ABSTRACT. Determination of the haemolytic comple- ment titres of serum from 24 foetal lambs bled at 77 to 149 days gestation failed to re- veal this activity ...
Haemolytic Complement Activity Of Sera Of Foetal And New-Born Lambs by Christine E. Rice' and A. M. Silverstein2
ABSTRACT Determination of the haemolytic complement titres of serum from 24 foetal lambs bled at 77 to 149 days gestation failed to reveal this activity earlier than 123 days. Titres near the end of the gestation period of 159 days were still very low. Among serum specimens collected from 17 newborn lambs before suckling, three were still without demonstrable complement activity. During the first week titres rose considerably in all of these lambs, were higher at three weeks but at ten weeks were still below the adult level. A number of foeti had been injected with antigen mixtures at 50 to 111 days' gestation and had responded with antibody development to one or more of these antigens. The rate of development of complement in these treated foetal lambs appeared similar to that in their untreated twin controls.
Introduction Although haemolytic complement activity has been demonstrated in the serum of the newborn of most animal species tested, the titre is relatively low in comparison with that of adults of the same species (1-6). As Nattan-Larriere et al (7) have pointed out, however, sera of certain newborn infants may contain anticomplementary substances the presence of which will result in abnormally low titre values. In the case of calves, lambs and piglets which absorb antibodies from the colostrum during the first day or two of suckling, there is evidence suggesting that their complement activity is also augmented. Foetal sera may likewise exhibit measurable com'Animal Pathology Laboratories, Health of Animals Branch, Canada Department of Agriculture, Animal Diseases Research Institute, Hull, Quebec. 2lmmunobiology Branch, Armed Forces Institute of Pathology, Washington, D.C., U.S.A.
plement activity (6-8) but little has apparently been done to determine at what stage of gestation this may develop. It seemed worthwhile therefore to obtain some information on this point with a group of sera collected at various periods during gestation from foetal lambs under immunization in connection with another experiment (9-10). Sera from serial bleedings from a number of newborn lambs that had been similarly treated prior to birth, were likewise titrated for complement activity.
Methods PRIOR TREATMENT OF FOETAL LAMBS At 50 to 90 days gestation, laparotomies were performed on 16 ewes and 14 of the 24 foeti so obtained were injected intramuscularly in the hind leg with a mixture of three antigens, ovalbumin, ferritin and bacteriophage (9), mixed with saline or Freund adjuvant as indicated in Table I. In the case of twins, one foetus usually served as the untreated control. Two foetal lambs were injected with trydimite, a powdered glass mixture which it was thought might act as a lymphoid stimulant. Two foeti received skin homografts. At intervals of 5 to 62 days thereafter, that is at 77 and 149 days gestation, the foeti were delivered by hysterotomy and bled from the cord. Sera were stored in the frozen state until complement titrations could be made. TREATMENT OF NEWBORN LAMBS Included in this group of 17 lambs were six which had been injected in utero with antigens in adjuvant, five with saline and adjuvant, one had received a homograft, and five had not been injected. All were
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bled on the day of birth before suckling and then injected with the multiple antigen mixture. They were bled at intervals thereafter for a period of up to 71 days. TITRATIONS OF COMPLEMENT ACTIVITY In this laboratory a suspension of rabbit red cells sensitized with antibody produced in a sheep is ordinarily used for the titration of haemolytic complement activity in sheep or cattle serum. Sheep red cells sensitized with antibody prepared in the rabbit have not proved satisfactory for this titration (11). A five-per-cent suspension of washed rabbit red cells was made up and mixed with an equal volume of suitably diluted sheep antiserum for rabbit red cells. To serial amounts of undiluted foetal lamb serum, 0.3 to 0.1 ml., sufficient buffered saline containing magnesium and calcium, was added to bring the volume to 0.3 ml. The newborn lamb sera, undiluted, 1:2 or 1:5, were titrated in 0.1 to 0.01 ml. quantities. After the addition of 0.2 ml. of the sensitized rabbit-red-cell suspension, the mixtures were shaken and incubated at TABLE I.
37°C. for 60 min. The percentage haemolysis was read and the amount required for 50 per cent haemolysis, the H5o unit, ascertained graphically. From this value the complement titre in H5o units per ml. was estimated. When 0.3 ml. of undiluted serum gave less than 50 per cent haemolysis, the result of the titration was recorded as "negative"
Results The haemolytic complement titres of sera of the 24 foetal lambs are given in Table I. It will be noted that none of these animals exhibited significant complement activity in their serum earlier than 123 days gestation. All of those bled at 77 to 122 days gestation had titres of less than three units per ml. which as stated above is classified as "negative". Two of the three lambs bled at 123 days gestation had a low complement titre, the third was negative. All six foeti bled at 142 to 149 days displayed complement activity, titres 4.4 to 7.7 units per ml. However, it did not appear that the particular treatment which
Complement titres of serum from foetal lambs that had been treated in different ways
Number of lamb
305-1 .................... 7013-1 .................... 348-1* .................... 348-2* .................... 7031-1* .................... 7031-2* .................... 7039-1* .................... 7039-2* .................... 7001-2 .................... 7023-1 .................... 326-1 .................... 326-2* .................... 7035-1 .................... 335-1 .................... 340-1 .................... 310-1* .................... 310-2* .................... 346-1 .................... 7007-1* .................... 7007-2* .................... 332-2* ....................
332-1*.................... 303-1* .................... 303-2*....................
Antigens & saline Antigens & adjuvant Antigens & adjuvant Antigens & saline Antigens & saline Antigens & adjuvant Antigens & adjuvant Not injected Not injected Antigens & adjuvant Antigens & saline Not injected Antigens & adjuvant Antigens & adjuvant Antigens & adjuvant Trydimite Trydimite Antigens & adjuvant Not injected Antigens & adjuvant Not injected Homografted Not injected Homografted
Days' gestation At time of At time of bleeding operation 48 70 50 50 72 72 91 71 50
89 92 93 111 111 94 92 122
77 80 80 80 83 83 97 97 97 100 112 112 118 122 123 123 123 124 142 142 143 143 149 149
Complement titres units/ml.
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. 5.3 4.5 4.4 6.2 7.7 6.7 5.7 5.9 4.4
the foetus had received, had had any effect on the rate at which serum complement developed, since injected and control twins at the same stage of development showed relatively comparable titres. Three of the 17 lambs bled on the day of birth had titres less than 3 units per ml. but were not anticomplementary. In the other 14 lamb sera, the complement titres ranged from 3.7 to 10 units per ml. In all of these animals there was a progressive increase in the complement activity of their serum during the first few weeks of life. (Table II), although in some individuals the rise was more rapid than in others. All of the lambs were injected with antigens on the day of birth but in the ones injected in utero this constituted the second exposure to antigen and was often accompanied by anamnestic antibody formation. Nevertheless this difference did not appear to be a factor related to the individual variation in the rate of complement titre increase. In one of the two bottle fed lambs, the rise was slower than in most of the suckling lambs. The complement titres appeared to increase more slowly in newborn lambs than
in piglets (5), which was probably to be expected since the titre of adult swine serum is usually higher than that of adult sheep. The different sensitized red cell systems used in titrating the serum complement of the two species should also be considered.
Discussion As Kekwick states in his review (12) more information is available concerning the rate of formation of serum proteins at various stages in the development of the human and bovine foetus than for any other species, but at least three papers presenting data on foetal sheep sera may be cited (13-15). Both groups of investigators reported a progressive rise in total serum protein and the relative proportion of albumin with increasing foetal age. Barboriak et al (15) found this change to be particularly marked between 64 and 128 days' gestation During this period they observed that the percentage of beta-globulin also increased, that of alpha,-globulin declined but the proportion of alpha2-globulin and "fetuin", a protein component migrating between albumin and alpha-globulin,
TABLE II. - Complement titres of serial serum specimens from newborn lambs that had been treated in different ways prior to birth (at 93 to 132 days gestation). All animals were bled prior to suckling on the day of birth, after which they were injected with multiple antigens.
Material injected before birth
Number of lamb
None 311-1 ............ None 333-1 .. 345-2* .................. None 345-1* ................ Saline & adjuvant 359-2* ......... None 359-1* ......... Saline & adjuvant Saline & adjuvant 332-2 ... 341-1 ..... Saline & adjuvant 337-1 ........... Antigens & adjuvant 340-1 ...... Antigens & adjuvant Antigens & adjuvant ......... 335-1** .... 346-1 ........ Antigens & adjuvant 314-2 ........ Saline & adjuvant . Antigens & adjuvant .. 314-1 .. 306-2* .................. None 306-1* ... Antigens & adjuvant 336-1** ................. Homografted .....
Complement titres (units/ml.) Age of animals when bled (days) 4-8 9-15 20-24 28-32 0 16.7 10.0 5.9 *** 8.5 8.0 14.3 11.1 12.5 8.6 6.3 20.0 20.8 18.2 10.0 7.1 15.4 11.1 10.0 12.8 9.9 10.0 18.2 16.7 8.5 22.2 13.3 10.0 10.0 7.1 Neg. 12.5 * 7.1 14.3 12.5 4.5 18.5 11.4 6.3 16.7 7.7 Neg. 13.7 10.9 Neg. 20.0 19.2 14.3 12.7 4.4 14.3 8.6 12.5 5.5 14.3 12.5 11.8 5.6 5.7
*-twin lambs. **-bottle-fed lambs; the second bleeding from No. 335-1 was at seven days of age, from No. 336-1 at four days of age. ***-sample unsatisfactory for testing: haemolysed or cloudy.
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fell only slightly. Reference to Table I will show that it was near the end of this period, i.e. 123 days, that the foetal lambs began to show complement activity iin their serum. No evidence was obtained in this series of experiments to suggest that antigen injection with its resultant antibody response and lymphoid hyperplasia had any influence on the relative rate of development of serum complement in individual lambs, nor would this be expected since different cell systems are presumably involved in the synthesis of antibody globulin and the globulin components of complement. It seems likely that the latter are formed in the liver along with many other serum globulins of the alpha- and beta-categories (16, 17). Since the haemolytic activity of complement is dependent upon a number of different protein components, it is clear that only one need be absent to furnish a negative titre. Thus, the present study may involve the measurement of only that component of complement which develops latest in the foetal lamb. It also seems possible that a certain proportion between the various components must be reached before the serum can function effectively as complement. After the ingestion of colostrum, the gamma- and beta-globulin content of the serum of the newborn lamb increases rapidly (13, 18-20) as it does in the newborn calf. In the day-old colostrum-deprived animal this abrupt change in the serum protein picture does not occur. Our experimental newborn lambs were all bled prior to suckling but the second bleeding was not made until four to eight days later. Hence this potential sharp rise in complement titre following colostrum ingestion would be missed. One of the bottle-fed lambs bled at four days of age showed little change in complement titre but the other artificially-fed lamb at one week of age had a titre higher than that of several of the suckling lambs. When the lamb is ten days to two weeks old its total serum protein level has usually risen to about that of adult sheep (1820), but certain qualitative differences may continue to be evident in the serum protein pattern. In our present group of newborn lambs, complement titres at eight to ten weeks of age were still below adult values of about 40 to 140 units per ml.
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Some decrease in the complement titre of the lamb sera may also have occurred during storage in the frozen state. REFERE N CE S 1. NATTAN-LARRIERE, L. LEPINE, P. and MAY J. Dosage comparatif de l'alexine dans le sang de la mere et dans le sang de l'enfant au moment de la naissance. Compt. rend., Soc. Biol. 97: 671-672, 1927. 2. WASSERMAN, P. and ALBERTS, E. Complement titre of blood of the newborn. Proc. Soc. Exper. Biol. Med. 45: 563-564, 1940. 3. RICE, C. E. and DUHAMEL, L. A comparison of the complement, conglutinin and natural anti-sheep red cell antibody titres in the serum of newborn and older calves. Can J. Comp. Med. 21: 109-116, 1957. 4. STERZL, J., KOSKA, J. and LANC, A. Development of bactericidal properties against Gram-negative organisms in the serum of young animals. Folia Microbiol. 7: 163-173, 1962. 5. RICE, C. E. and L'ECUYER, C. Complement titres of naturally and artificially raised piglets. I. In piglets of different birth weights. Can. J. Comp. Med. 27: 157-161, 1963. 6. PIERCE, A. E. Electrophoretic and immunological studies on sera from calves from birth to weaning. I. Electrophoretic studies. II. Electrophoretic and serological studies with special reference to the normal and induced agglutinins to Trichomonas foetus. J. Hygiene, 53: 247-260 and 261-275, 1955. 7. NATTAN-LARRIERE, L. GRIMARD, L., and DUFOUR, J. L'alexine chez le nouveau-ne. Compt. rend. Soc. Biol. 125: 358-361, 1937. 8. SAGE, H. J. and LEON, M. A. Isolation and characterization of bovine conglutinin. Fed. Proc. 21: 22, 1962. 9. SILVERSTEIN, A. M., UHR, J. W., KRAMER, K. L. and LUKES, R. J. Fetal response to antigenic stimuius. II. Antibody production by the fetal lamb. J. Exper. Med. 117: 799-812, 1963. 10. SILVERSTEIN, A. M., , THORBECKE, G. J. KRANER, K. L.. and LUKES, R. J. Fetal response to antigemic stimulus. III. Gamma globulin production in normal and stimulated fetal lambs. J. Immunol. 91: 384, 1963. 11. RICE, C. E. and BOULANGER, P. The interchangeability of the complement components of different animal species. IV. In the hemolysis of 12.
rabbit erythrocytes sensitized with sheep antibody. J. Immunol. 68: 197-206, 1952. KEKWICK, R. A. The serum proteins of the fetus and young of some mammals. In: Advances in Protein Chemistry. Vol. 14: 231-254, 1959. DALGARNE, A., GODDEN, W., and McCARTHY, E. F. The effect of high- and low-plane feeding on the serum protein levels of pregnant ewes, foetuses and young lambs. Biochem. J. 46: 162-167, 1950. McCARTHY, E. F. and McDOUGALL, E. I. Absorption of immune globulins by the young lamb after the infestion of colostrum. Biochem. J. 55: 177-182, 1953. BARBORIAK, J. J., MESCHIA, G., BARRON, D. H. and COWGILL, G. R. Blood plasma proteins in fetal goats and sheep. Proc. Soc. Exper. Biol. Mcd. 98: 635-637, 1958. RICE, C. E. Relative effects of various agents on complement and antibody production. J. Immunol., 73: 375-382, 1954. RICE. C. E.. PLUMMER, P. J. G., ANNAU, E. and ROBERTSON, A. The effect of prolonged injection of ethionine on rabbits and guinea pigs on a high cholesterol diet. J. Immunol. 81: 506-512, 1958. EARLE, I. P. Influence of the ingestion of colostrum on the proteins of the blood of young foals, kids, lambs and pigs. J. Agric. Res. 51: 479-490,
1935. 19. CHARLWOOD, P. A., and THOMSON, A. Electrophoretic patterns of lamb sera before and after transfer of colostrum. Nature 161: 59-60, 1948. 20. SMITH, E. L. and HOLM, A. The transfer of immunity to the newborn calf from the colostriim. J. Biol. Chem. 175: 349- 1948.