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Thrombocyte Antigens of Chickens
1632
RESEARCH NOTES
the diet was supplemented with methionine. REFERENCES Dean, W. F., and H. M. Scott, 1966. Use of free amino acid concentrations in blood plasma of chicks to detect deficiencies and excesses of dietary amino acids. J. Nutr. 88: 75-83.
Dean, W. F., and H. M. Scott, 1968. Ability of arginine to reverse the growth depression induced by supplementing a crystalline amino acid diet with excess lysine. Poultry Sci. 47: 341-342. Douglas, C. R., H. J. Hochreich and R. H. Harms, 1958. Glycine in broiler nutrition. Poultry Sci. 37: 620-624.
THROMBOCYTE ANTIGENS OF CHICKENS
(Received for publication July 17, 1968)
This report describes the demonstration in chickens of thrombocyte specific antigens and further shows that such antigens may exhibit isoantigenic specificity. The cell preparations were derived from the buffy coat layer obtained by differential centrifugation of peripheral blood (Sengar et al., 1968). Eight ml. of EDTA-treated blood were centrifuged in 13 X 100 mm. tubes at 80 g for six to seven minutes. The buffy coat was then aspirated with as little as possible of the underlying red cell layer. The cells thus obtained were washed three times with cold saline (8 ml. each time) and finally suspended in saline. Such preparations consisted of approximately 60 percent thrombocytes and 40 percent leukocytes (less than five percent granulocytes). The red cell contamination did not exceed two percent. Because no suitable method for the separation of chicken thrombocytes was known, these mixed cell preparations were used for immunizations and agglutination tests. These are hereafter referred to as 'thrombocyte preparations.' Adult White Leghorn males were immunized. The immunization procedure consisted of an initial intradermal injection, followed by four intravenous injections at Partially supported by NRC grant No. A-1977.
five to seven day intervals. Cells harvested from 8 ml. of blood were injected each time. The sera were obtained seven days after the last injection. The agglutination tests were carried out in non-siliconized round-bottomed tubes (10 X 75 mm.) by placing one drop of a saline suspension of cells from the buffy coat (10,000 to 15,000 cells/mm.3) and one drop of heat-treated serum (56°C. for 30 minutes) in each tube. The suspensions were then incubated at 37°C. for one hour and the tubes gently agitated three times during the first twenty minutes. Appropriate negative and positive controls were included. The suspensions were examined under the phase contrast microscope for agglutination. When difficulty was encountered in recognizing the type of cell involved in agglutination, Wright-Giemsa stained smears were prepared and examined. The red cells used for the absorption studies were prepared by repeated differential centrifugation of EDTA-treated blood and the removal of buffy coat layer each time. The red cell preparations contained on an average no more than a single thrombocyte or leucocyte per 1000 cells. The thrombocyte preparations for absorptions were also prepared by differential centrifu-
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 10, 2015
D. P. S. SENGAR, R. J. DOUGLAS AND F. N. JEROME Departments of Poultry Science and Microbiology, University of Guelph, Guelph, Ontario, Canada
1633
RESEARCH NOTES TABLE 1.—Cross-absorption experiment with reagent — 3f obtained from serum no. 930 Agglutination of thrombocytes from donor no.:
Before absorption
After absorption with 945* (1) Lymphocytes (2) Red Blood Cells
918
919
920
935
937
939
940
945*
927
951
+
+
+
+
+
+
+
+
+
-
-
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
— —
— —
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
_ —
_ —
f Reagent —3 was obtained as a result of absorption of serum no. 930 with thrombocytes of chicken no. 927. * Donor of cells for the production of serum no. 930. + Positive reaction, ^ 50% of thrombocytes observed to be clumped when examined by phase contrast microscopy.
gation, however, a superficial layer of red cells was also aspirated along with the buffy coat in an effort to increase the yield of cells. The red cell contamination was subsequently reduced by centrifuging the thrombocyte-rich suspensions in sealed pasteur pipette tubes (Szenberg and Shortman, 1966; Senger et al., 1968). Lymphocytes for absorption were prepared using heparinized blood by a procedure (Terasaki, 1959) that yielded 99 percent pure lymphocyte preparations. RESULTS One of the immune sera gave a titer of 1:16,000 (four-fold dilutions) with buffy coat cells and a hemagglutinating titer of only 1:2. From this serum no. 930, three reagents (1,2,3) were prepared each thought to react with but a single thrombocyte antigen. These reagents had titers of 256, 16 and 256 respectively when tested
with buffy coat cells derived from the donor used for immunization. The results of cross absorptions studies (van Rood and van Leeuwen, 1963) with reagent —3 are given in Table 1. All three reagents gave highly reproducible reactions. When cells were washed with cold saline and excessive washing avoided, no difficulty was encountered with non-specific clumping of cells. In the preliminary studies agglutination-negative-absorption-positive cells (van Rood and van Leeuwen, 1963) were not encountered. The reaction patterns for the three reagents with a panel of White Leghorn thrombocytes are given in Table 2. When the 'thrombocyte preparations' were treated with the highly agglutinating reagent 1 or 3 the thrombocytes were preferentially clumped; the residual cell suspension consisted almost entirely of lymphocytes. These preliminary studies suggest
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 10, 2015
After absorption with thrombocytes from donor no.: 917 918 919 920 93S 937 939 940 945* 927 951
917
1634
RESEARCH NOTES
TABLE 2.—Reaction patterns of reagents 1, Z and 3 (obtained from serum no. 930*) with thrombocytes of a part of the panel of White Leghorns Thrombocytes Reagent-1 Reagent-2 Reagent-3 from
+ — —
+ + — + — — — —
+ + + — + + + + — — — — — —
+ + — + — + + + + — + — + + + + + + + — —
—
+ + +
•
+ — + + — + — + + + + + + — — —
+ — — —
+ + + +
* Immune serum no. 930 was obtained from chicken no. 930 immunized with the 'thrombocyte preparations' of chicken no. 945. f Reagents 1, 2, and 3 were originally obtained as a result of absorption of serum no. 930 with thrombocytes of chicken no. 940,919 and 927 respectively.
the presence of thrombocyte specific antigens in chickens. However, since the preparations used for agglutination test did not contain granulocytes to any significant extent, the possibility that some of these antigens may also be shared by granulocytes is
SUMMARY
Three chicken thrombocyte antigens which are not shared by red blood cells or lymphocytes have been demonstrated by means of a microscopic agglutination test. REFERENCES Rood, J. J. van, and A. van Leeuwen, 1963. Leukocyte grouping. A method and its application. J. Clin. Invest. 42: 1382-1390. Sengar, D. P. S., F. N. Jerome and R. J. Douglas, 1968. A simple method for separation of buffy coat from peripheral blood of chickens. Can. J. Comp. Med. In press. Szenberg, A., and K. Shortman, 1966. Fractionation of fowl blood lymphocytes by their buoyant density: Localization of cells active in graft-versus-host reactions. Ann. New York Acad. Sci. 129:310-326. Terasaki, P. I., 1959. Identification of the type of blood cell responsible for the graft-versus-host reaction in chickens. J. Embryol. Exp. Morph. 7: 394-408.
RESPONSE OF CHICKS TO ALCOHOL TREATED FEMALES HAKKI S. TAMIMIE Thudicum Psychiatric Research Laboratory, State Research Hospital, Galesburg, Illinois 61401 (Received for publication August 12, 1968)
Tamimie (1968) observed that one-year old White Rock cocks orally treated with 5 or 10 ml./kg. body weight of 33 percent grain alcohol showed more maternal behav-
ior to day-old and one-week old White Rock chicks than water treated cocks. This observation confirmed the findings of Kovach (1967).
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 10, 2015
94S 930 940 919 927 917 951 937 938 918 920 939 922 952 924 925 929 944 923 931 950 780 783 941 926
not completely ruled out. The distribution of thrombocyte antigens may vary from one breed to another; an examination of eighty Columbian Rock chickens indicated all to be reagent — 3 negative and reagent — 2 positive. Studies are in progress to investigate the distribution of thrombocyte antigens, their genetic behaviour and their relationship to skin grafting. Possible relationships between thrombocyte antigens and some desirable economic characteristics in chickens are also being studied.
RESEARCH NOTES
the diet was supplemented with methionine. REFERENCES Dean, W. F., and H. M. Scott, 1966. Use of free amino acid concentrations in blood plasma of chicks to detect deficiencies and excesses of dietary amino acids. J. Nutr. 88: 75-83.
Dean, W. F., and H. M. Scott, 1968. Ability of arginine to reverse the growth depression induced by supplementing a crystalline amino acid diet with excess lysine. Poultry Sci. 47: 341-342. Douglas, C. R., H. J. Hochreich and R. H. Harms, 1958. Glycine in broiler nutrition. Poultry Sci. 37: 620-624.
THROMBOCYTE ANTIGENS OF CHICKENS
(Received for publication July 17, 1968)
This report describes the demonstration in chickens of thrombocyte specific antigens and further shows that such antigens may exhibit isoantigenic specificity. The cell preparations were derived from the buffy coat layer obtained by differential centrifugation of peripheral blood (Sengar et al., 1968). Eight ml. of EDTA-treated blood were centrifuged in 13 X 100 mm. tubes at 80 g for six to seven minutes. The buffy coat was then aspirated with as little as possible of the underlying red cell layer. The cells thus obtained were washed three times with cold saline (8 ml. each time) and finally suspended in saline. Such preparations consisted of approximately 60 percent thrombocytes and 40 percent leukocytes (less than five percent granulocytes). The red cell contamination did not exceed two percent. Because no suitable method for the separation of chicken thrombocytes was known, these mixed cell preparations were used for immunizations and agglutination tests. These are hereafter referred to as 'thrombocyte preparations.' Adult White Leghorn males were immunized. The immunization procedure consisted of an initial intradermal injection, followed by four intravenous injections at Partially supported by NRC grant No. A-1977.
five to seven day intervals. Cells harvested from 8 ml. of blood were injected each time. The sera were obtained seven days after the last injection. The agglutination tests were carried out in non-siliconized round-bottomed tubes (10 X 75 mm.) by placing one drop of a saline suspension of cells from the buffy coat (10,000 to 15,000 cells/mm.3) and one drop of heat-treated serum (56°C. for 30 minutes) in each tube. The suspensions were then incubated at 37°C. for one hour and the tubes gently agitated three times during the first twenty minutes. Appropriate negative and positive controls were included. The suspensions were examined under the phase contrast microscope for agglutination. When difficulty was encountered in recognizing the type of cell involved in agglutination, Wright-Giemsa stained smears were prepared and examined. The red cells used for the absorption studies were prepared by repeated differential centrifugation of EDTA-treated blood and the removal of buffy coat layer each time. The red cell preparations contained on an average no more than a single thrombocyte or leucocyte per 1000 cells. The thrombocyte preparations for absorptions were also prepared by differential centrifu-
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 10, 2015
D. P. S. SENGAR, R. J. DOUGLAS AND F. N. JEROME Departments of Poultry Science and Microbiology, University of Guelph, Guelph, Ontario, Canada
1633
RESEARCH NOTES TABLE 1.—Cross-absorption experiment with reagent — 3f obtained from serum no. 930 Agglutination of thrombocytes from donor no.:
Before absorption
After absorption with 945* (1) Lymphocytes (2) Red Blood Cells
918
919
920
935
937
939
940
945*
927
951
+
+
+
+
+
+
+
+
+
-
-
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
— —
— —
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
_ —
_ —
f Reagent —3 was obtained as a result of absorption of serum no. 930 with thrombocytes of chicken no. 927. * Donor of cells for the production of serum no. 930. + Positive reaction, ^ 50% of thrombocytes observed to be clumped when examined by phase contrast microscopy.
gation, however, a superficial layer of red cells was also aspirated along with the buffy coat in an effort to increase the yield of cells. The red cell contamination was subsequently reduced by centrifuging the thrombocyte-rich suspensions in sealed pasteur pipette tubes (Szenberg and Shortman, 1966; Senger et al., 1968). Lymphocytes for absorption were prepared using heparinized blood by a procedure (Terasaki, 1959) that yielded 99 percent pure lymphocyte preparations. RESULTS One of the immune sera gave a titer of 1:16,000 (four-fold dilutions) with buffy coat cells and a hemagglutinating titer of only 1:2. From this serum no. 930, three reagents (1,2,3) were prepared each thought to react with but a single thrombocyte antigen. These reagents had titers of 256, 16 and 256 respectively when tested
with buffy coat cells derived from the donor used for immunization. The results of cross absorptions studies (van Rood and van Leeuwen, 1963) with reagent —3 are given in Table 1. All three reagents gave highly reproducible reactions. When cells were washed with cold saline and excessive washing avoided, no difficulty was encountered with non-specific clumping of cells. In the preliminary studies agglutination-negative-absorption-positive cells (van Rood and van Leeuwen, 1963) were not encountered. The reaction patterns for the three reagents with a panel of White Leghorn thrombocytes are given in Table 2. When the 'thrombocyte preparations' were treated with the highly agglutinating reagent 1 or 3 the thrombocytes were preferentially clumped; the residual cell suspension consisted almost entirely of lymphocytes. These preliminary studies suggest
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 10, 2015
After absorption with thrombocytes from donor no.: 917 918 919 920 93S 937 939 940 945* 927 951
917
1634
RESEARCH NOTES
TABLE 2.—Reaction patterns of reagents 1, Z and 3 (obtained from serum no. 930*) with thrombocytes of a part of the panel of White Leghorns Thrombocytes Reagent-1 Reagent-2 Reagent-3 from
+ — —
+ + — + — — — —
+ + + — + + + + — — — — — —
+ + — + — + + + + — + — + + + + + + + — —
—
+ + +
•
+ — + + — + — + + + + + + — — —
+ — — —
+ + + +
* Immune serum no. 930 was obtained from chicken no. 930 immunized with the 'thrombocyte preparations' of chicken no. 945. f Reagents 1, 2, and 3 were originally obtained as a result of absorption of serum no. 930 with thrombocytes of chicken no. 940,919 and 927 respectively.
the presence of thrombocyte specific antigens in chickens. However, since the preparations used for agglutination test did not contain granulocytes to any significant extent, the possibility that some of these antigens may also be shared by granulocytes is
SUMMARY
Three chicken thrombocyte antigens which are not shared by red blood cells or lymphocytes have been demonstrated by means of a microscopic agglutination test. REFERENCES Rood, J. J. van, and A. van Leeuwen, 1963. Leukocyte grouping. A method and its application. J. Clin. Invest. 42: 1382-1390. Sengar, D. P. S., F. N. Jerome and R. J. Douglas, 1968. A simple method for separation of buffy coat from peripheral blood of chickens. Can. J. Comp. Med. In press. Szenberg, A., and K. Shortman, 1966. Fractionation of fowl blood lymphocytes by their buoyant density: Localization of cells active in graft-versus-host reactions. Ann. New York Acad. Sci. 129:310-326. Terasaki, P. I., 1959. Identification of the type of blood cell responsible for the graft-versus-host reaction in chickens. J. Embryol. Exp. Morph. 7: 394-408.
RESPONSE OF CHICKS TO ALCOHOL TREATED FEMALES HAKKI S. TAMIMIE Thudicum Psychiatric Research Laboratory, State Research Hospital, Galesburg, Illinois 61401 (Received for publication August 12, 1968)
Tamimie (1968) observed that one-year old White Rock cocks orally treated with 5 or 10 ml./kg. body weight of 33 percent grain alcohol showed more maternal behav-
ior to day-old and one-week old White Rock chicks than water treated cocks. This observation confirmed the findings of Kovach (1967).
Downloaded from http://ps.oxfordjournals.org/ at University of Bath, Library on June 10, 2015
94S 930 940 919 927 917 951 937 938 918 920 939 922 952 924 925 929 944 923 931 950 780 783 941 926
not completely ruled out. The distribution of thrombocyte antigens may vary from one breed to another; an examination of eighty Columbian Rock chickens indicated all to be reagent — 3 negative and reagent — 2 positive. Studies are in progress to investigate the distribution of thrombocyte antigens, their genetic behaviour and their relationship to skin grafting. Possible relationships between thrombocyte antigens and some desirable economic characteristics in chickens are also being studied.