., . f , ‘i ”a 4;?! ;’{‘}§‘{ \Ei‘h‘) {:1 m ‘I - 74" iltu. -i*‘ ';-,*\).:t r I; f [3.7 wt} ,ft . 11, “- Q; " k x), .57" ‘34 .- ‘ ' -.‘/ '\ ‘.b x F’s-'7 1. " 1.:- . - a‘ft- .- * a. 1“; Vi.“- WW3“ ‘ . - *‘l ‘ — ~ A 0‘ \ ‘ .‘ “ ‘ K -‘ I. - " ' .- ' ‘ V . . i . ' " I ' . h .v u ’ h . ‘ -;‘- I A V v C . ‘. ' . - -1 « ,' . ‘ I I h. - " ' _ v w‘ A l ' > ’ . ' . a.» ’24", v I «Q «5' ‘ .‘< 2 . 5.1.1 9,5 '4??? {3"}? BLOOD GROUPS IN SHEEP by Girdhari L._§§arma .A Thesis Submitted to the School of Gradunte Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Pathology 1950 THESiS ACKNOWLEDGEEEN I am very grateful to the Government of India for granting a scholarship for studies in the U. S. A. wnicn has made this in- vestigation possible. With deep feelings of gratitude I wish to acknowledge the guidance of Dr. Frank Tharp, Jr. wnose training in the use of literature has furnished the most valuable tool for use in researcn. His keen interest and readiness to help have provided a constant inspiration and feeling of self confidence. To Mr. M. L. Gray I owe my indebtedness for the help and valuable suggestions in preparing the manuscript and phot05raphs. My appreciation is extended to Miss Sylvia Laine for cor- recting the manuscript and to Dr. R. A. Runnells for encouragement and help in this work. I also wish to thank all the friends in this department who have helped from time to time and I am obliged to the Department of Animal Husbandry for providing the sheep for this study. 239292 TABLE OF CONTENTS INTRODUCTION REVIEW OF LITERATURE MATERIAL AND METHODS Natural isoagglutination Results Isoimmunization Results Heteroimmunization Results' BLOOD TYPING OF HEALTHY SHEEP DISCUSSION HISTOGRAMS SUMMARY AND CONCLUSIONS BIBLIOGRAPHY LITERATURE CONSULTED BUT NOT CITED PAGE lb lb 18 22 30 3b “7 5b b8 72 80 81 35 TABLE 10 11 13 1n 15 lb 17 18 19 20 21 22 LIST OF TABLES Natural isoagglutination reactions in Rambouillet sheep. Natural isoagglutination reactions in Shropshire . Sheepe Natural isoagglutination reactions in Oxford sheep. Natural isoagglutination reactions of Shrapshire erythrocytes with Rambouillet pooled serum. Schedule of isoimmunization of sheep. Sheep used for analysing isoimmune sera and hetero- immune sera. Hemolytic reactions with isoimmune sera. Analysis of isohemslysin No. 3. Analysis of isohemolysin No. 5. Presence of natural Forssman like antibodies in rabbit serum. Record of transfusions for prouucnion of hetero- immune sera. Hemolytic titres of heterohemolysins. Hemolytic reactions of three anti sheep rabbit immune sera after absorption with heated sheep cells. (Forssman antigen) Hemolytic reactions of heteroimmune sera after ab- sorption with heated sheep red cells. Analysis of heteroimmune serum No. 1. Analysis of heteroimmune serum No. 2. Analysis of heteroimmune serum No. 3. Analysis of heteroimmune serum fig. 4. ‘J1 0 Analysis of heteroimmune serum No. .Analysis of heteroimmune serum No. b. Analysis of heteroimmune serum No. 8. Analysis of heteroimmune serum No. 10. PAGE 19 21 21 24 32 33 35 37 39 us an 47 M8 #9 50 51 52 53 55 LIST OF TABLES (continued) TABLE FAG» 23 List of sheep used for blood typing. 59 24 Results of blood typing by isoimmune sera. bl 25 Results of blood typing by heteroimmune sera. b3 Eb Percentages of different factors in the seven breeds of sheep. bb 27 Percentages of different factors in the whole population of sheep tested. 67 28 Method of analyzing antibodies. 70 INTRODUCTION It has been known for a long time that there are differences in the blood of animals of different species. Some of these differ- ences are in the size and shape of the red blood corpuscles. the relative proportion of different kinds of leukocytes and the chemical constitution of respiratory pigments. It is also known that with various infectious diseases such as small-pox and typhoid fever. changes of certain properties of the blood occur more or less permanently by the formation and accumulation of antibodies. In the beginning of this century it was discovered that in addition to the above men- tioned morph010gical. serological and accidental differences, there existed hereditary and constitutional differences of individual and racial significance. Since the discovery of blood groups in man, attempts have been made to investigate the question of constitutional blood factors in animal... The work of Bruner gt al_(l9h8, 1949) had shown that an icteric condition in foals and baby pigs might be explained on the basis of a hereditary factor in the red blood cells. Muraschi 2_ El (1939) had shown that sheep blood varied in its nemolytic activity. Among 13h samples of sheep sera tested. seventeen showed evidence of lysine for red blood cells of other sheep. It seems to be fairly well established that constitutional blood factors are hereditary; these blood factors serve as good guides in studying inheritance of important characters due to genes linked on the same chromosome pair. In view of (l) the increasing importance of blood factors in animals (2) relatively scanty literature on blood factors in sheep (3) the availability of different pure breeds of sheep at the farms of Michigan State College. it was decided to investigate the problem of blood groups in sheep. REVIEW OF LITBRATURE There was a close relationship in the history of constitutional blood differences and that of blood transfusion. Blood transfusions were practicegdas early as the fifteenth century in cases of excess- ive hemorrhage. Blood of lambs or heifers was used in suCh cases and the results were so disastrous that in the 17th century by an act passed in Paris. transfusion of blood was made illegal in France. Landois (1875') showed that agglutination of red blood cells of one species of animals would occur when mixed with the serum from animals of another species. This was called heteroagglutination and was due to the presence of antibodies ... heteroagglutinins. tAs a result of this information blood transfusions for humans was practised again but only with human blood. By the end of 19th century it hadbeen observed that serum from certain diseased humans would agglutinate the blood cells of apparently normal individuals. This pehnomenon was regarded as an indication of a pathological condition. Landsteiner (1900) discovered that isoagglutinins could exist in normal human beings and that the properties of isoagglutination were not the same in all individuals. For this discovery of fundamental importance Landsteiner was awarded the Nobel Prize. Further invesigation into this phenomenon disclosed that there were two antigens: 4A and B in human red blood cells and two corresponding h antibodies in the sera. The antibodies were called 'a' and I'b". The four blood groups in human beings were mainly based on the distribution of the antigens and the antibodies as tabulated below: Blood group AgglutinOgen in Agglutinin in cells plasma A. A. b. B. B. a. A.B. A.B. - o. - .ObO Ehrlic and Morggnrothp(l90G) demonstrated individual differences in the blood of goats. for this work they used immune isoantibodies. 2393's d Ehitg (1910) examined the sera of cattle which were being used for the production of Rinderpest serum at The Hygieneic Institute. Public Health Department. Cairo. The authors came to the conclusion that cattle, not closely related genetically, could be differentiated from other animals of the same species by the use of immune isohemolysins. Walsh (192%) in a study of blood inter-relationship of horses, asses and mules arrived at the following conclusions: a. ‘Ass serum agglutinated the erythrocytes of horses in a high percentage of cases but did not agglutinate the red blood cells of mules. b. 0. d. 5 {Ass serum hemolysed the erythrocytes of both horses and mules in a high.percentage of cases. Ass serum invariably either agglutinated or hemolysed the erythrocytes of horses. but mule serum did not alter the ery- throcytes of the horses. The blood of the mule had characteristics of both the horse and of the ass blood; the serum resembled that of the horse and the corpuscles resembled the cells of the ass. Baker (1925) in his search for a preservative for blood found that 0.1 cc. of a 5% solution of boric acid gave good results with 2.5 cc. of blood. Ireund (1927) studied the eventual accumulation of antibodies in the organs of rabbits which had received injections of rabbit blood containing agglutinins. Ill. 2. 3e The conclusions were: Agglutinins accumulate in the organs of rabbits which have been passively immunised by an injection of rabbit blood con- taining agglutinins. The rote of accumulation of agglutinins in liver, spleen, kidney and lungs. was greater than in uterus and skin. If the animal was killed within from five to 17 hours after injection of the serum containing agglutinins. all or a large part of the agglutinins can be removed from the uterus and skin by perfusion; after 17 hours the agglutinins became perfusion fast. b h. Agglutinins accumulate in the organs of rabbits which have been injected with killed 2. typhosus. The distribution of agglutinins in the serum and organs of such actively immunized rabbits was similar to that found in passively immunized animals, one or more. days after the injection of immune serum.“ Little (1929) found that isoagglutinins in bovine serum would deteriorate after a few days storage in an ice chest. The serum with the highest titre lost its agglutinative properties after nine to lb days storage under similar conditions. The inactivated agglutin- ative properties could be restored by the addition of fresh serum from a normal cow. Eiiils (1929 a) examined blood from 209 cows and 31 bulls and established the fact that isoagglutinins were present in bovine blood. Three principle groups were identified. although anomolous reactions suggested additional grouping. The agglutinins were labile and dis- appeared on standing at room or refrigerator temperature. Different blood types seemed to have no relation to the various breeds of cattle. Little (1929 b) further found that the agglutinative substance of group I was generally inactivated at 60° C after one hour. He also found that all sera were not completely inactivated at 56° 0 in one hour. With the addition of fresh unheated serum from certain cows. the component was restored. Laigr (1930) examined the blood of forty dogs. 1All the canine sera agglutinated human group 0 blood corpuscles. The human blood 7 group A.B. sera agglutinated canine blood corpuscles. The pooled canine serum was shown to possess a B receptor with regard to human blood. Canine blood was found to resemble the Ba human blood group. Thomoff (1930) demonstrated four blood groups in 100 horses: 0ab..Ab, Ba and.L.B.o. as in man. Agglutinins and isohemolysins had». at lower titre and it was often difficult or impossible to demonstrate them by the usual methods. The percentages of the four groups were: 0. 12;.A. 70; B, 5; and AB. 13. Attempts to produce test sera by immunization were not useful because of the appearance of non-specific agglutinins. Todd (1930) produced isoimmune sera by injecting blood from one bird into another bird. Absorption experiments showed that the exhausted serum. though absolutely without action on the cells with which it was exhausted. in most cases had lost comparatively little of its agglutinating power for the corpuscles of other individuals. A.high1y polyvalent isoagglutinating serum. exhausted with the corpuscles of a particular fowl was still capable of agglutinat- ing the corpuscles of any other individual fowl (not a close blood relative) and so behavel as a specific reagent for the individual in question. Landsteiner (1931) found that agglutinin agglutinogen reactions were found in animals. These reactions were definite only in certain species and were seldom as consistent as in man. 8 giglgr (1931) immunized rabbits with paratyphoid bacilli which contained Forssman antigen: the serum produced, caused agglutination of human red blood cells of group A and.L.B, but did not agglutinate cells of group 0 or B. Andersen (1932) demonstrated four blood groups in 61 horses. His results were as follows: Group 0 21h59 75 Group.A 31-15 % Group B 37-70 % Group 1.13. b.5o % Eight animals could not be grouped in the above scheme. The author suggested the possibility of differentiating the breeds of horses by means of blood groups. Lehnert (193%) in a study of blood groups in horses, pointed out the difficulties in determining their blood groups. The very low content of isoagglutinins, in normal horse, was suggested as a cause of the difficulty in the blood grouping. Group specific serum was produced by injecting serum of one group into horses of another blood group. Buscher (193N) examined the blood of 98 horses and on the basis of agglutinin-absorption demonstrated five distinct groups. innderson (1935) divided sheep into three blood groups on the basis of consistent reactions and pointed out the relationship between 9 sheep blood agglutinogen B and human blood agglutinogen A. Human A blood would absorb anti R agglutinin; sheep blood R agglutinogen would partially absorb anti A agglutinin. Paniseft (1935) reported the presence of different blood groups in horses. cattle. sheep and pigs but the groups were not as distinct as those in man. Four groups were found in horses. Semanowski (193%) pointed out that blood groups in animals were not as clear as these were in man because of the great frequency of atypical agglutinins in the blood of animals. Hggmgg (1936) studied blood groups of horses and concluded: a. a comparison of the blood of the horse and of man demonstrated a resemblance between the blood groups of the two species with regard to agglutinins and agglutinogens. b. After removal of the species specific agglutinins by adsorp- tion. sera from the horse and man still showed group specific agglutination of human and horse erythrocytes. c. This reciprocal interaction showed the similarity of the ag- glutinogens and agglutinins in man and in the horse. Hardot (1937) performed cross agglutination tests with blood from 250 Danish pigs. It was not possible to set up a simple system of blood types. These experiments confirmed the results obtained by Jettmar (1930) who worked with blood of swine from Manchuria. Ferguson (19N1) made intensive studies in typing of bovine blood. Immune sera were produced by four intravenous injections. 10 at weekly intervals. of one titre of citrated blood from one cow injo another. Nine antigens were established in red blood cells of cattle by hemolytic tests with absorbed and unabsorbed sera. .L genetic study of seven of the antigens in the red blood corpuscles of 10” animals showed that each antigen was inherited as if controlled by a single gene. Ierguson suggested that certain of these cellular antigens might be of value in cases of disputed parentage and in the inheritance of complete physical characteristics such as milk yield. lhploying the same technique. in 1942. Ferguson 33 a; were able to demonstrate 23 additional antigens in the bovine. ‘91: g (19M3) discovered three different types of agglutination patternsin chicken erythrocytes. by the use of bovine serum which normally contains agglutinins for chicken erythrocytes. Red blood cells of type I removed all agglutinins from the bovine serum. Cells of type I! removed agglutinins fer type II only. so that bovine serum ab- sorbed by cells of group II. was still capable of causing agglutination of cells of type I and III. Chicken erythrocytes of type III absorbed agglutinins from serum of type II and III, but left antibodies for type I cells. Genetic studies showed that the progeny of type I was produced when both parents belonged to the same group. Similarly parents of group II produced progeny of group II. Out of 157 individuals produced as a result of mating of parents of type III, about half had blood of group III, one fourth had type I cells and one fourth had cells of type II. 11 Bruner gt_gl (19M8) sensatized mares to certain types of horse erythrocytes. These mares were later bred to stallions which trans- mitted the corresponding genotype in the red blood cells of the off- spring. ¢Apparently normal foals developed hemolytic icterus when they received the corresponding antibodies through the milk of their dams. Susceptible foals when put on nurse mares, immediately after birth. did not show this condition. Bruner £t_§l (19M9) injected gestating sows with erythrocytes of the boar to which they were bred. Baby pigs developed anemia and died after suckling the sow. Muraschi £t_§1 (19u9) pointed out the importance of sheep red blood cells in the standardization of complement fixation tests. They found that sheep blood varied in its hemolytic activity. Among 134 samples of sheep sera tested. seventeen (12%) showed some other evidence of lysine for red blood cells of some other sheep. In view of this in- formation the authors checked the compatibility of different sheep blood before pooling them as a source of red blood cells. used exten- sively in their laboratories for complement fixation tests. The amount of complement required for 50% hemolysis (hemolytic units) of cells from seven different sheep showed marked variation. Yoggg gt a; (1949) demonstrated four different isohemoagglutinins in sera of dogs by isoimmunization. The antigenic factors were called "canine.A. B. C and D“ and the corresponding antibodies ”canine anti.A. B. C and D“. "A" antibodies were most easily produced. Dog cells containing "A” factor were designated as “Do-positive” and those which 12 lacked this factor were called "DOQnegative" cells. A Do-negative bitch was immunised by Do-positive cells and was then bred to a Do-positive male. The litter of eight from this bitch had four Do-positive and four Do—negative pups. One of the Do—positive pups showed icterus and bilirubin concentration in the plasma was markedly increased. Another Do—positive pup showed severe anemia but did not deve10p jaundice. The other two Do—positive pups showed neither anemia nor jaundice. ”The severely anemic whelp and a normal litter male were sacrificed for histologic studies -—-. The affected animal showed splenomegaly and more marked erythrOpoiesis in the liver and spleen. The erythroid elements in the bone marrow. liver and spleen of the affected puppy were more immature than those in the normal litter mate." 19.925 31 a}; (1949) immunized a dog after a series of transfusions from several donors. The immune serum contained hemoagglutinins and hemolysins for cells of about 2/3 of dogs selected at random. Bed cells which reacted with this immune serum were called Do-positive and those which did not show any reaction were labelled Do-negative. Incompatible whole blood was transfused into 13 different re- cipient dogs. The main manifestations during this period of immuniza- tion were: Restleness. salivaticen. vomiting. incontinence and fever occurred in all cases. but the manifestation of shock was variable. Three dogs showed synptoms of hives and one died immediately after the injection. ' The concentration of hemoglobin. in the plasma of recipient dogs 13 was maximum within 10 minutes after transfusion of incompatible blood. Concentration of bilirubin was maximum 3 to b hours after such a transfusion but the bilirubin level in plasma returned to normal range within 2H hours. The authors tagged the donated red blood cells with radio-active iron and demonstrated that the transfused cells completely disappeared from the recipients circulation within the first hour after the trans- fusion was completed. The complement in the plasma of the recipient dog abruptly de- creased during the transfusion of incompatible blood and in some dogs was barely detectable five hours after the injection of the blood. Bappaport 53’31 (l9h9) in their work on rapid macroscopic test for infectious mononucleosis used the following technique for preserving sheep red blood cells: Reagent 1. 5f Borax solution. 10 grams of crystalized borax were dissolved in 150 m1. of dis- tilled water and concentrated citric acid solution added to ob- tain a pH of 7.u - 7.b. The solution was made up to a volume of 200 m1. and was sterilised. Eeagent g. One part of reagent one was mixed with 3 parts of 0.9 per cent saline solution. Equal parts of reagent 1 and defibrinated sheep blood were mixed and stored at refrigerator temperature. For use. to one part of the stored blood was added, drop by drop. in three parts of reagent 2. The resulting mixture was centrifuged and washed with borax saline solution. Traum (l9b9) obtained successful results in treating hemolytic icterus foals by repeatedly injecting 300 cc. of blood at 3 hour in- tervals and recommended that the icteric foals should preferably be fed on milk sources other than its dam. Lt—wae—aleo—euggested—thai—léOO—ee. of—blood—might_ha_ramoxed_from—icteric_faals—and—replaced—with—QGG—ec. of—eompatible—bloods Bruner £2.2l,(1950) established that foals between the ages of 2” to 36 hours lost their ability to absorb antibodies from the digestive system when fed horse serum. One-day old foals were injected intravenous- ly with incompatible high titred serum from a sensatised mare; the foals developed hemolytic icterus and showed the severest anemias 3 to 6 days after the injections. No icteric foals were produced when ertythrocytes of the mares were compatible with those of the stallions to which the mares were bred. Bzitgn (1950) reported that during the years l9hb to l9h9. nine foals developed icterus neonatorum. These foals were born from four thoroughbred mares out of a band of b5. Within 12 to 9b hours after birth the foals showed the following picture: somnolence, inappetence. weakness. jaundice and hemoglobinuria. The temperature of the foals was normal. In view of the findings of Bruner 3£_gl (19N9). the foals were treated as follows: 1. The blood of all foals born from mares known to have produced icteric foals previously was typed. 2. 3. 15 The foals were not allowed to suckle their dams until the results of a blood examination were known. This was achieved by muzzling the foals. In cases where the serum of the dam agglutinated the red cells of the foal. the latter was kept muzzled and the dam was milked at hourly intervals for a period of M8 hours. The foals. during this period. were bottle fed a mixture of cow milk and lime water in a ratiorof 2:1. At the end of the M8 hours. the foal was allowed to suckle the dam for only five minutes and was then watched for symptoms of icterus - neonatorum. If there were no untoward symptoms the foal was allowed to nurse freely but was kept under observation. MATERIAL AND METHODS The problem of typing sheep blood was approached from three different angles. outlined as follows: I. Natural Isoagglutination II. Isoimmunization III. Heteroimmunization In an attempt to avoid complications. a description of each method will be followed by the results. b. 1. Natural Isoagglutination Sheep used for collecting blood samples: The sheep used were from Michigan State College Animal Husbandry Department and represented the following breeds. 1. Rambouillet 2. ShrOpshire 3. Oxford The animals. at the time of bleeding. were apparently healthy. Bleeding technic: The sheep were bled from the jugular vein. usually in the morning. The site of operation was swabbed with tincture of iodine. The vein was rendered prominent by thumb pressure applied to the jugular furrow at the base of the neck. A 1” guage California bleeding needle was used and 10 ml quantities of blood were collected in tubes containing 20 mgm of potassium oxalate. To insure thorough mixing of the anticoagulant with the blood the vials were gently agitated. Not more than 12 animals were bled on one day. Co 17 Separation 2; red blood cells and plasma: The blood samples were brought to the laboratory and centrifuged at 1500 r.p.m. for 10 minutes and the plasma was pipetted off into another sterile tube and was inactivated at 56° C for half an hour. The red blood cells were washed twice with sterile physiological saline solution and finally a 2.5 per cent suspension (by volume) was made in saline. To avoid mixing of one reagent with another. separate Pasteur pipettes were used for each sample of plasma and red blood cells. Isoagglutination tests: Each sample of plasma was diluted with an equal volume of physiological saline solution and was distributed in 0.5 cc quantities. into as many agglutination tubes as there were samples of red blood cells to be tested. .A 0.5 cc quantity of 2.5 per cent red blood cell suspension from each blood sample was then added to the plasma contained in the agglutination tubes. The contents of the tubes were thoroughly mixed and were incubated at 37° 0. in an electric water bath. for two hours. The tests were not performed at room temperature in order to avoid chances of autoagglutination (Weiner 1948). At the end of the incubation period. to facilitate the reaction of agglutinins if present. the tubes were spun at 1000 r.p.m. for not more than five minutes and were then examined for evidence of ag- glutination. 18 RESULTS From the data in tables 1 and 2 it was apparent that a de- finitely positive agglutination reaction was not found. In some cases. indicated byl: sign. the cells appeared to have agglutinated but slight tapping of the tubes. showing such a reaction. or incubating them at 37° 0 for about 10 minutes. would break these clumps. resulting in a suspension indistinguishable from the control tube containing no plasma. Microscbpic examination of these doubtful reactions confirmed the results of naked eye examination. This difficulty was met with throughout and it was felt that under the conditions of the experiments. the agglutinins if present. wese too weak to be detected by this method. Positive controls. using human.L and B sera and human red blood cells of known blood group. were run in parallel for purposes of comparison. TABLE 1 Natural isoagglutination reactions in Rambouillet sheep. Cells of S e r a o f 5.9 e egp, 4 Sheep 1 2 3 5’ 5 B 7 8 9 '10 ll 12 Control 1 - - - - - - - - - - - .. - 2 - - - - - - - - - - - - - 3 .. - - - - - - - - - - - - u - .. - - - - - - - - - - - 5 - - - - .. - .. - - - - - - b - - .. - - - - .. - .. - - - 7 - - - .. .. - - - - - - - - 8 - - - - - - - - - - - - - 9 - .. - .. - - - .. .. - .. - - 10 - - - - - - - - - - - - - ll - - - - - - - - - - - - - 12 - - - - - - - - - - - - - - t No reaction TABLE 2 Natural isoagglutination reactions in Shr0pshire sheep. Cells of Sheep 1 2 3 r_§, o f S h e e p 5 b 7 8 9 10 ll 12 Control :0) 1 c a n n - o n n n - I l+ I I I I I I I I I I I l+ I I 00 N 0‘ kn #3 Dr! I I I I I I I I I I I I I ----------:-- 9--------:-:-- 10 a------------ 11 - - - - - - - - - - - - - 12 - - - - - - — - - - - - - - = No reaction '1 = Doubtful 21 mm 3 Natural isoagglutination reactions in Oxford sheep. Cells of Sera of Sheep ‘44 sheep l 2 3 E17 5 b ’7' Control 2--+++_~t: - 3 - - - - .1 + + - u----::e- s-----:- - b - - - - - - z; - 7-:--:.:- - TABLE h Natural isoagglutination reactions of Shrapshire erythrocytes with.Rambouillet pooled serum. Erythrocytes of Shrgpshire Sheep _¢_ 1 2 3 h 5 b 7 S 9 10 11 12 Control Bambouillet pooledserum-t-u---z+-::- - .8 No reaction _'_0-_ = Doubtful agglutination +=Agglutination _-—--—.5—-.—.——. m- .— I .‘"x 22 II, ISOIMMUNIZATION Szgmanowsg (193M). £51.39; (1938). gigs.“ (1935) and Vanguoth (1938) found that natural insoavalutihation reactions were not as specific for animals as for humans. According to £31333 (l9h8), "—-- in many animal-species. such as lower monkeys, rabbits. guinea pigs. dogs, cats. mice and fish, isoagglutination reactions are either rare or entirely absent. Even in those species of animals in which isoagglutination reactions occur. these do not. as a rule. determine as sharply defined groups as in man and apes." Lehnert(1934) determined blood groups of horses by the use of group-specific high value sera. obtained from horses by means of iso- immunization. The author employed guinea pig serum for complement and produced characteristic hemolysis. Hemolytic reactions were found to be more uiiform than hemoagglutination reactions and for this reason the hemolytic method was chiefly used for typing blood. Wilson and Elleg.(l9ub) in the discussion on unity and diversity of antibodies stated that immunization with a single antigen yielded an antiserum which could display a variety of specific phenomena ——. precipitation. neutralization of a toxin and protection of an infected 9.1.111. '. 1 etc. In light of these facts the problem of blood groups in sheep was studied by using immune bsohemolysins. 23 Isoimmunization g£_§hggp: The procedure has been tabulated in Table 5. Apparatus l. Erlenmyer flask marked to approximately 110 cc. 2. Intravenous outfit for blood transfusion as illustrated in Figure l. 3. Anticoagulent: Sodium citrate 8.0 gm. Dextrose 20.0 gm. Distilled water to make 100.0 cc. One part of this solution was used with nine parts of blood (Kolmer. 19M5). Technic 11 cc. of the sterile anticoagulant was put in the sterile Erlenmyer flask. The donor sheep was bled. as described previously. from the Jugular vein and the blood collected in the flask as aseptically as possible. lAt the time of the collection of blood. the flask was continuously but gently shaken to insure thorough mixture of the anti- coagulant with the blood until the required quantity of blood was obtained. The neck of the flask was then carefully slipped into the rubber cup (Fig. l) and the blood allowed to flow to the needle by opening the pinch cock. Air bubbles or clots could be detected through the translucent rubber tubing. The blood was then transfused into the recipient sheep intravenously through the Jugular vein after applying tincture of iodine to the site of operation. Fig. 1 2“ TABLE 5 Schedule of isoimmunization of sheep. No. of Donor Dates of blood Reactions. if any. shown by the Sheep transfusions into recipient sheep. and remarks. recipient sheep 3 .August 8. 19U9 August 1h About 10 minutes after injection. the sheep showed increased respiration and became listless and dull. stood in one corner with the head down and eyes closed. The animals appeared normal next morning. August 22 August 29 Mild reaction. September u On September 15. 19u9. 150 cc. of blood was collected from the immunized sheep. The blood was allowed to clot and the serum was pipetted off into a sterile bottle. The serum was markedly pink in color. u August 3. 19u9 August 1” Mild reaction. August 22 August 29 The animal could not be prOperly re- strained as a result only about half the quantity of blood could be injected. September N ' On September 15. 19h9. 100 cc. of blood was collected from the recipient sheep. Blood allowed to clot and serum separated. The serum was clear and of natural color. 5 August 8. 1949 August 14 August 22 Augus t 29 September h Mild reaction. On September 15. 19u9, 150 cc. of blood was collected from the recipitnt sheep. The serum was of natural color. 25 Collection and preservation g: isoimmune sera: Blood from the immunized sheep. was kept for approximately two hours at room temperature and was then transferred to the re- frigerator at t 5° C and kept overnight. I The overnight refrigeration was adopted to (a) facilitate maximum retraction of the blood clot and thereby to get the largest - possible volume of isoimmune serum. (b) avoid or minimize the chances of carrying with the serum irregular of "cold" agglutinins - Weiner (1948). The next morning the serum was removed by means of a Pasteur pipette7placed in a sterile Erlenmyer flask and was inactivated in a water bath at 5b° C for half an hour. The inactivated serum was pre- served by the addition of a phenol-ether mixture. recommended by Michigan Department of Health. Lansing. Michigan. The preservative consisted of Ether 5.0 cc. Helted Phenol Crystals 5.0 cc. The mixture was added at the rate of 12 cc. per liter of serum. The preserved serum was then distributed in 25 cc. quantities in rubber stoppered bottles and stored in the refrigerator. Collectigg and preservation pf sheep red blood cells: Blood samples were collected from sheep using the same anti- coagulant and technic as indicated under natural isoagglutination. The samples were centrifuged and the red cells washed twice with physiological saline solution and were stored in the refrigerator in 0.85% sodium chloride solution. 2b The difficulty with this method of storing of red cells was that after 2 to h days of refrigeration. the cells would show slight to appreciable sign of hemolysis and would also give unreliable results. In view of this difficulty. the following preservation teChnique was employed: Anticoagulant: Sodium citrate 3.8 gm. Sodium chloride 0.5 gm. Distilled water 100.0 CO. 1.5 cc. of this sterilised anticoagulant was added to sterile test tubes marked to a capacity of about 15 cc. Blood samples were collected in separate tubes up to this mark and the tubes were gently inverted a few times for a uniform distribution of the anticoagulant. These tubes were stored in the refrigerator without centrifuging. washing and suspending in physiological saline solution. In the hemolytic tests. red blood cells were obtained from a refrigerated sample of blood not more than four days old. Complement_- its collection and preservation: Six apparently healthy adult guinea pigs were maintained for this purpose and were bled directly from the heart at weekly intervals. The pooled blood was allowed to coagulate. as indicated previously. and the serum was separated. Bhamy's sodium acetate method. as modified by Sonnenschein. was employed for preserving complement serum (Kolmer. 1945). 27 Sodium acetate 12.0 gm. Boric acid 4.0 gm. Distilled water 100.0 cc. The salts were dissolved in water and the solution was sterilised be- fore use. Bqual volume of this preservative was added to the complement serum and the latter was stored at': 5’ C. Every week. old complement was discarded and was replaced by fresh. Hemolytic_Tests: Isoimmune hemolysin. complement and the blood sample were kept cold. until required for use. Separate Pasteur pipettes were used for complement and for each sample of blood and isoimmune sera. Dilution of reagents. where indicated. was done by refrigerated sterile physiological saline solution. The latter precaution was taken to minimise chances of hemolysis due to thermal variation. Bed blood cells were separated from the stored blood samples and were washed twice with physiological saline solution by centrifugation at 1000 r.p.m. for a period of about 10 minutes. Hemolysin was diluted 1:5 with physiological saline solution. Hamolytic tests were carried out iriclean Kahn tubes. The quantities of reagents used for each test were as follows: 28 Hhmolysin (1:5) 1.0 cc. Complement (1:30) 0.2 cc. Cell suspension (2.5%) 1.0 cc. Contents of each tube were gently shaken and the tubes set in Kahn rack for water-bath incubation at 37° C for 30 minutes. ‘At the end of this period, the tubes were examined for indications of hemolysis. Degrees‘g£_Hemolysis: Those which showed 50% or more of hemolysis were designated as + (positive). and those which showed no hemolysis. when compared to control tube, were declared as - (negative). In between these two limits were the tubes which did not show clear hemolysis: these reactions were assigned 3 (doubtful) sign. Analysis 2£_isoimmune sera: Analysis of the isoimmune sera was done by differential absorption technic; for this purpose. red blood cells of 19 different sheep were used as listed in table 6. Absorption of an immune serum was done according to the technic employed by Ferguson (l9ul). ‘.~(s e; \. N TABLE 6 Sheep used for analysing isoimmune sera and hetero immune sera. Serial No. Animal No. Breed 1 b99 Rambouillet 2 b8} Rambouillet 3 649 Shrapshire u b50 Shropshire 5 753 Oxford 6 75b Oxford 7 713 Hampshire 8 71b Hampshire 9 424 ShrOpshire lO b35 Shropshire ll b08 ‘ Shropshire 12 b09 Shropshire 1} b32 Shropshire 1” 630 Shropshire 15 629 Shropshire lb b5l Shropshire 1? b42 Shrapshire 18 bBb Shrapshire 19 one Shropshire 30 RESULTS In table 7 are tabulated the data of hemolytic reactions with red .blood cells of nineteen different sheep. using the three different iso- hemolysins and guinea pig serum (1:30) is complement. §g§g§.§g. 1, It was noticed that cells of sheep No. 1. 11 and l! were definitely lysed by henolysin/s contained in the isoimmune serum. With this serum cells of sheep No. 5 and It did not give consistent results and the hemolysis. if produced. was not very marked. Red blood cells of l the other fourteen sheep were not lysed. Serum .1120 5. In the case of isoimmune serum No. 5. cells of sheep No. 11 were definitely lysed. thereas red blood cells of sheep No. 5 and 7 gave variable results. Cells of other sixteen sheep gave negative hemo- lytic reaction. Serum g3, E, Under the conditions of this investigation. serum No. R could not be shown to contain any detectable hemolysis for the cells of any of the 19 sheep. Interpretation of the data in table 1, —~*" Comparison of these data for isoimmune sera No. 3 and 5 showed: (a) Serum lo. 3 has hemolysins for red blood cells of sheep No. 1. 11 and 17. (b) Serum No. 5 has hemolysins for the cells of sheep No. 11 but not for the cells of sheep Ho. 1 and 17. (c) Serum No. 3 and 5 contained a common hemolysin for cells of sheep He. 11. (d) Serum No. 3. also contained qualitatively differenchemolysin/s for cells of sheep No. 1 and 17. 'T ._- a , '1 A ... r‘ V , - -.. .. .f , - . ‘1 .— a , " -..... ...._. ' I. .- e u..- .-_. ....-. H... . A Q ._ . 31 Analysis of isoimmune Serum No.u3. To further investigate into the nature of plurality of hemolysins of the hmmune sera No. 3 differential absorption was resorted to. The results of hemolytic reactions of these absorbed sera have been tabulated in table 8. Observation of the table indicated that: (a) Absorption of the immune serum with sheep cells, which were not lysed by the serum did not take out hemolysins for the cells of sheep No. l. 11 and 17. which were lysed by the unabsorbed serum. (b) The serum absorbed by cells of sheep No. 1. gave + hemolytic reaction with cells of sheep Ho. 11 and 17 but cells of sheep No. 1 were 1.1%. unhemolyzed. (c) The isoimmune serum. absorbed by cells of sheep 11. had hemolysins for cells of sheep No. l and 17 but none for the cells with which it was absorbed. (d) Cells of sheep No. 17 absorbed out hemolysins for cells of all the three sheep 1. 11 and 17. In the light of these facts it was assumed that the cells of sheep No. 1 had an antigenic factor qualitatively different from the one present in the cells of sheep No. 11 because of the fact that the cells of sheep No. 1 did not absorb out the hemolysin for the cells of sheep Ho. 11. Since the cells of sheep No. 17 absorbed out the two qualitatively different hemolysins for cells of sheep No. l and 11. it was inferred that cells of sheep No. 17 had two antigenic factors - one corresponding to that present in the cells of sheep No. l and the second factor corresponding to the one present in cells of sheep No. 11. Hemolytic reactions with isoimmune sera. TABLE 7 Cells of sheepgno. 3, 1 2 cos: 1o 11 12 13 1n 15 lb 17 18 19 20 u Isohemolysin number‘gA‘ 5 l+ |+ |+ No hemolysis Doubtful hem Hemolysis olysis 32 Analysis of isohemolysin no. 3. TABLE 8 H e m o 14y t i c R e a c t i o n s Cells of Unabsorbed __:f§erum absorbed in cells o£;___ sheep no. serum 7 15__ 1 ll 17 1 + + + - + - 2 - . - - - - 3 .. - - - - - H 9 - - - - - 5 z :2 - - - - b .' .. - - - - 7 - - .. .. - - 8 a. - .- .. .. - 10 - - - - - - 11 + + + + - - 12 - - - - - - l3 - - - - - - 1M - q - - - - 15 - - - - - - 1b 2. r. z - - - 17 + + + + + «p 13 - - . - - .' l9 - - - - - - 20 - - - - - - = No hemolysis = Doubtful hemolysis = Hemolysis 33 34 Tentative designation to these two antigenic factor and to the corresponding antibodies were according to the following plan; Sheepio. .Antigen Present COrresponding Antibodig; 1 31 81 17 31' s2 '1' '2 Analysis 22 isoimmune serum go. 2‘ The immune serum was absorbed with the cells of sheep No. 7, ll. 15 and 18. As shown in table 9 the reactions of these cells with the un- absorbed serum were: '3. +, - and - respectively. Results of hemolytic reactions of the absorbed sera with cells of the different sheep have also been given in table 9. It was observed that the isoimmune serum contained hemolysins for cells of sheep 11 but not for the cells of any of the other sheep used. At first it appeared as if the serum contained antibody s2 corresponding to 82 antigen of cells of sheep No. 11. But further observation revealed that if the hemolysin responsible for the lysis of cells of sheep 11 was s2 , then it should hemolyze the cells of sheep No. 17 also because the antigenic constitution of these cells was proved to be 8152. This pointed to the possibility of a third type of antibody (s3) which caused hemolysis of cells of sheep No. 11. Under the circumstances it seemed logical to revise the previously prepared constitution of the cells of sheep No. 11 and represent it by the formula 81, 82, S3. 35 mm: 9 Analysis of isohemolysin no. 5. H e m o 1_y t i c R e a c t i o n s Cells of Unabsorbed Serum absorbed by cells of sheep no. serum 7 11 15 18M4¥ 1 - - - - - 2 - - - - - 3 - - - - - I4. - .. - .- .. 5 .1- - - - - b - - - - - .1 - - - - 10 - - - a - 11 + + - + + 12 - - - - - l3 — - - - - 1H - - - - - l5 - - - - - lb - - - - - 17 - - - - - 18 - - - - - l9 - - - - - 20 - .. .. - - - = No hemolysis z :- Doubtful hemolysis + = Hemolysis .Aoona Bonn couscoam sue. oqsaadoaa and: aHHoo can noose no coauoeea owuhaoaon Hooanme m .3.— 3b III. HETEROImMUNIZATION ‘Anderson (1938) immunized rabbits with human type A blood cells and produced anti-A immune rab.it sera. It was demonstrated that hemolysins for sheep blood cells. contained in the antinA sera, were qualitatively different. It was felt that with suitable absorption technic, individual differences in sheep blood cells could be detected. Heteroimmunization of rabbits with sheep red cells presented the problem of the presence of Forssman antigen. This thermostable antigen was known to be preent in red cells of sheep (Boyd. 1947). and in addi- tion sheep cells might also contain antigenic factors other than the Fores- man. It was therefore obvious that serum from rabbits immunized by sheep cells would contain Forssman antibodies and antibodies corresponding to the additional antigenic factors, if present in the cells used for immuniza- tion. Demonstration g£_paturally_occurring|lorssmag like antibonEEDin the serum 25 rabbits: Sera of three rabbits 1. M and b. selected at random from a group of ten. were inactivated at 56 C for half an hour. Sheep cells were heated. in a boiling water bath. for half an hour. Equal volume of 50% suspension of these heated cells was used for the absorption of the three rabbit sera. Absorbed sera were diluted 1:10 with 0.85% sodium chloride solution. Hemolytic reactions were run using (a) the absorbed sera (b) sheep cells and (o) 1:10 guinea pig serum as complement. For purposes of 3? comparison the tests were run with unabsorbed sera. TABLE 10 Presence of natural Forssman like antibodies in rabbit serum. —§abbit no. Absorbed Serum Unabsorbed Serum 1 - + u - + b - + Hemolysin control - - Complement control - - Physiological saline contrOI . - In view of these preliminary findings rabbits were immunized with red blood cells from various sheep to determine (a) whether absorp- tion of heteroimmune sera with Forssman antigen (heated sheep cells) would leave other antibodies and (b) to analyze these absorbed sera for the presence of principal qualitatively different hemolysins. Heteroimmunization g£_rabbitp‘with cells from different sheep: The tabulations in table 11 presents the source of red blood cells. the number of the sheep from which the cells were obtained and the dates of inoculations. A 25% suspension (by volume) or red blood cells. was made in sterile physiological saline solution. and rabbits were immunized by re- peated intravenous inoculations (into ear vein) of h cc. of the fresh suspension. The blood used for making the suspension was stored in the 38 refrigerator for not more than four days before use. Nine days after the last injection. the rabbits were bled from the heart. The blood was allowed to clot at room temperature and stored overnight in the refrigerator. The following day the serum was separated by centrifugation. The hemolysin was then inactivated at 56 C for half an hour and was distributed into 25 cc. sterile rubber stoppered vials. The immune serum was preserved by the ether-phenol preservative previously indicated. {A stock solution (1:100) was prepared from the undiluted serum. The 1:100 concentration was made as follows: Undiluted serum 1 cc. Phenol 5% (in 0.85% sodium chloride) u cc. Physiological solution 95 cc. Both undiluted serum and the stock solution were stored in the refrigerator. Hemolytgg titre of hgteroimmung sera: The technic employed was similar to those used for the demonstra- tion of isohemolysins, except for the dilutions of the immune sera. These data are shown in table 12. ~ I (a J _ 1 4 . ‘4 - 7 F a v \. .‘ ‘ A Z 'O'Y ‘1‘! ‘f". r » V , J '1 t .0 \« v v e 9' . . -‘ . ‘s ' a f 1 . ' : a . - ;" I — t ' . I. ‘— ' ‘ 1 I w P , s - v r . _ - I 1 ‘ .. f O ., ' ' I . \‘ C " ‘ I 1 . .- ‘- . .I . V 39 TABLE 11 Record of transfusions for production of heteroimmune sera. Quantity of Date of I/V blood Remarks ijection iniected 1 Donor - Sheep No. 1 Recipient - Rabbit No. 1 July b, 19u9 u cc. of 25% ‘ suspension of red blood cells July 9 ' hfly12 N July lb s July 19 fl JU1V 23 ' Marked difficulty in standing; no control over neck muscles: the head wobbled when the animal attempted to walk. August 1 - Bled from the heart. Donor - Sheep No. 2 Recipient - Rabbit No. 2 July b. 1949 M cc. of 25% cell suspension July 9 ” July 12 ' July 1b ' July 19 " July 23 ” Reaction similar to rabbit No. 1. August 1 and 3 - Bled from the hCBrte Donor - Sheep No. 3 Recipient - Rabbit Ho. 3 July b, 19u9 n cc. of 25% suspension of red blood cells July 9 ' July 12 ' July 1b ” Symptoms of incoordination of limbs and no control over the muscles of the neck; attempted to walk but could not take more than a few steps at a time. Considerably improved the next morning. M0 Donor - Sheep No. 3 Recipient - Rabbit No. Donor - Sheep No. R TABLE 11 (continued) Quantity of Date of IIV blood Remarks Injection. injected 3 July 19 4 cc. of 25% No symptoms of incoordination. suspension of but passed bloody urine a few red blood cells minutes after the injection. afly23 ' August 1 and 3 - Bled from the hQErta u Recipient - Rabbit No. July b. Jul? 9 July 12 July 1b July 19 mny23 Donor - Sheep No. 5 Recipient - Rabbit Ho. July be July 9 July 12 July 1b Mfiyl9 July 23 Donor - Sheep No. b Recipient - Rabbit Ho. July b. July 9 July 12 July lb July 19 mfly23 1949 u cc. of 25% cell suspension s Mild reaction. August 1aand 3 - Bled from the heart. 5 19u9 u cc. of 25% cell suspension e August 1 and 3 - Bled from the ”arts 6 1949 h cc. of 25% cell suspension August 1 and 3 - Bled from the heart. sV #1 TABLE 11 (continued) Quantity of Date of I/V blood Remarks Injection Donor - Sheep No. 7 Recipient - Rabbit No. July b. July 9 MflylZ July lb Jflyl9 hfly23 Donor - Sheep No. 8 Recipient - Rabbit No. July be July 9 hflylZ July lb hfly19 July 23 Donor - Sheep No. 9 Recipient - Rabbit No. July 12 July lb Mfly19 Donor - Sheep No. 10 Recipient - Rabbit No. July 12 July 1b fifly19 Mfly23 7 1949 8 1949 10 injected H cc. of 25% cell suspension s August 1 and 3 - Bled from the hOBI'te h cc. of 25% cell suspension s August 1 and 3 - Bled from the hurts h cc. of 25% cell suspension s July 20 - The rabbit died: showed a large abscess in the left lung. u cc. of 25% cell suspension August 1 and 3 - Bled from the hCQrte 42 TABLE 12 Hemolytic titres of heterohemolysins. Heteroimmune Hemolysin Dilution Hemolysin Complement Serum 1:500 1:1000 1:2000 Control Control 1 + + + - .- 2 + + + .. - 3 + + + - - u + + ‘3 - - 5 + .+ ‘1 - _ b + + I. - - 7 + + I - - 8 + + ‘3 - - 10 + .1 '3 - - + = Hemolysis 1: = Slight hemolysis No hemolysis Reagents used for each test: Hemolysin 1.0 cc. Complement 1:30 0.2 cc. Cell suspension (2.5%) 1.0 cc. Absogptigp_g£_Forssm§n_antibodyqpy heated sheep cells: Reteroimmune sera No. 1. 3 and h were selected at random and were absorbed by sheep cells which had been heated in a boiling water bath for half an hour. The procedure followed was the same as previously given. Hemolytic tests were run with these three absorbed sera. Unabsorbed sera were used as controls. 43 It was observed from table 13.that the cells of sheep 10 and 11 gave positive reactions with unabsorbed but doubtful or negative reactions with absorbed sera No. l and h. In many cases the absorption did not influence the hemolytic properties. The latter were believed to be due to antibodies other than Forssman and these antibodies were further expected to correspond to the anti- genic factor (other than Rorssman) present in the cells used for immunisa- tion of the rabbits. After absorption the reaction of some of the sera often varied. From the results of the absorption. as shown in table 10. it was concluded that antibodies related to Forssman antigen could be absorbed by heated sheep cells. TABLE 13 Hemolytic reactions of three anti sheep rabbit immune sera after absorption with heated sheep cells. (Forssman antigen) Hemolytic __j I m m u.n e S e r u m _~j test with Anti Sheep 1 Anti Sheep 3 Anti Sheep—h cells of Absorbed Unabsorbed Absorbed Unabsorbed Absorbed Unabsorbed argon 1 + + + + + + 2 z. + z z r. + 3 '0‘ 2: § '0‘ o I ’+ .4. .2: .4. 2: .t - 5 z - :5 + .1 2: b I - l+ I I I I 8 .1 ‘ .1 1 .1 .1 10 ‘3 + .3 '3 _: + 11 - + .. .. ..' 1 13 ' 1 .1 .1 .1 1 13 + .3 - e + - 1H + 6 - - 4 e 15 + 4 e + + + lb 1 + + 4 - .1 17 - .1 + 4 - .1 18 ‘3 - + .t - .3 19 . . .‘ 4 + . ’45 In view of the information above the heteroimmune sera were absorbed with heated sheep cells. to remove as much Forssman like antibodies as possible. .Analysis‘gf absorbed_£5teroimmune sera: Hemolytic tests were conducted using the absorbed immune sera and cells of the 19 sheep listed on page 29 with guinea pig serum (1:30) as com- plement. The format of the tests has been illustrated in table 1”. Examination of table 1% revealed that the heteroimmune sera fell into four distinctly different groups on the basis of their hemolytic prOperties. Group one included heteroimmune sera 1. 2 and 10: group two. 3 and 5; group three. u and 8; and group four. 6 and 7. From this it was assumed that the hemolytic activity of members of each group was due to similar hemolysin or hemolysins. .Analyses of these im- mune sera were carried out by absorption with some of the red cells which had given +.': and - hemolytic reactions. The absorption technic was as previously described. Results of hemolytic tests of these absorbed sera with sheep cells have been tabulated in tables 15 to 22. Examination of the data showed: (a) Absorption with any cell which showed hemolysis. removed the anti- bodies for all other cells which gave positive reactions with the un- absorbed serum. (b) In general. sheep cells which were not hemolysed with unabsorbed serum. failed to remove the hemolytic antibodies. Mb The similar hemolytic properties of each.group of immune sera ap- peared to be due to one type of antibody or antibodies closely related to each other. It was therefore concluded that. under the conditions of the investigation. four groups of immune sera represented four different types of antibodies. TABLE in 1i7 Hemolytic reactions of heteroimmune sera after absorption with heated sheep red cells. Cells of ._§eteroimmune sera corresponding to cells of shegp - 1 sheep 2 3 U 6 10 l + + + + + + + + + 2 + + .1 + - - - + .’ 3 1 I. + .. + - - .' 1 u 1 3 1 e .. .. - + - 5 ~01 + 4 ..' + 1 - 1 _+_ b .t 4 -' . - .t. .1 .t - 4 1 - .. .. - - e e - .. 8 - - + + + 1 - + .1 10 + + .3 1 A + + 4 + 11 4- + - + 1 - 1 + + 12 + + ‘1 - - - - - + 13 .1 " " " " ' " 1 " 1M + e - - - - ‘3 - + 15 1 1 1 .1 1 ' 1 " .2: lb _-I_- - + _-_I_- 4- .. - .. :- 17 3: - r .2: 4 r: - - z. 18 - z. .z - .t .t - - z 19 + + + - + + + - + 20 + + + - -I- 1 _+_ _I_- +' us TABLE 15 Analysis of heteroimmune serum No. 1 Cells of Unabsorbed Serum absorbed chell; of sheep iheep serum 1 2 ll 5 8 10 ll l3 15 lb 18 l9_2_0 1 + - - . .. - - .. - —- - 4 _+_ .- 2 '0' - - - - - - - .- 3 + + 4» .. 3 .1 " " " " .1 " "' " ' " 1 ‘- '- 1 ' " ' " .1 .1 " " .1 " .1 " " 5 1 " "' v " 1 1 " " " "’ " " ' o 1 - — - - - - - - - - - - .- 7 - - - - .. .. .. - .. - .. - - - g .. - - .. - .. .. - - - - .. - - 10 + .. - + 11 + u o + - + n o n c o + n n 12 + - .. - - .. .. - .. .. - + - .. 13 .1 - - - - 2. z - - + - .+. - - 1).: + - .. .. - + + - + - .. «I- - - l5 3 .. - - - + - - - .. - + .. - 1b :2 - - - - .t 1: - - - - - - - 17 l - - «- - + - - - .. - z .. - 18 - .. - - - - - - - .1 - - - - 19 + - - .. 3-. + - - - - - + - - 20 + a u o + c n - o a C * n - I+9 mu: 1b Analysis of heteroimmune serum No. 2 Cells of Unabsorbed Serum Epsorbed by cells of shgep sheep serum 1 2 a 5 12 1’4 15 1L 19 20 1 + - - - - - - - - - - 2 1 - - - - - - + + ; 1. 3 .t - - - - - - - - - - 4 .3 ¢ - - - - - + + + + 5 + I - - - - «- + + .. e o - .. - - - - - - - - .1 7 - - - - - - - - - - - 3 - - - - - - - - - - - 10 + - - - - - - - - - - 11 + + ,3 - * - - r r r - 12 + - .. .. - .. - + + l: - 13 2: - - z + - - - - 14 + - + + - - - + '1 - - 15 t - - - - - - - - .. - 1b - - - - - -. - - - - - 17 - - - - .1 - - - - - - 18 .3 - - - - - - .3 + 7; - 19 + - - - .- .. - - - - - Cells of Unabsorbed Analysis of heteroimmune serum No. 3 TABLE 17 50 Serum absorbed by cells of shegp_ sheep serum 5_g 8 10 13__l5, 16 17 18 19 20 1 + . - - -' .e - - .3 .. - 2 z. - ._+. _+_ 5 - - - - - 3 + - - + + - - + - - M .3 - - - .1 - - - - - 5 + - - + - - - .1 1- - b - - - - - - - - - - g + '3 I Q~ + - - + .3 - 10 .1 - - J 1 - - + - - 11 .. .. .. - - - - - - - 12 .1 1; - - g - - + - - l3 - - - - - - - - - - 1h - - .3 - - - - - - - 15 e - - + + - - + - - 15 + - - e + - - e - - 17 + - - + + - - ; - - 18 _+_ - - - - - .. .. - - 19 + - - + + - - 3: - - 20 + .3 + + - - + - - '5“- ,-.—.——- A I o .— up. A veg-.- e» - ‘— I ‘- --.- .— P—-_ .1 e.--“ a-. A A a... < w —- __ ~<.—---—- -. I I a. h. I p. Cells of Unabsorbed Analysis of heteroimmune serum No. R TABLE 18 51 u A .- Ssrum absorbed by cells ofggheep sheep serum 1 :2: 3 u 11 1 + - - + - - 2 + - - + _: - 3 - - - . .1 - u + + a. .3 - .2 5 - - - .' - - b - - - - - - 7 - - - - - .2 8 + - - 2: I - 10 - .t - . .. .. 11 + - - + - - l2 — .1 - - - - 13 - - - - - - 11+ 1 - - z. .1 - 15 :2 - - .+. - - 1b .1 - - - '1 - 17 .3 - - .3 .3 - 18 - - - - - - l9 - - - - - - 2O - - - - - .2 Analysis of heteroimmune serum No. 5 mun 19 52 Cells of Unabsorbed Serum absorbed by cells of sheep sheep serum 1 2 3 5 b 11 15 lb 17 29‘ 1 + .- + .- - + + .. .- .. .. 2 .. - - - .. .. - - .. - .. 3 + .. + .. .. + + - - .. .. it - - - - .. - - - - - - + - + - .. + + - .. .. .. b I - - - - - .. .. - - - 7 - .. - - - .. - - .. .. - 8 + - + - .. _1_-' + - .. - - 10 - - - - - - - - - - - n .r - .2 4- - - 1 - .+_ - - 12 - .. .1 .. .. - .. .. .. + - 13 - .+. - - - - - - - - - 1h - 4 - - .- - .- - - - - 15 + .. + - - + + - - - - 15 + - 9- - .. + + - .. .. .. 17 + - + .. l + + .. .. - .1 18 :- - - - - a _+_ - - - - 19 + - + - - + + 1 - .. . 20 + .1 + .. - + .1 - - .. .. 'TABLE 20 Analysis of heteroimmune serum No. b Cells of Unabsorbed Serum absorbed by cells of sheep sheep ggrum 2 5 10 15 18 19 l + L: .1 - + + - 2 - - - .. .. .. - 3 .. - .. - .. - - u - - - - q - - 5 :s - l - .I 3. - b ‘3 - - — - - - 7 + '+ + - + + - 8 - - - - - .3 - 10 + + + - + + - ll - - - - - - - l2 - - - - - _ .1 13 - - - - - - - 11+ - - - '. - - - 15 - - - - - - - 1b - - - - - - - 17 .+. - .2: - - :c z 13 .1 I - - 1 - - 19 + + + .. + - .. 20 .3 - - - .3 - - TABLE 21 Analysis of heteroimmune serum No. 8 5% Cells of Unabsorbed Serum absorbed by cells of sheep sheg serum 1 2 L b 8 10 11+ 15 1 + - - + + - - + - 2 + - - r + - - t + 3 .. - - - - .. - - - u + .1 - t _: '1 + + - 5 .+. - - 3- - - - - - b .1 ' .1 1: ' ' ' .1 v 3 + - - + + - - + - 10 «i- - - 4 + .. .. «I; 1 11 + - - + + - - + - 12 - - - - - - ‘3 - - 13 .1 ' ' .1 ‘ ' ' ‘ ‘ 1H - - - - — - - - - 15 2: - - + l - - a - lb - - - - - - + - - 17 - - - - - - - .. .. 18 - - - - - — - - - l9 - - - .. - - - - .. 20 3 - - - - I - - .. 55 TABLE 22 Analysis of heteroimmune serum No. 10 Cells of Unabsorbed ___ Serum absorbed by cells of sheep sheep, serum 1 M 10 12 13¥ 15 lb 13 1 + - 1- .. -‘ + + + - 2 + .. + - - + 4, + .. 3 :2 :c - - - - - - - u - - - - - - - - - 5 r: - z - - - - - - b - .. .' - - - - - - 7 - - - - - - - - - S 1 - - - - I. - .. - 10 + 3; + - - + - + .. 11 + k - ‘+ z - + - .t I z 12 + - + - .3 + - + - l3 - . - - - - + - - - 14 + - + - - + I + - 15 I - .t - - :0: - + .- 1. :1 - I - - g - .~: - 17 I - - - - .r. :. .t - 18 j; - - - - - - .1; - 19 + - + .3 - + - + '1 BLOOD TYPING 3F EEhLTHY SHEEP As indicated above three different types of antibodies were detected by analyzing isoimmune sera and four qualitatively different antibodies were recognized by analyzing heteroimmune sera. Three and four different hemolysins were prepared by absorption of isoimmune and heteroimmune sera respectively. These seven reagents were employed to type blood of apparently healthy sheep maintained at the Michigan State College Farms. Results 3: blood typing: Each type of antibody would cause hemolysis of the cells containing the corresponding antigenic factor. Isoimmune sera could therefore show 51, $2 and S3 factors in red cells and the four reagents from heteroimmune I sera could show R51, RSa C, R53 and RS“ antigenic factors. "B“ indicated that the factors were related to heteroimmune sera produced in rabbits. The various possible combinations of the $1, $2 and S3 factors were: a. $1. $2, S} b. 51, 52 c. 51, 53 d. 32, 33 e. 81 f. S2 g. 83 and Hum. u awupoaw nosowwauo Heaoawon on ouoav Hon cuppa swab boaonopaacbo new» waoacoeo muoa absence. 57 Similarly the possible combinations of the four "E” antigenic factors were: a. RSI, $2, 53, Su b. RBI, 52' S3 0. 332. 53, Su d. 382, Su, 81 0. R33, Su, 51 f. R51, 32 g. R31 , 83 h. RS 1. R82, S3 3. RS k. RS3, Sn 1. R51 m. R52 n. R83 0. RSu and I)... The total number of sheep tested was 260 and represented the following breeds: 1. Shropshire 2. Hampshire 3. Oxford h. Rambouillet 5. Southdown 6. Dorset-Horn 7. Suffolk a 58 All the animals in each breed were listed with their serial and identification number as shown in table 23. Results of blood typing by isoimmune and heteroimmune reagents were tabulated in table 2k and 25 respectively. Table 2b shows the total number of sheep of each breed together with the percent which were positive for a particular antigenic factor or group of factors. 59 TABLE 23 List of sheep used for blood typing. Serial Animal Serial Animal Serial .Lnimal Serial Animal No. No. No. No. No. No. No. No. SEROPSEIIRE 5111102511131: SEBDPSHIRE HAMPSHIRE 1 3n #6 517 91 0uu 132 707 2 202 “7 552 92 659 133 (31 3 153 ”8 25 93 - 134 733 u 503 u9 555 94 050 135 738 5 7 50 501 95 b52 136 720 0 0n 51 522 9b 635 137 719 7 812/ 991 52 551 97 651 138 (no 8 35/108 53 27 98 000 139 10/9 9 509 54 ’+211» 99 783 1110 728 10 509 55 521 1H1 - 11 5u5 50 502 . 1u2 732 12 333 57 5% 1111125111121: 1143 7/9 13 3”? 58 558 111 73a in H3 59 61 100 110 15 250 60 50a 101 313 16 0 01 - 053 102 u10 oxroan 17 821?992 02 007 103 440 18 302 63 b3? 10a 7 1M5 1820n8 19 39 04 639 105 #21 1&6 197 20 19 05 005 100 113 1u7 379 21 737 00 030 107 105 1H8 908 22 2 67 002 108 302 1H9 71 23 1M 08 61 109 425 150 u59 2H 11 69 00 110 u08 151 35h 25 539 70 0H2 111 130 152 989 26 30 71 0n1 112 uu0/002 153 102 27 30 72 011 113 u16 15h u98 28 221 73 048 11u 10n 155 186033 29 - 7a 020 115 81 156 M25 30 487 75 619 116 u20 157 112 31 171 76 017 117 10k 158 nun 32 21 77 050 118 #39 159 u90 33 519 78 608 119 66 100 571 3n 212 79 bh7 12o uiu 101 108 35 213 80 - 121 303 102 752 36 355 81 621 122 - 103 119/7147 37 215 82 018 123 313 10k 759 38 5&1 83 629 12u 160 105 758 39 232 8h 6M5 125 121 160 755 no 206 85 609 126 100 107 (53 u1 526 80 058 127 293 108 706 12 209 8? b31 128 713 1b9 757 “3 551 88 057 129 723 170 7b} an 513 89 610 130 710 171 750 #5 223 90 632 131 72” 172 75a 60 TABLE 23 (continued) List of sheep used for blood typing. Serial .Animal Serial Animal Serial Animal __322_ __122_. ._lEb_. ._188._ __§g;_. __§g;_. OXFORD SOUTHDowN DORSET HORN 173 750 212 331 251 48/589 174 780 213 342 252 40/518 175 705 214 01 253 48/572 170 758 215 501 254 48/575 177 7b7 21b 597 178 7b“ 217 563 . 218 509 SUFFOLK 219 500 RAHBOUILLET 220 570 255 501 221 141 250 583 179 5 222 000 257 500 180 48/574 223 4 258 582 4181 898 224 145 259 570 182 40/95 225 137 200 558 183 47/271 220 505 184 46/204 227 554 185 41/134 228 504 186 48/591 229 789 187 40/92 230 Jenkin 05 188 40/213 231 4003 189 176 232 800 190 46/522 233 791 191 212 234 782 192 40/210 235 793 193 1+b/520 23b 792 194 “8/590 237 798 195 “8/595 238 795 190 40/102 239 790 197 42/301 240 788 198 41/142 241 785 199 085 242 717 200 090 201 688 202 684 DORSET HORN 203 682 204 695 243 144 205 691 244 - 200 677 245 131 207 700 240 120 208 079 247 334 209 ‘ 080 248 47/277 210 781 249 48/584 211 797 250 45/585 bl TABLE 24 Results of blood typing by isoimmune sera. c t o r s A n t i g e n i c f a S 32 $153 5233 - Breed 51 82 S3 $15253 -# l SHROPSHIRR 1, 8,08 21, 11,18, 10,14, 20,35 71,74, 2,3,4,5,0,12, (.9. 24. 34.53: 19.25. 78 13.15.16.17. 22.51. {3. 7b9859 21.32. 33.26.28.29. 33.31. (7. 90.92. 45.59. 30.39.39.40. 38.42. (9 94.96 b4.b9. 41.43.94.96. 95.99 “7.43.99.50. 51:52:53.5”. 55.59.57.50. 01.02.03.05, 00.07.70.72. 75.30.81.32. 83.84.80.87, 88.89.91.93. 97.98- 11 2 5 "10 10 2 "3“" 50‘ HAMPSHIRE 102, 113 134 117,121, 135 100,101,104, 103, 130 105,100,107, 139 108,109,110, 111,112,114, 115,110,118, 119,120,121, 122,123,124, 125,127,128, 129,130,131, 132.133.131. 138,140,141, 142,143,144 3‘ 1 1 3 ’15' 30 OXFORD 171 170. 150.153. 100 145.14b.147. 177, 104,109, 148,149,151, 178 174 152.154.155. 150,157,158, 159,100,101, 102,103,105, 107,108,172, 113.175.17b 1 3 5 1 24 62 TABLE 24 (continued) Results of blood typing by isoimmune sera. .A n t i gge n i c F a c t O r s RAMBOUILLET 194 190, 210 203 208 179.180.181.182. 183.184.185.180, 187.188.189.190. 191.192.195.19b. 197.198.199.200. 201.202.204.205, 200.207.209.2112 1 2 1 1 28 SCUTHDOWN 230, 223. 232 212.213.214.215. 231 229 210.217.218.219, 220.221.222.224, 225.226.227.228, 253.254.235.239. 239.240.241.238, 251.242 2 2 1 20 DORSET HORN 243 253 240 244.245.247.248. 249.250.251.252, 2 —~ —— _ “‘ 1 1 l 9 SUFFOLK 255. 257 250. 260 8 63 m m H n a m :H m m w 7777 .7777 7777 .7777 - 77. 7. .ImmH .amH .mmH OMH HRH.o:H .HNH .omH msH .NmH.OMH.anH .omH 22H .mmH . .me me .mHH.~HH.mHH mmH .OHH .RMH naH .sHH .nnH .wHH .NHH.HHH.moH mmH .mmH .NOH .RoH .mMH moH .mHH .NOH .oHH .moH.m0H.HoH .mmH .ooH .OOH amHmmmzam .77H H m H m : .77m77 77 m m 0: m7 .77m m NH 777 .7 7 7 . .mm4mm.am 7 .mm.:m.mm.Hm .om.ww.:m.mw .Hm.ow.o~.:~ .H~.mo.wo .No.oo.mo .mm.wm.mm .om.m:.~:.mz mm.ow mm mm .::.m:.oz .o~.oo .mm.- .ao.mm .mm.mm.Hm.~m .mm.mm .n~.m: .zm.mm .mm.~m.om m: ma .mm.om ma H: ~m.m~ .am.om .0:.m: .o~.mH.mH.HH .om .H0.:H .mm.:m am mm .mo.mo Hm .Hm.m .mH.~H ma .zm.mH .mm.mH .oH.~.m.~ mm .o.m .:.H .oH.a amHmmmommm :mmm 3% 3 N . m..mn. 11H} m H . N H . .2 “a h z m N," hm a. on m H . mu... N” Hmm I 609nm Hmm mmm u a o a o s h o H a e a H p a 4 .saee eqsaaHoaepon mp mnHmha ocean no euHsoem mm aqmaa 61+ 7hr. 7mr1 7m1 .H m 7-.HM777 7m1 7dr. 1m. a. 7 7 .mww ozm .mmm .mmm.~mm .Hmw .omm.mmm .amm .mmm.mmm 0mm .mmm mHm .Hmm.~Hm me .omm .mmm 3mm mam .nHm .HRN www.mHm .sz.mHm .mHm mmm .zmm .mom zzonmspom .uM7 .7w1 m H Mr1. m m 7hw7. mH ‘ .11w1 me.HHm .mom.~om .mom.~mH .omH.:mH .mmH.mmH .HmH.omH maH .mmH.wwH mmH How .mom com .amH.mmH .owH mom .mmH owH mom .Hom .mmH.HmH Rom .zmH.me com emHHHpomzqm m m .1m1 H m H m m o .1M7 .Lw1 m 1m1 mMH .mOH HNH .mmH mNH ooH.mmH HoH NNH .ooH .omH wNH .moH.moH .zmH.mmH .mmH NmH .NNH .on moH mmH .anH oaH .oaH .mzH.@:H .HmH.w:H NOH HNH NOH .mzH .omH amoaao :mmm :mmmm :mNmm nmmmm :mHmm mmHmm mmHmm mmm mmm Hmm, 1 venom n .u Hmm O ammmHmm :mmmmmm nmw H 1 m mmHmm, 1mm anew posh o Gouda.“ 203 mm H.349 “and b5 omm.mmm mzm m 7hr. wmm.owm smm OH mmm mmm .mmm.Hmm .omm.mzm .8zm.mzm .Ram.m:m mackhbm zmom ammmon :mmmHmm zmnmm :mmmm mammm :mHmm mmHmm mmHmm :mmmHmm :mmmmmm mmNmHmm :mmmNmHmm :mm H o e h hlu'l' o » Haaqusqoov mm Haney 0 a a e 7w H a q .4 mmm doeam 60 TABLE 26 Percentages of different factors in the seven breeds of sheep. 7Antigenic Shropshire. Hampshire Oxford_ Rambouillet §gpthdown Qorset Horn §pff01k Constitution 71 38 + + :8 + + j + f + ‘ Based on.isoimmune sera 81 .11 11.2 3. 0.7 1 2.9 1 3.0 2 0.4 2 33.3 32 2. 2.0 1 2.2 2 6.1 S 5. 5.1 1 2.2 3 8.8 2 0.4 l 8.3 375283 10 10.1 3 6.7 1 3.0 1 8.3 $182 10. 10.1 5 14.7 1 3.0 1 8.3 1 10.7 S38} 2 2.0 l 3.2 8283 3 3.0 1 2.2 l 2.9 2 33.3 - 56 50.0 30 80.0 4 70.0 28 84.8 20 78.8 9 75.0 1 10.7 Total 99 45 34 33 31 12 0 Based on heteroimmune sera R51 5 5.1 3 0.7 3 8.8 3 9.0 1 8.} R82 4 4.0 2 4.4 1 2.9 1 3.0 l 3.2 R53 1 1.0 1 2.9 2 0.4 RS” 1 2.9 351523354 46 40.7 14 31.1 0 17.0 18 54.5 11 35.5 10 83.3 1 10.7 RSISZS3 9 9.1 3 6.7 5 14.7 1 3.0 2 33.3 3525354 8 8.1 4 8.9 2 5.9 3 9.0 2 0.4 l 8.3 2 33.0 3513354 1 1.0 1 2.9 ‘3 9.1 1 3.2 R8182 4 4.0 5 11.1 5 14.7 2 6.4 RS1183 3 3.0 1 2.2 1 2.9 l 3.0 R513” 1. 1.0 l 3.0 1. 3.2 1 10.7 R3283 3 3.0 1 2.9 2 6.1 R828“ 1 1.0 2 4.4 R535” 1 1.0 3 8.8 l 3.0 l 3.2 - 12 12.1 8 17.8 2 5.9 7 22.0 Total 99 45 34 33 31 12 0 + = Number of sheep 97 TABLE 27 Percentages of different factors in the whole population of sheep tested. Antigenic Number Constitution Positive Percentageg Based on isoimmune sera SI 20 7.7 $2 5 1.9 $3 12 4.6 518283 15 5.8 8182 18 6.9 $183 3 1.2 8283 7 2.7 - 180 09.2 Total 260 Based on R51 15 5.8 heteroimmune R52 9 3.5 sera R83 4 1.0 R571 1 O. 4 R81$QS3SR 106 40.8 Rslszs; 20 7.7 RSQS3Sfi 22 8.4 3.3153311 6 2.3 R3182 16 6.2 R8183 0 2.} R515“ 4 1.6 R8233 6 2. 3 R8254 3 1.2 R535“, 6 2.3 35132571 7 2. 7 - 29 11.2 DISCUSSION Ever since the discovery of four blood groups in man at the be- ginning of this century, attempts had been made to investigate into the problem of blood groups in various species of animals. The literature revealed that the problem had been approached from three different angles: 1. Natural isoagglutination 2. Isoimmunization 3. Heteroimmunization Most of the work to date has been done with the first two methods. 1. Natural Isoagglutination Paniseft (1935) recognized blood groups in horses, cattle, sheep and pigs. the groups, however, were not as distinct as those in man. Blood groups in sheep, by natural isoagglutination technic, could not be detected in this study. In natural isoagglutination tests, clumps of red cells were ebtained only after centrifugation. These clumps transformed into sus- pension, indistinguishable from control saline suspension. when the tubes were slightly tapped or were incubated at 37 G. Lehnert (193M), Szymanowsgi_(l935) and.Eardt (1937) could not recog- nize blood groups in animals by natural isoagglutination technic. 2. Isoimmunization Ehrlich and Morgenroth (1900) discovered that for the formation of antibodies, the red corpuscles need not be from an animal of different species. They produced hemolysins for goat red blood cells by injecting a goat with b9 erythrocytes from another goat. They used the term ”isolysins" to designate these antibodies in order to distinguish them from 'heterolysins" formed after the injection of foreign cells. Egg§_(1930), Lehnert (193k) and Ferguson (19M1) employed the technic of isoimmunizetion and demonstrated blood groups in the horse, fowl and cattle respectively. In the present study three antigenic factors were distinguidhed by the technic of isoimmunization. Eight different combinations, from these three factors, were possible; of these eight possible constitutional com- binations, Shrapshire, showed 8; Hampshire, 6; Oxford, 5; Rambouillet, 5; Southdown, h; Dorset Horn, M and Suffolk, N. The percentages of these differ- ent antigenic combinations were given in table 26 and histograms 1 to 9, pages 12. to 7‘}. To illustrate the basic principle underlying the technic of reCOgnizing qualitatively different antibodies, the following example could be con- sidered. if an animal (free from x factor) is inoculated with cells containing X antigen, the animal would produce X antibodies. Similarly, cells with Y antigen, when inoculated into an animal without this factor, would cause the production of Y antibodies. Cells of XY antigenic structure, would produce XI antibodies in the serum. Absorption of the latter with I, Y and KY cells would leave Y, I and no antibodies respectively, in the serum. Reactions of these three absorbed sera with the three different cells have been illustrated in table 28. 70 TABLE 28 Method of analyzing antibodies. Unabsorbed __§erum absopbed by cells. Cells Sera X Y XY X + - + - Y + + - - KY 4 4 + - 3. Heteroimmunization Human A and AB red blood cells were agglutinated by anti—sheep blood immune rabbit serum. This was due to Forssman like antigen in sheep red cells. Guinea pigs which had Forssman antigen in their tissues, could not deve10p lysins for sheep cells, when immunized with A cells. (Wiener, 19us). In preliminary experiments conducted during this investigation, it was shaman that in rabbit sera natural hemolysins for sheep cells could be absorbed by heated sheep cells. This was in conformity with the thermo- stable nature of Forssman antigen present in sheep erythrocytes as shown Heteroimmune sera were produced in rabbits and from these sera Forssman like antibodies were removed by absorption with heated sheep cells. Analyses of these absorbed sera revealed four qualitatively different anti- bodies which would correspond to the corresponding number of antigenic fac- tors. Sixteen different combinations were possible out of these four fac- tors and percentages of animals corresponding to different combinations _-...- 71 were given in table 26 and histOgrams 10 to 26, pages 15.671 Iseki gt_§1 (IQHO) and Eero ££_gl'(l9u9) employed heteroimmune sera from rabbits for determining blood groups in dogs and cattle re- spectively. ‘Ability of sheep cells to stimulate the production of different antibodies in addition to those corresponding to Forssman antigen, pointed to the differences in antigenic constitution of sheep red blood cells. Muraschi (19u9) indicated the possibility of antigenic differences in sheep blood cells. In laboratories, where large amounts of blood are required for daily serological work, it would be advantageous to pool blood from different sheep after performing tests for compatibility. Evidence put forth in this study would indicate that heating of sheep red cells densensitissd the internal structure of antigenic factors other than the thermostable Forssman antigen, to the extent that absorption of heteroimmune sera with heated sheep red cells would have antibodies other than Porssman. Ferguson (l9h1) and 91332.(19M3) showed that antigenic factors in cattle and chicken were inherited according to Mendel'e law of heredity. Genetical analysis of the data from the sheep used in this study formed a separate problem in itself and such a treatment was not attempted in this thosiie Percentages 35 25 15 10 72 _ HI 11H 5‘. $1. ‘ 535%.? "'5 33.1"; 53:55.23 Einifisa 5 5 3 1+ 5.51:1}. mstopsS-s showing percentages of different factors, based on ieei-une sera, in the seven breeds of sheep tested. 73 $61. 10 31S}. listens showing percentages ef different factors, based ea teem ee ”a. m .eueaaeouem mhthemnbmdsefebep 60s”. 70 b5 \ n “N K...) \J‘ X Percentages N \n 20 15 10 Jen JV.“ 56:. $3, 9: 97.. $\S3. SL5; Histogram showing percentages of different factors, based on isoimmune sera, 1n the whole population of sheep tested. 71+ Percentages Percentages 35 25 15 10 20 15 10 f 75 II IO 'RSI iOtOéé; 0 0.0.00 aiaaggfi 12 nfl i153. R32. 8" 535 Egg 5335322 :3 fl 5151-}. Histograms showing percentages of different factors, based on heteroianune sera, in the seven breeds of sheep tested. 76 r” 75 1“ 35 15 r... 65 30 .55 25 3 "l 5'65 r—' 20 '3 “.35 —-n 15 F fir 25 10 15 _ 5 5 —‘I 0 . RS618“ 3561.93 . ‘ ' ' .w mama “-4.333 ginzgza $3315.: 35 16 if" 17 30 5” 820 E is 15 10 5 o , a , , , - _ M193 Sq. R953 Ss- Hietea-sas showing percentages of different factors. based on heterei-une sera, in the seven breeds of sheep tested. 77 I9 35 18 30 .25 .5: 320 2’. 9e 15 .1 1b *‘ 5 [—1 o , RSt51 he: 9 433‘s 5335332 35 20 y: 025 5;“ 320 De —— 15 10 5 RfiSq RSv?5 d°§s§é§ éinagga fin R9193. Histogrens showing percentages of different factors, based on heteroi-nne sera. in the seven breeds of sheep tested. .__.—_* ___4__—.__.___ ' .- 78 22 23 35 30 .25 3 e I:20 .1 15 10 5 0.11 g 7 R519. R8354 O ..0.' .235” mamas 8' 0P5 .8 g on eN aisaggz Snags; 35 24 25 30 _ Percentages 8 {2 H U 5 "171 —l o , RSI $15!.- .. Histograms showing percentages of different faster-s, based on heterei-me sera, in the seven breeds of sheep tested. 79 26 $.53 . .3 an . :maea mm 4.3 :m .3. mag .55. e eama.ma :mwm awa mm .3 .3. -iwaaa :3. .5. “Ava 9. .3 O 3 5 0 2 7. wowmoamoamm (a 10 .3. ested. 6. based on heteroimmune sera, in the whole population Histogram showing percentages of different factors, of sheen t 80 SUMMARY AND CONCLUSIONS In view of the increasing importance of blood factors in animals, investigation was made to determine whether or not blood groups existed in healthy sheep. Apparently healthy sheep of Michigan State College, Animal Husbandry Dept. were used for this investigation. 1. Typing of red blood cells by naturally occurring antibodies did not appear to be a reliable means of approach to this problem. 2. Isoimmune sera were produced in sheep and by absorption technique three qualitatively different antigenic factors were detected. 3. Hetereimmune sera were produced by immunizing rabbits with sheep cells. Forssman antibodies were removed by absorption with heated sheep cells (Iorssman antigen theemostable). Analyses of these absorbed sera con- taining no or a little of rorssman antibodies. indicated the presence of four different types of antibodies, pointing to the possibility of four corresponding antigenic factors. BIBLIQGRABEY Abderson, J. (Original not seen) cited b; Wiener. A. S. Blood Groups and Transfusion, ed. 3. Charles C. Thomas. Springfield, Illinois. 19nd. Anderson, J. Blood groups in man ané animals with special reference to its occurrence in the equine. Vet. Rec. 12: b9l-b38, 1932. Ab- stracted in Vet. Bull. 3: 715, 1333. - ‘ Bdmr,A.H. A useful preservative for whole blood. Brit. Jour. Expt. Path. 6: 201-202, 1325. Boyd, W. C. Fundamentals of Immunology. ed. 2. Interscience Publishers Inc. New York, 19u7. Briton, J. w. A method of handling hemolytic icterus of new born foals. Jour. Amer. Vet. Med. 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