not exclude the possibility of, such a mechanism. A nonspecific hemagglutination factor (for ovine erythrocytes), however, appears to be more prevalent in the serum of Surcocystis-infected calves than in that of control calves. One of the four calves fed infective S. bovicunis sporocysts in this study failed to become anemic (fig. 1). Confirmation of infection in the calf was elevated indirect hemagglutination titers to S. bovicanis antigen (table I). No immediate explanation for the different response of this calf was available from the findings in this study. The decrease in total leukocyte count by reduction in neutrophil and lymphocyte numbers paralleled the crisis of anemia in the infected calves. Mechanisms for cell destruction might have been similar; neutrophil and lymphocyte counts also decreased, however, in the nonanemic calf. Neutropenia and lymphopenia are, in general, characteristic response to acute infections in cattle [ 141. Lymphocytosis, or an increase in lymphocyte count, occurred after the anemic crisis period. The overall pattern of leukocyte response during experimental acute sarcocystosis was similar to that described for bovine trypanosomiasis [ 161. Acknowledgements Supported in part by the University of Georgia Veterinary Medical Experimental Station. We thank Merrill Lago, D.C. Davis and L.T. Young for their excellent technical assistance,
Hematology of Surcocysris in Calves. I. 357 M.N. Lunde for assistance with the indirect hemagglutination test, and A.J. Guidry for providing rabbit anti-bovine immunoglobulins. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. References 1 BREMMER, K. C.: The reticulocyte response in calves made anemic by phlebotomy. Aust J Exp Biol Med Sci U251-258, 1966 2 CORNER, A. H.; MITCHELL, D.; MEADS, E. B.; TAYLOR, T. A.: Dalmeny disease. An infection of cattle presumed to be caused by an unidentified protozoon. Can Vet J 4252-264, 1963 3 FAYER, R.: Multiplication of Sarcocystis bovicanis in the bovine bloodstream. J Parasitol 65980-982, 1979 4 FAYER, R.; JOHNSON, A. J.: Development of Sarcocystis fusiformis in calves infected with sporocysts from dogs. J Parasitol59: 1135-1 137, 1973 5 FAYER, R.; LYNCH, G. P.: Pathophysiological changes in urine and blood from calves experimentally infected with Sarcocystis cruzi. Parasitology 79325-336, 1979 6 FRELIER, P.; MAYHEW, I. G.; FAYER, R.; LUNDE, M. N.: Sarcocystosis: A clinical outbreak in dairy calves. Science 1951341-1342, 1977 7 FRELIER, P.; MAYHEW, I. G.; POLLOCK, R.: Bovine sarcocystosis: Pathologic features of naturally occurring infection with Sarcocystis cruzi. Am J Vet Res 40:65 1-657, 1979 8 JOHNSON, A. J.; HILDEBRANDT, P. K.; FAYER, R.: Experimentally induced Sarcocystis infection in calves: Pathology. Am J Vet Res X995-999, 1975 9 LANDSVERK, T.: An outbreak of sarcocystosis in a cattle herd. Acta Vet Scand 20238-244, I979 10 LUNDE, M. N.; FAYER, R.: Serologic tests for antibody to Sarcocystis in cattle. J Parasitol 63222-225, 1977 11 MAHRT, J. L.; FAYER, R.: Hematologic and serologic changes in calves experimentally infected with Sarcocystis fusiformis. J Parasitol61:967-969, 1975 I2 MEADS, E. B.: Dalmeny disease-another outbreak-probably sarcocystosis. Can Vet J 17: 27 1-276, 1976 13 PRASSE, K. W.; FAYER, R.: Hematology of experimental acute Sarcocystis bovicanis infection in calves. 11. Serum biochemistry and hemostasis studies. Vet Pathol 18358-367, 1981 14 SCHALM, 0. W.; JAIN, N. C.; CARROLL, E. J.: Veterinary Hematology, 3rd ed., pp. 122-144. Lea and Febiger, Philadelphia, 1975 15 SCHNAPPAUF, H.; STEIN, H. B.: SIPE, C. R.; CRONKITE, E. P.: Erythropoietic response in calves following blood loss. Am J Vet Res 2&275-278, 1967 16 VALLI, V. E. 0.; FORSBERG, C. M.; LUMSDEN, J. H.: The pathogenesis of Trypanosoma congolense infection in calves. 111. Neutropenia and myeloid response. Vet Pathol 1696-107, 1979 17 VALLI, V. E. 0.; FORSBERG, C. M.; MCSHERRY, B. J.: The pathogenesis of Trypanosoma congolense infection in calves. 11. Anemia and erythroid response. Vet Pathol 15732-745, 1978 Request reprints from R. Fayer, Animal Parasitology Institute, U.S.D.A., Beltsville, MD 20705 (USA).