The Blood Group Systems in the Chickens' Received March 29, 1961 Kyuki MATSUMOTO and Ikuo OKADA Department of Animal Science, Faculty of Agriculture, Hokkaido University, Sapporo In 1924, Landsteiner and Miller distinguished eight different blood types in ten chickens by means of heteroagglutinating sera prepared by injecting chicken blood into rabbits. Since then, the existence of numerous individual serological differences -in chicken blood has been reported by Todd (1930), Kozelka (1933), Thomsen (1934, 1936), Boyd and Alley (1940) and Hayashida (1942), though their inheritance mode had not been clarified. In accounting for the probable genetic relationships of the genes responsible for such antigenic differences, Wiener (1934) hypothesized the existence -of multiple alleles at some loci. Briles, McGibbon and Irwin (1950) found twelve cellular antigens in the chicken using reagents prepared by differential absorption of the isoimmune sera. They presented evidence that these antigens were determined by multiple allelic genes belonging to two autosomal loci, of which one consisted of nine alleles, the other of five -alleles. After that, Briles, Briles and Quisenberry (1950) and Briles (1951, 1958) reported the existence of three additional loci, C, D and E. The serological and genetic relationship of these loci were described by Briles, McGibbon and Irwin (1959). Scheinberg (1956) found two linkage groups of genes for cellular antigens in the -chicken, and showed that one of them might belong to the A system of Briles, McGibbon and Irwin (1950). Recently, two additional blood group systems, L and N, were reported by Gilmour (1959). The present paper deals with the serological and genetic identification of ten cellular antigenic factors of the fourteen reported previously by the authors (1958 a, b), in the chicken. The evidence that these antigenic factors were determined by.genes at three loci will be presented. Materials and Methods All of the chickens used for these experiments were Single Comb White Leghorns, kept at the Experimental Farm of Hokkaido University. This flock has been closed since it was introduced from the Takikawa Livestock Breeding Station to this farm in 1957. Antisera used for this experiment were prepared by isoimmunization between 1) This investigation was supported in part by a Grant in Aid from the Scientific Research Fund of the Ministry of Education (67004 in 1960).
258 K. MATSUMOTO AND I. OKADA chickens. The citrated whole blood withdrawn from the wing vein of a donor was injected into the wing vein of a recipient. About 4 ml of whole blood was injected at intervals of four or five days until trial bleeding gave serum of satisfactory antibody titer. Usually five or six injections were sufficient and antibody titers were 1:160 to 1:320. The antisera were collected six or seven days after the last injection, The sera were inactivated by heating for 30 minutes at 56 C and stored in a refrigerator (0-5 C) after 1% Merthonine2' solution had been added in such quantity as to give a final concentration of 0.05%. The agglutination tests were performed by adding one drop of an approximately five percent suspension of washed red cells in 0.85 percent saline to 0.1 ml of reagent or antiserum in test tubes 10 x 105 mm. The mixture of cells and reagent was shaken and incubated at 37 C in a water bath, and shaken again after one hour. The reaction was read at two hours, and after the tubes remained overnight at room temperature was checked again. To prepare reagents specific for each antigenic factor, absorptions were performed. An antiserum was diluted with saline according to its titers-usually 1:3 to 1:5--and mixed with a suitable quantity of washed blood cells. The mixture was allowed to incubate at 37 C for two to three hours with frequent agitation and was stored overnight in the refrigerator, after which it was centrifuged. The absorptions were repeated until the supernatant produced a negative reaction with the absorbing cells. The reagents were kept in a deep freezing box (about -10 to -20 C) if they were not used immediately. Terminology The symbols, A, B, C, F, G, H, I, K, L and M, used to designate the antigenic characters, were used in order of their detection. Thus, they have no relationship to the A, B, C, D, E, L and N systems of Briles et al. (1950, 1951, 1958) and Gilmour (1959). The relationship of the three systems shown in the present paper to the seven systems of Briles et al. and Gilmour is entirely obscure. Therefore these are tentatively designated F, G and H systems. The terms, " antigenic factor ", " antigen " and " blood type " used in the present paper have essentially the same meaning as they did in the reports of Briles, McGibbon and Irwin (1950) and of Stormont, Owen and Irwin (1951). " Antigenic factor" is the specific substance on the red blood cells, which reacts with the respective reagent. For example, blood cells reacting with A reagent are said to carry the antigenic factor A. The term " antigen " refers to one or more antigenic factors produced by a single gene. " Blood type " consists of all the known antigenic factors of the red blood cells of a bird. In particular cases, this term means frequently only the type of special antigenic factors under discussion. 2) Sodium Ethylmercurithiosalicylate.
THE BLOOD GROUP SYSTEMS IN THE CHICKEN 259 Results Preparation of reagents The antisera prepared by isoimmunization reacted usually with the cells of various individuals which had the different antigenic substances. Then, absorption tests of each antiserum were performed. The birds used for absorptions were chosen either at random or because of their content of antigenic substances. To illustrate the general method for the preparation of reagents typical example of the analyses of antisera will be given. The antisera to be used as examples do not necessarily correspond to the order of the detection of the various antigenic factors. The serological analysis of antiserum 128 (G622), which was produced from the injection of the blood of bird G622 (the donor) into 128 (the recipient), is presented in Table 1. G622 was chosen as donor because of having none of the first five antigenic factors (A, B, C, F and G), while 128 had the A antigen. The antibody titer of this serum was 1:160. Twenty birds were chosen at random for the absorption test. The unabsorbed serum reacted weakly or strongly with the cells of 16 birds among 20 birds and gave a negative reaction with four birds. When this serum was absorbed by the cells of bird 14 which reacted weakly, agglutinins not only for the absorbing cells but also for the weakly reactive cells were removed. The results of following absorptions by the mixtures of 14 cells and powerfully reactive cells showed that the serum absorbed by 14 cells contained specific antibodies reactive with two antigenic factors. That is, the absorption with the pooled cells of 14 and a bird of the first group (35, G667 and G826) provided a reagent which was reactive with the cells of the donor G622 and of the second group birds 61, 166 and G837, while the absorption with the pooled cells of 14 and a bird of the second group left an agglutinin reactive with the cells of the donor and the first group. Then it was postulated that the former reagent had an agglutinin specific for the antigenic factor designated K and the latter had anti-l. Finally, the absorption by the pooled cells 14 and G622 (or G667 and G837) removed, as expected, all antibodies from the antiserum. Thus, the reagents prepared by absorbing antiserum 128 (G622) with the pooled cells of 14 and 35 and 14 and 61 were used as the K and L reagents, respectively. After these antigenic factors were identified, birds whose cells possessed the antigenic factors were chosen as donors and birds lacking them as recipients, in order to obtain duplicate reagents. For example, to obtain a second source of G reagent the bird 88 (blood type AG) as donor and 142 (AL) as recipient were chosen for immunization. These birds were half sisters. The antibody titer of the immune serum was 1:320. As shown in Table 2, the antiserum reacted powerfully with cells containing the antigenic factor G, while with cells lacking the G factor it reacted weakly. When the pooled cells of 901 and 124 were used in absorbing the antiserum, a reagent was produced which reacted only with the cells of birds possessing the antigenic factor G.
260 K. MATSUMOTO AND I. OKADA
THE BLOOD GROUP SYSTEMS IN THE CHICKEN 261
262 K. MATSUMOTO AND I. OKADA All of the reagents were prepared by the same procedure. The details of absorptions carried out by the authors will be furnished on request. Genetic analyses Many blood group workers have reported that an antigenic character is inherited as dominant over its absence. If an antigenic character is determined by a completely dominant gene, matings between positive individuals or between a positive individual and a negative individual will result in either positive or both positive and negative offspring, while matings between negative individuals will yield only negative offspring. The results of matings are summarized in Table 3. The birds which were believed to be heterozygous for the gene determining the antigenic character were chosen for positive ones. In consequence, the expectation from matings between positive and negative is a 1:1 ratio of positive to negative progeny. In the matings between positive types the ratio expected is 3:1. The differences between the observed and expected numbers were small and not significant as shown by the x2-values. In the matings between negative types, all of the offspring have to be negative. The data from many matings involving each antigenic character presented evidence supporting the hypothesis. That is, all offspring from matings between individuals negative for each of the antigenic factors A, B, G, H, I, K and M were negative. In C-negative matings, however, one of 156 offspring was C-positive; in F-negative matings, one of 146 was positive; and two of 142 offspring in L-negative matings were positive. It seems likely that these exceptions were due to errors in technique or pedigree. It was also of interest to determine whether the genes determining the antigenic factors were independent or whether any of them were linked or allelic to one another. For this purpose matings of birds positive for two antigenic factors and birds negative for both were performed. Positive ones believed to be heterozygous for both antigenic factors were chosen. The expectation for the progenies, therefore, was 1:1:1:1 with a hypothesis of independence. The data observed are shown in Tables 4 and 5. As shown in Table 4, it appeared that eight antigenic factors, A, B, C, G, I, K, L and M, were determined by genes at the same locus or by closely linked genes in the repulsion phase, except for B and M in the coupling phase. The antigen B was found always to be coupled with the antigen M, while the latter could be found without accompanying the antigen B. However, in checking on individual cases considered to be results of recombination if there were linkage, the cases with the superscript "t"-that is, four cases in the AC, BC and CK rows-were considered to be results of errors in technique, although it was not possible to make further tests. Of the three cases with superscript "*", the two cases in the Al and LM rows came from bird B313 (blood type LM). Its sire was G667 (LM) and its dam A370 (AI). By progeny test they were believed to be heterozygous for the antigenic factors. Therefore, if the progeny resulted from crossing over, the crossing over must have occurred not only in the gamete from the sire but also in the gamete from the dam. The probability of this must be very low. It seems plausible that this case
THE BLOOD GROUP SYSTEMS IN THE CHICKEN 263
264 K. MATSUMOTO AND I. OKADA resulted from errors in pedigree, though the possibility of crossing over can not be ignored. The bird shown as the last case in the GK row had an antigenic factor which was not found in either parents; therefore this case was also considered as. being due to errors in pedigree. Thus, in the present data there was no good evidence for crossing over. The. authors, therefore, adopted as a working hypothesis the idea that these antigenic: factors are determined by multiple alleles at the same locus. According to this hypo thesis, the antigenic complex BM is an antigen which is determined not by two genes,, but by a single gene. If the hypothesis is true, all birds must have two or less. Table 4. Distribution of progenies in testcrosses of hypothesis 1:1:1:1 on the antigenic factors A, B, C, G, I, K, L and M
THE BLOOD GROUP SYSTEMS IN THE CHICKEN 265 Table 5. Distribution of progenies in testcrosses of hypothesis 1:1:1:1 on the antigenic factors F and H antigenic factors (or three including BM complex) with reference to this locus. Unpublished data obtained from about 800 chickens of the Hokkaido University the Takikawa Livestock Breeding Station strongly supported the hypothesis. Farm andd The relationships among F, H and the other eight antigenic factors are shown in Table 5. In these cases the antigenic factors segregated with a ratio 1:1:1:1 as expected. Thus it was considered that there were three loci on three different chromosomes determining antigenic factors. These loci were designated F, G and H, which consisted of two, nine and two alleles, respectively,-that is, FF and Ff; GA, GB", GO, GG, Gr, GK' Gz, GM and G9; HH and H". At the G locus, an allele such as. GB was never found in this experiment. However, the relationship between F and H requires further study because in these matings only one dam 139 (blood type AFHL) was available. Discussion It has been reported by many workers (Ferguson 1941; Matsumoto 1949; Matsumoto and Watanabe 1954; Andresen 1957) that the antigenic characters in erythrocytes are inherited as dominants. The present data agree with that conclusion, although several exceptional cases were found. They were considered to be due to, errors in technique or pedigree. However, the possibility of different explanations might be presented. One of them is that some of the antigenic characters may be
266 K. MATSUMOTO AND I. OKADA inherited as recessives. Thomsen (1934) and Boyd and Alley (1940) found several exceptional cases, in which the offspring had antigenic characters not found in their parents. They pointed out the possibility of recessiveness in an antigenic character. An obvious example of a recessive blood group was found by Stormont (1951) in sheep. In the data presented here, however, the explanation of recessive inheritance cannot explain the results. Another possible explanation is that an antigenic factor may be determined by the interaction of two or more genes. Examples of antigenic characters resulting from interactions of genes at the same or different loci were reported by Cohen (1956, 1960) in the rabbit, and by Rendel, Neimann-SqSrensen and Irwin (1954) in sheep. However, the anomalous cases in the present data cannot be explained by the interaction of genes. The various antigens of the G series were believed to reflect a multiple allelic system. However, the results may be accounted for by assuming that the antigenic factors are determined by genes on very closely linked loci. For the data presented here, there may be two explanations for linkage hypotheses. One of them is that there are eight multiple loci in repulsion phase, except B and M. The other is that there are two loci, of which one consists of genes determining seven antigenic factors A, B (or M), C, G, I, K and l., and the other only one antigenic factor M (or B). These hypotheses presuppose that crossing over occurs between the genes responsible for these antigenic factors. Therefore, there must be an approach to the randomness of combination expected among these antigenic factors if there were multiple loci, even at rates of crossing over comparable in rarity to those of mutation. However, there was no such approach in the data. Similar results were reported by Briles, McGibbon and Irwin (1950), Briles, Allen and Millen (1957) and Gilmour (1959) in chicken; and by Stomront, Owen and Irwin (1951) in cattle. They hypothesized that the antigenic factors were controlled by multiple alleles. Stormont (1955) supported the hypothesis of multiple alleles on the basis that there was no randomness of combination among antigenic factors, as expected from the hypothesis of closely linked multiple loci, and no correspondence between the incompatibilities among the antigenic factors and the proposed sets of linked genes, with reference to the B system in cattle and the Rh system in man. Thus, without the obvious evidence for crossing over it seems reasonable to accept the hypothesis of multiple alleles, although it should not be forgotten that there is still discussion between English and American investigators about the human Rh blood group (cf. Cushing and Campbell 1957). Summary Ten different antigenic factors were found by isoimmunization in the erythrocytes of the chicken. Genetic analyses showed that these antigenic factors were controlled by genes belonging to three non-linked loci, which were designated F, G and H. One of these, namely G, consisted of nine alleles GA, GEM, Gc, GG, GI, GK, GL, G" and G9, and the other two loci consisted of two alleles each-ff and Ff, and H$ and Ha, respectively.
THE BLOOD GROUP SYSTEMS IN THE CHICKEN 267 Literature cited Andresen, E. 1957 Investigations on blood group of the pig. Nord. Vet. Med. 9: 274-284. $oyd, W. C. and O. E. Alley 1940 Individual blood differences in chickens. J. Hered. 31: 135-136. Briles, C. 0., W. H. McGibbon and M. R. Irwin 1959 Additional alleles affecting red blood cell antigens in the chicken. Genetics 44: 955-965. Briles, W. E. 1951 A new blood group in chickens (Abst.). Poult. Sci. 30: 907-908. - 1958 A new blood group system, E., closed linked with the A system in chickens (Abst.). Poult. Sci. 37: 1189. C. P. Allen and T. W. Millen 1957 The B blood group system of chickens, I. Heterozygosity in closed populations. Genetics 42: 631-648. C. O. Briles and J. H. Quisenberry 1950 Three loci affecting the blood group antigens of the chicken (Abst.). Poult. Sci. 29: 750. W. H. McGibbon and M. R. Irwin 1950 On multiple alleles effecting cellular antigens in the chicken. Genetics 35: 633-652. Cohen C. 1956 Occurrence of three red blood cell antigens in rabbit as the result of interaction of two genes. Science 123: 935-940. 1960 A second example of a rabbit red blood cell antigen resulting from the interaction of two genes. J. Immunol. 84: 501-506. Gushing, J. E and D. H. Campbell 1957 Principles of immunology, pp. 344, McGraw-Hill, New York. Ferguson, L. C. 1941 Heritable antigens in the erythrocytes of cattle. J. Immunol. 40: 213-242. ^Gilmour, D. G. 1959 Segregation of genes determining red cell antigens at high levels of inbreeding in chickens. Genetics 44: 14-33. Hayashida, S. 1942 Studies on blood groups in the chicken (Japanese). Jap. J. Criminol. 16: 266-277. -K ozelka, A. W. 1933 Individuality of the red blood cells of inbred strains of fowls. J. Immunol. 24: 519-530 Zandsteiner, K. and C. P. Miller 1924 On individual differences in the blood of chickens and ducks. Pro. Soc. Exp. Biol. & Med. 22: 100-102. Matsumoto, K. 1949 Studies on the erythrocyte-type of horses (Japanese). Jap, J. Zootech, Sci. 19: 50-54. and I. Okada 1958a Blood groups in the chicken, I (Abst. Japanese). Jap. J. Zootech. Sci. 29: (Suppl.) 20. _ and _-_ 1958b Blood groups in the chicken, II (Abst. Japanese). Rept. Hokkaido Soc. Anim. Hush. 1, 4. and Y. Watanabe 1954 Inheritance mode of cellular antigens in rabbits (Japanese). Memoi. Fac. Agr. Hokkaido Univ. 2: 162-168. Rendel, J., A. Neimann-S~rensen and M. R. Irwin 1954 Evidence for epistatic action of genes for antigenic substances in sheep. Genetics 39: 398-408. 'cheinberg, S. L. 1956 Genetic studies of cellular antigens in the chicken. Genetics 41: 834-844. Stormont, C. 1951 An example of a recessive blood group in sheep (Abst.). Genetics 36: 577-578. 1955 Linked genes, pseudoalleles and blood groups. Amer. Nat. 89: 105-116. R. D. Owen and M. R. Irwin 1951 The B and C systems of bovine blood groups. Genetics 36: 134-161. 'Thomsen, O. 1934 Untersuchungen uber erbliche Blutgruppenantigene bei Huhnern. Hereditas 19: 243-258. 1936 Untersuchungen uber erbliche Blutgruppenantigene bei Huhnern. II. Ibid., 22: 129-144. Todd, C. 1930 Cellular individuality in the higher animals, with special reference to the individuality of the red blood corpuscle. Proc. Roy. Soc. Lond. 106: 20-44. Wiener, A. S. 1934 Individuality of the blood in higher animals, II. Agglutinogens in red blood cells of fowls. Jour. Genet. 29: 1-8.