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Endogenous Production of Immunoglobulin lgG1 in Newborn Calves1
Endogenous Production of Immunoglobulin IgG1 in Newborn Calvesz J. E. DEVERY, C. L. DAVIS, and B. L. LARSON Department of Dairy Science University of Illinois Urbana 61801
ABSTRACT
INTRODUCTION
There is a decrease in the specific activity of labeled IgG1 of serum over 3 wk following the feeding of iodine125 labeled immunoglobulin lgG1 in colostrum to calves at birth. This decrease indicated the appearance of new IgG1 from some source. To determine if this new IgG1 came from endogenous production in the calf or from continued small amount of intestinal absorption from milk, labeled IgG1 was added to normal milk and fed to calves of various ages up to 3 wk after an initial feeding of colostrum at birth. Labeled lgG1 was also added to colostrum fed to calves at birth, and the calves were maintained on a normal milk diet or fed a synthetic milk diet. Determination of iodine-125 in the serum protein fractions of these calves indicated that there was no apparent intestinal absorption of labeled IgG1 from the milk in the period from 2 days to 3 wk. Furthermore, comparable decreases occurred in the specific activity of labeled IgG1 in serum in the calves fed the labeled IgG1 in colostrum at birth and subsequently maintained either on a diet including milk or on the synthetic milk diet devoid of IgG1. The results support the conclusion that the origin of new IgG1 in the calf after about 36 h and up to about 3 wk of age arises from endogenous production at a rate of about 1 g of IgG1 per day.
The neonate of the bovine species is born with little or no detectable immunoglobulins in its serum, and the transfer of passive immunity to the offspring is through immunoglobulins (chiefly IgG1) transferred from the maternal blood serum via the mammary gland to the colostrum (1, 3). After ingestion of the colostrum by the calf, immunoglobulins are absorbed intact across the intestinal epithelium and appear in the blood stream within several hours (2, 4). The ability to absorb colostrum immunoglobulins without degradation is believed to last up to 24 to 36 h after birth, at which time closure of the intestinal lining and activation of the digestive system occurs (5, 16). In studies to measure the half-life of maternal IgG1 in the blood of newborn calves, Sasaki et al. (14) fed colostrum containing [12s I] IgG1 to unsuckled calves. Although decreases in the plasma concentrations of IgG1 and [12sI] IgG1 were found over 3 wk with half-lives of 20 days and 12 days, respectively, the validity of these and other such determinations (8 to 10) with regard to the kinetics of IgG1 metabolism was brought into question when it was demonstrated that the specific activity of [12sI] IgG1 in the plasma decreased at a linear rate up to 21 days after birth, suggesting that there was either endogenous production and/or intestinal absorption of IgG1 after 36 h of life (14). From the decay curve of specific activity of [12Sl] IgG1, Sasaki et al. (14) calculated the half-life of plasma IgG1 was 26 days. The decrease in specific activity indicated that IgG1 must be entering the existing pool in the calf after day 1. It was suggested that the most likely source of this new IgG1 was endogenous production; however, since the milk consumed by the calves contains some IgG1 (about .25 g/liter), the possibility could not be eliminated that some continued intestinal absorption of IgG1 from milk after 36 h was the source of the new IgG1.
Received February 12, 1979. 1Taken from M.S. thesis ofJ. E. Devery. Supported by Hatch Project 35-351 of the Illinois Agricultural Experiment Station. 1979 J Dairy Sci 62:1814-1818
1814
TECHNICAL NOTE Our studies were designed to evaluate these two possibilities by: 1) feeding [12sI] lgG1 in milk to calves of ages from 2 days to 3 wk after an initial feeding of colostrum to determine if labeled lgG1 appeared in the IgG1 fractions of the blood serum proteins, and 2) feeding [12Sl] IgG1 labeled colostrum to calves at birth and subsequently maintaining them on a diet including either milk or a synthetic milk devoid of IgG1 to determine if a decrease in specific activity of serum[12Sl] IgG1 occurred in both situations. MATERIALS A N D METHODS
Eleven calves, weighing 35 to 50 kg, were separated from their dams immediately after birth. Within 4 h, each of the animals was fed 1.4 to 1.8 kg of colostrum (38.4 g/kg body weight) from its respective dam. Calves 1 through 8 (Table 1) were given an initial feeding of colostrum and then maintained on a normal milk diet for the duration of the experiment. On the day indicated in Table 1, the calves were given one feeding of milk containing [12Sl1 IgG1. Blood samples were taken from the jugular vein prior to the radioisotope feeding and 24 h afterwards for determination of the iodine-125 labeled protein in the serum protein fractions. Calves 9 and 11 were given [12sI] IgG1 in
1815
their initial feeding of colostrum. Thereafter, they were fed normal bovine milk for the remainder of the measurement period. Blood samples were taken prior to the colostrum feeding, 24 h later, and every 3rd day for 21 days. Calf 10 was given [12s I] IgG1 in the initial colostrum feeding, and afterwards was fed a synthetic milk devoid o f IgG1. The synthetic milk contained 8% sodium caseinate, 6% lard oil, 9% lactose, 6% dextrose, .2% vitamin mix, .2% lecithin, and 2% mineral supplement. Blood samples were taken on the same schedule as for calves 9 and 11. Procedures were similar to those used b y Sasaki et al. (13, 14) to allow direct comparison of results. These included isolation and purification of lgG1 from either bovine serum or from the pseudoglobulin fraction of colostrum by salting-out, ion-exchange chromatography, and gel-filtration chromatography (3, 11). The IgG1 was labeled with 12si by the modified chloramine T method (7) with an overall efficiency of labeling of approximately 50%. The 12% TCA precipitable radioactivity was always greater than 90% of the total activity o f the final preparation. The IgG1 was quantitated from serum, colostrum, and milk by a radial immunodiffusion method (6) with monospecific antisera. The nonspecific precipitating components of
TABLE 1. Background data on experimental calves.
Calf no.
Dose agea (days postpartum)
1 2 3 4 5 6 7 8 9 10 11
2 4 7 8 11 12 15 19 Birth Birth Birth
Total lgG1 fed
Specific activity of (12 s l)lgG1 added to milk or coiostrum
Total 22s I consumed
(g)
(cpm X 10 s/mg)
(cpm × 107)
114.5 193.0 105.7 117.4 201.4 180.5 258.8 195.7 25.8 58.7 270.0
8.67 4.91 8.67 4.91 1.50 8.67 4.91 2.64 2.64 2.64 13.50
6.3 8.7 5.1 7.0 19.5 12.4 4.6 1.8 .26 1.38 2.76
aDose age reported in days indicates animal received (l as l)lgG1 in milk. Dose age at birth indicates received (i 2s l)lgG1 in colostrum. Journal of Dairy Science Vol. 62, No. 11, 1979
1816
DEVERY ET AL.
the rabbit anti-bovine IgG1 serum were removed by precipitating out the c o m m o n subclass determinants with IgG2 heavy chains. The heavy chains of the lgG2 subclass were prepared by the reduction and alkylation method of Utsumi and Karush (18). Radioactivity in serum or whey fractions was determined by direct count in an automatic gamma well counter. Localization of the radioisotope in the serum protein fractions of representative serum samples was by counting fractions eluted from a DEAE-cellulose (DE-52) column by ionexchange chromotography (13, 14). The rate of IgG1 synthesis was calculated from the data collected for calves 9, 10, and 11. Turnover time of IgG1 was calculated according to the method of Sterling (17). The turnover time (T t) is the time required for an amount o f IgG1 equal to that in the total body pool on day 1 to be replaced by new IgG1. In the equation:
2.0 - (o)
1
Sets
-8000
Albumin
II II L=" - -
I! It
--
II It
II
i.o -
- 4ooo
I I
I. I =
I~
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1', 'o°.
tgG,
,,_
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Serum Albumin 15--
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1 Tt = Ty:z X l n ~ T w is the observed half-life o f the specific activity of the serum [12Sl] IgG1 and In is the natural logarithm. The body pool size of lgG1 was calculated from the amount of IgG1 in the serum if 8% of the calf weight was blood of which half represented serum. The body pool size of IgG1 divided by T t gave the daily production of lgG1.
RESULTS
AND
DISCUSSION
Typical data are in Figure 1 obtained by the localization of [12sI] lgG1 in the serum protein fractions of a calf (No. 9) fed the labeled IgG1 in colostrum at birth (Figure la) and those obtained from the 8 calves (Nos. 1 to 8) fed the labeled IgG1 in milk at various ages from 2 days up to 3 wk, When the labeled lgG1 was fed in colostrum at birth (Figure la), it appears concentrated in the serum IgG1 eluted from the column in fractions 30 to 35 with much smaller amounts associated with the other serum protein fractions. Some scattering of the S2Sl label is expected in such experiments due to small amounts of free iodine-125 and possible other impurities in the original labeled preparation of [ t2s I] IgG1 plus the continuing catabolism of the [12st] IgG1 in the calf with release of the iodine-125. In all of the eight calves fed Journal of Dairy Science Vol. 62, No. 11, 1979
0.5
0
~gG=
2000
20
4O
FRACTION NUMBER
60
80 0
Figure 1. Comparison of the location of [S2Sl} lgG1 in the serum protein fractions of calves fed the labeled protein at two ages. Calf 9 (Figure l a ) w a s fed lSZSl]lgG1 in colostrum at birth. Calf 2 (Figure lb) was fed unlabeled colostrum at birth and [*=Sl] lgG1 in milk at 4 days. The serum samples were taken from calf 9 at 4 days and from calf 2 at 5 days. Samples of 4 ml of each serum were chromatographed on a DE-52 column. Fractions were eluted by changing phosphate buffer, pH, and sodium chloride concentration and collected in 6-ml aliquots as noted in the Methods and described previously (13). The serum of calves varied considerably in their blood protein content chiefly because of differences in the amount of immunoglobulins in and absorbed from the colostrum at birth. The amount of IgG1 in the serum on the days indicated were 9.5 mg/ml for calf 9 and 31 mg/ml for calf 2 with the former lower than usual and the latter higher. The amount of igG2 is low in both calf serum samples compared to maternal serum since colostrum contains only about one-tenth as much igG2 as IgG1 whereas they are about equal in adult blood (3). Figure la shows that the s 2s I-label was concentrated in the lgG1 serum protein fraction of calf 9. Figure lb shows that the s2s I-label is scattered throughout the serum protein fractions of calf 2. Calf 2 consumed 33 times as many epm of 12s I as did calf9 (Table 1). If comparable amounts had been used, the cpm's for S2Sl in Figure lb essentially would disappear into the base line.
TECHNICAL NOTE the labeled lgG1 in milk at various ages after 36 h up to 3 wk, there was no concentration of label in the IgGl-containing fractions with only small amounts of iodine-125 scattered throughout the various fractions (Figure lb). These experiments indicate that significant [12s I] IgG1 is absorbed from the gut into the blood stream in calves fed colostrum at birth, but it is not absorbed when fed in milk in the period from 2 days to about 3 wk. Data in Figure 2 show changes in the specific activity of [12SlllgG1 in the serum of two calves which were fed the labeled lgG1 in colostrum at birth with one (No. 10) maintained on a synthetic milk diet devoid of IgG1 and the other (No. 11) on a normal diet with milk. The specific activity of [12sI]IgG1 decreased in both calves indicating the entry of IgG1 into the pool. In the case of calf 10, this IgG1 could not have come from the diet, thus providing further evidence that the source of the new IgG1 appearing in the serum must have come from endogenous production. The calculated production rates of new lgG1 in these three calves averaged .84 g per day which represents 4.2% of the calf's original pool on day 1 (see Figure 2 legend for calculation data). This is a small but significant amount of IgG1 easily overlooked by other methods for the determination of changes with time in the blood concentration of IgG1 as an index of the production of new lgG1 (see Introduction). Our methods utilizing changes in the specific activity of [12Sl] IgG1 have provided a means to make a reasonable estimate of the rate and a m o u n t of this production, and most significantly, that it does occur. The results of this investigation confirm many previous studies (1) that the newborn calf is incapable of absorbing colostrum immunoglobulins (specifically lgG1) across the intestinal barrier after about 36 h of age. The tracer methods involved in reaching this conclusion are capable of locating minute amounts of labeled IgG1 in blood and would have detected any significant amount of labeled IgG1 in the blood signifying intestinal absorption. The results indicate that the newborn calf has the capability for the endogenous production of IgG1 during the first 3 wk of life. Even though a limited capability for IgG production develops in the fetal calf (12, 15), previous studies have provided conflicting evidence for
-
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Figure 2. Changes in the specific activity of serum [laSlllgG1 with time in two calves after both had received an initial feeding of 112s1] lgG1 in colosrcum within 4 h after parturition. Calf 11 was subsequently fed a diet with milk containing lgG1 and calf 10 was fed a synthetic milk diet free of lgG1. The values at each sampling time are expressed as the log percent of the values at day 1. The day 1 values for calf 11 were 22.2 mg/ml of lgG1 in the serum and 10,092 cpm/ml. The day 1 values for calf 10 were 6.79 mg/ml of lgG1 in the serum and 6,862 cpm/ml. The calculated halfqives for the decrease in specific activity of [a2s I] lgG1 from these two animals (see below) differed but is not significant since this figure shows results from individual animals. It is important that the specific activity of |lZSlllgG1 showed a significant decrease in both animals. The rates of lgG1 synthesis were calculated from the turnover times of lgG1 (see Methods for formula) for calves 9, 10, and 11 that had been fed colosrrum at birth containing {t2SlJlgG1. The pertinent data for these three calveswere, respectively: body weights of 35.0, 48.6, and 40.9 kg; day 1 pool size for lgG1 of 11.6 g, 13.2 g, and 36.11 g; and half-lives for the decrease in activity of [t2Sll IgG1 of 28, 11, and 18 days. The calculated production rates of new lgG1 for calves9, 10, and 11 were, respectively, .28, .83, and 1.4 g/day (average .84). This represents a daily production, respectively, of 2.4%, 6.3%, and 3.9% (average 4.2%) of the original day 1 pool of lgG1.
significant IgG1 production immediately after birth (1, 8, 14). The rate of this production is not large, approximating 1 g/day of new IgG1 and probably is dependent on the antigenic stress to which the ca/f is subjected. The cumulative amount produced is undoubtedly Journal of Dairy Science Vol. 62, No. 11, 1979
1818
DEVERV ET AL.
significant in p r o t e c t i n g t h e calf during t h e s e difficult 3 wk a f t e r birth.
REFERENCES
1 Brambell, F.W.R. 1970. The transmission of passive immunity from mother to young. Page 201 in Frontiers of biology. Vol. 18. A. Neuberger and E. L. Tatem, ed. North Holland Publishing Co., Amsterdam. 2 Brandon, M. R., and A. K. Lascelles. 1971. Relative efficiency of absorption of IgG1, lgG2, IgA, and lgM in the newborn calf. Australian J. Exp. Biol. Med. Sci. 49:629. 3 Butler, J. E. 1974. lmmunoglobulins of the mammary secretions. Page 217 in Lactation: A comprehensive treatise. Vol. III. B. L. Larson and V. R. Smith, ed. Academic Press, NY. 4 Comline, R. S., H. E. Roberts, and D. A. Titchen. 1951. Route of absorption of colostrum in the newborn animal. Nature 167:561. 5 Comline, R. S., H. E. Roberts, and D. A. Titchen. 1951. Histological changes in the epithelium of the small intestine during protein absorption in the newborn. Nature 168: 84. 6 Fahey, J. L., and E. M. McKelvey. 1965. Quantitative determination of serum immunoglobulins in antibody-agar plates. J. Immunol. 94: 84. 7 Hunter, W. M., and F. C. Greenwood. 1962. Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature 194:495. 8 Logan, E. F. 1974. Quantitative studies on serum immunogiobulin levels in suckled calves from birth
Journal of Dairy Science Vol. 62, No. 11, 1979
to five weeks. Vet. Rec. 94: 367. 9 MacDougall, D. F., and W. Mulligan. 1969. The distribution and metabolism of fast lgG immunoglobulins in neonatal calf. J. Physiol. 201:77P. 10 McEwan, A. D., E. W. Fisher, and I. E. Selman. 1968. The effect of colostrum on the volume and composition of the plasma of calves. Res. Vet. Sci. 9:284. 11 Milstein, C. P., and A. Feinstein. 1968. Comparative studies of two types of bovine immunoglobulin G heavy chains. Biochem. J. 107: 559. 12 Olson, D. P., and G. L. Waxier. 1976. Immune response of the bovine fetus and neonate to Escbericbia coli: quantitation and qualitation of the humoral immune response. Amer. J. Vet. Res. 37:639. 13 Sasaki, M., C. L. Davis, and B. L. Larson. 1976. Production and turnover of lgG1 and IgG2 immunoglobulins in the bovine around parturition. J. Dairy Sci. 59:2896. 14 Sasaki, M., C. L. Davis, and B. L. Larson. 1977. lmmunoglobulin IgG1 metabolism in newborn calves. J. Dairy Sci. 60:623. 15 Sehultz, R. D. 1973. Development aspects of the fetal bovine immune response: a review. Cornell Vet. 63:507. 16 Smith, E. L., and A. Holm. 1948. Transference of immunity to the newborn from colostrum. J. Biol. Chem. 175: 349. 17 Sterling, K. 1951. The turnover rate of serum albumin in man as measured by 131 I-tagged albumin. J. Clin. Invest. 30:1228. 18 Utsumi, S., and F. Karush. 1964. The subunits of purified rabbit antibody. Biochemistry 3:1329.
ABSTRACT
INTRODUCTION
There is a decrease in the specific activity of labeled IgG1 of serum over 3 wk following the feeding of iodine125 labeled immunoglobulin lgG1 in colostrum to calves at birth. This decrease indicated the appearance of new IgG1 from some source. To determine if this new IgG1 came from endogenous production in the calf or from continued small amount of intestinal absorption from milk, labeled IgG1 was added to normal milk and fed to calves of various ages up to 3 wk after an initial feeding of colostrum at birth. Labeled lgG1 was also added to colostrum fed to calves at birth, and the calves were maintained on a normal milk diet or fed a synthetic milk diet. Determination of iodine-125 in the serum protein fractions of these calves indicated that there was no apparent intestinal absorption of labeled IgG1 from the milk in the period from 2 days to 3 wk. Furthermore, comparable decreases occurred in the specific activity of labeled IgG1 in serum in the calves fed the labeled IgG1 in colostrum at birth and subsequently maintained either on a diet including milk or on the synthetic milk diet devoid of IgG1. The results support the conclusion that the origin of new IgG1 in the calf after about 36 h and up to about 3 wk of age arises from endogenous production at a rate of about 1 g of IgG1 per day.
The neonate of the bovine species is born with little or no detectable immunoglobulins in its serum, and the transfer of passive immunity to the offspring is through immunoglobulins (chiefly IgG1) transferred from the maternal blood serum via the mammary gland to the colostrum (1, 3). After ingestion of the colostrum by the calf, immunoglobulins are absorbed intact across the intestinal epithelium and appear in the blood stream within several hours (2, 4). The ability to absorb colostrum immunoglobulins without degradation is believed to last up to 24 to 36 h after birth, at which time closure of the intestinal lining and activation of the digestive system occurs (5, 16). In studies to measure the half-life of maternal IgG1 in the blood of newborn calves, Sasaki et al. (14) fed colostrum containing [12s I] IgG1 to unsuckled calves. Although decreases in the plasma concentrations of IgG1 and [12sI] IgG1 were found over 3 wk with half-lives of 20 days and 12 days, respectively, the validity of these and other such determinations (8 to 10) with regard to the kinetics of IgG1 metabolism was brought into question when it was demonstrated that the specific activity of [12sI] IgG1 in the plasma decreased at a linear rate up to 21 days after birth, suggesting that there was either endogenous production and/or intestinal absorption of IgG1 after 36 h of life (14). From the decay curve of specific activity of [12Sl] IgG1, Sasaki et al. (14) calculated the half-life of plasma IgG1 was 26 days. The decrease in specific activity indicated that IgG1 must be entering the existing pool in the calf after day 1. It was suggested that the most likely source of this new IgG1 was endogenous production; however, since the milk consumed by the calves contains some IgG1 (about .25 g/liter), the possibility could not be eliminated that some continued intestinal absorption of IgG1 from milk after 36 h was the source of the new IgG1.
Received February 12, 1979. 1Taken from M.S. thesis ofJ. E. Devery. Supported by Hatch Project 35-351 of the Illinois Agricultural Experiment Station. 1979 J Dairy Sci 62:1814-1818
1814
TECHNICAL NOTE Our studies were designed to evaluate these two possibilities by: 1) feeding [12sI] lgG1 in milk to calves of ages from 2 days to 3 wk after an initial feeding of colostrum to determine if labeled lgG1 appeared in the IgG1 fractions of the blood serum proteins, and 2) feeding [12Sl] IgG1 labeled colostrum to calves at birth and subsequently maintaining them on a diet including either milk or a synthetic milk devoid of IgG1 to determine if a decrease in specific activity of serum[12Sl] IgG1 occurred in both situations. MATERIALS A N D METHODS
Eleven calves, weighing 35 to 50 kg, were separated from their dams immediately after birth. Within 4 h, each of the animals was fed 1.4 to 1.8 kg of colostrum (38.4 g/kg body weight) from its respective dam. Calves 1 through 8 (Table 1) were given an initial feeding of colostrum and then maintained on a normal milk diet for the duration of the experiment. On the day indicated in Table 1, the calves were given one feeding of milk containing [12Sl1 IgG1. Blood samples were taken from the jugular vein prior to the radioisotope feeding and 24 h afterwards for determination of the iodine-125 labeled protein in the serum protein fractions. Calves 9 and 11 were given [12sI] IgG1 in
1815
their initial feeding of colostrum. Thereafter, they were fed normal bovine milk for the remainder of the measurement period. Blood samples were taken prior to the colostrum feeding, 24 h later, and every 3rd day for 21 days. Calf 10 was given [12s I] IgG1 in the initial colostrum feeding, and afterwards was fed a synthetic milk devoid o f IgG1. The synthetic milk contained 8% sodium caseinate, 6% lard oil, 9% lactose, 6% dextrose, .2% vitamin mix, .2% lecithin, and 2% mineral supplement. Blood samples were taken on the same schedule as for calves 9 and 11. Procedures were similar to those used b y Sasaki et al. (13, 14) to allow direct comparison of results. These included isolation and purification of lgG1 from either bovine serum or from the pseudoglobulin fraction of colostrum by salting-out, ion-exchange chromatography, and gel-filtration chromatography (3, 11). The IgG1 was labeled with 12si by the modified chloramine T method (7) with an overall efficiency of labeling of approximately 50%. The 12% TCA precipitable radioactivity was always greater than 90% of the total activity o f the final preparation. The IgG1 was quantitated from serum, colostrum, and milk by a radial immunodiffusion method (6) with monospecific antisera. The nonspecific precipitating components of
TABLE 1. Background data on experimental calves.
Calf no.
Dose agea (days postpartum)
1 2 3 4 5 6 7 8 9 10 11
2 4 7 8 11 12 15 19 Birth Birth Birth
Total lgG1 fed
Specific activity of (12 s l)lgG1 added to milk or coiostrum
Total 22s I consumed
(g)
(cpm X 10 s/mg)
(cpm × 107)
114.5 193.0 105.7 117.4 201.4 180.5 258.8 195.7 25.8 58.7 270.0
8.67 4.91 8.67 4.91 1.50 8.67 4.91 2.64 2.64 2.64 13.50
6.3 8.7 5.1 7.0 19.5 12.4 4.6 1.8 .26 1.38 2.76
aDose age reported in days indicates animal received (l as l)lgG1 in milk. Dose age at birth indicates received (i 2s l)lgG1 in colostrum. Journal of Dairy Science Vol. 62, No. 11, 1979
1816
DEVERY ET AL.
the rabbit anti-bovine IgG1 serum were removed by precipitating out the c o m m o n subclass determinants with IgG2 heavy chains. The heavy chains of the lgG2 subclass were prepared by the reduction and alkylation method of Utsumi and Karush (18). Radioactivity in serum or whey fractions was determined by direct count in an automatic gamma well counter. Localization of the radioisotope in the serum protein fractions of representative serum samples was by counting fractions eluted from a DEAE-cellulose (DE-52) column by ionexchange chromotography (13, 14). The rate of IgG1 synthesis was calculated from the data collected for calves 9, 10, and 11. Turnover time of IgG1 was calculated according to the method of Sterling (17). The turnover time (T t) is the time required for an amount o f IgG1 equal to that in the total body pool on day 1 to be replaced by new IgG1. In the equation:
2.0 - (o)
1
Sets
-8000
Albumin
II II L=" - -
I! It
--
II It
II
i.o -
- 4ooo
I I
I. I =
I~
o.5 -
1', 'o°.
tgG,
,,_
"( bl
o
/
<
-
o
,-
Serum Albumin 15--
- 6000 ~tqG,
1 Tt = Ty:z X l n ~ T w is the observed half-life o f the specific activity of the serum [12Sl] IgG1 and In is the natural logarithm. The body pool size of lgG1 was calculated from the amount of IgG1 in the serum if 8% of the calf weight was blood of which half represented serum. The body pool size of IgG1 divided by T t gave the daily production of lgG1.
RESULTS
AND
DISCUSSION
Typical data are in Figure 1 obtained by the localization of [12sI] lgG1 in the serum protein fractions of a calf (No. 9) fed the labeled IgG1 in colostrum at birth (Figure la) and those obtained from the 8 calves (Nos. 1 to 8) fed the labeled IgG1 in milk at various ages from 2 days up to 3 wk, When the labeled lgG1 was fed in colostrum at birth (Figure la), it appears concentrated in the serum IgG1 eluted from the column in fractions 30 to 35 with much smaller amounts associated with the other serum protein fractions. Some scattering of the S2Sl label is expected in such experiments due to small amounts of free iodine-125 and possible other impurities in the original labeled preparation of [ t2s I] IgG1 plus the continuing catabolism of the [12st] IgG1 in the calf with release of the iodine-125. In all of the eight calves fed Journal of Dairy Science Vol. 62, No. 11, 1979
0.5
0
~gG=
2000
20
4O
FRACTION NUMBER
60
80 0
Figure 1. Comparison of the location of [S2Sl} lgG1 in the serum protein fractions of calves fed the labeled protein at two ages. Calf 9 (Figure l a ) w a s fed lSZSl]lgG1 in colostrum at birth. Calf 2 (Figure lb) was fed unlabeled colostrum at birth and [*=Sl] lgG1 in milk at 4 days. The serum samples were taken from calf 9 at 4 days and from calf 2 at 5 days. Samples of 4 ml of each serum were chromatographed on a DE-52 column. Fractions were eluted by changing phosphate buffer, pH, and sodium chloride concentration and collected in 6-ml aliquots as noted in the Methods and described previously (13). The serum of calves varied considerably in their blood protein content chiefly because of differences in the amount of immunoglobulins in and absorbed from the colostrum at birth. The amount of IgG1 in the serum on the days indicated were 9.5 mg/ml for calf 9 and 31 mg/ml for calf 2 with the former lower than usual and the latter higher. The amount of igG2 is low in both calf serum samples compared to maternal serum since colostrum contains only about one-tenth as much igG2 as IgG1 whereas they are about equal in adult blood (3). Figure la shows that the s 2s I-label was concentrated in the lgG1 serum protein fraction of calf 9. Figure lb shows that the s2s I-label is scattered throughout the serum protein fractions of calf 2. Calf 2 consumed 33 times as many epm of 12s I as did calf9 (Table 1). If comparable amounts had been used, the cpm's for S2Sl in Figure lb essentially would disappear into the base line.
TECHNICAL NOTE the labeled lgG1 in milk at various ages after 36 h up to 3 wk, there was no concentration of label in the IgGl-containing fractions with only small amounts of iodine-125 scattered throughout the various fractions (Figure lb). These experiments indicate that significant [12s I] IgG1 is absorbed from the gut into the blood stream in calves fed colostrum at birth, but it is not absorbed when fed in milk in the period from 2 days to about 3 wk. Data in Figure 2 show changes in the specific activity of [12SlllgG1 in the serum of two calves which were fed the labeled lgG1 in colostrum at birth with one (No. 10) maintained on a synthetic milk diet devoid of IgG1 and the other (No. 11) on a normal diet with milk. The specific activity of [12sI]IgG1 decreased in both calves indicating the entry of IgG1 into the pool. In the case of calf 10, this IgG1 could not have come from the diet, thus providing further evidence that the source of the new IgG1 appearing in the serum must have come from endogenous production. The calculated production rates of new lgG1 in these three calves averaged .84 g per day which represents 4.2% of the calf's original pool on day 1 (see Figure 2 legend for calculation data). This is a small but significant amount of IgG1 easily overlooked by other methods for the determination of changes with time in the blood concentration of IgG1 as an index of the production of new lgG1 (see Introduction). Our methods utilizing changes in the specific activity of [12Sl] IgG1 have provided a means to make a reasonable estimate of the rate and a m o u n t of this production, and most significantly, that it does occur. The results of this investigation confirm many previous studies (1) that the newborn calf is incapable of absorbing colostrum immunoglobulins (specifically lgG1) across the intestinal barrier after about 36 h of age. The tracer methods involved in reaching this conclusion are capable of locating minute amounts of labeled IgG1 in blood and would have detected any significant amount of labeled IgG1 in the blood signifying intestinal absorption. The results indicate that the newborn calf has the capability for the endogenous production of IgG1 during the first 3 wk of life. Even though a limited capability for IgG production develops in the fetal calf (12, 15), previous studies have provided conflicting evidence for
-
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I0 AGE (DAYS)
i
15
,
i
20
Figure 2. Changes in the specific activity of serum [laSlllgG1 with time in two calves after both had received an initial feeding of 112s1] lgG1 in colosrcum within 4 h after parturition. Calf 11 was subsequently fed a diet with milk containing lgG1 and calf 10 was fed a synthetic milk diet free of lgG1. The values at each sampling time are expressed as the log percent of the values at day 1. The day 1 values for calf 11 were 22.2 mg/ml of lgG1 in the serum and 10,092 cpm/ml. The day 1 values for calf 10 were 6.79 mg/ml of lgG1 in the serum and 6,862 cpm/ml. The calculated halfqives for the decrease in specific activity of [a2s I] lgG1 from these two animals (see below) differed but is not significant since this figure shows results from individual animals. It is important that the specific activity of |lZSlllgG1 showed a significant decrease in both animals. The rates of lgG1 synthesis were calculated from the turnover times of lgG1 (see Methods for formula) for calves 9, 10, and 11 that had been fed colosrrum at birth containing {t2SlJlgG1. The pertinent data for these three calveswere, respectively: body weights of 35.0, 48.6, and 40.9 kg; day 1 pool size for lgG1 of 11.6 g, 13.2 g, and 36.11 g; and half-lives for the decrease in activity of [t2Sll IgG1 of 28, 11, and 18 days. The calculated production rates of new lgG1 for calves9, 10, and 11 were, respectively, .28, .83, and 1.4 g/day (average .84). This represents a daily production, respectively, of 2.4%, 6.3%, and 3.9% (average 4.2%) of the original day 1 pool of lgG1.
significant IgG1 production immediately after birth (1, 8, 14). The rate of this production is not large, approximating 1 g/day of new IgG1 and probably is dependent on the antigenic stress to which the ca/f is subjected. The cumulative amount produced is undoubtedly Journal of Dairy Science Vol. 62, No. 11, 1979
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DEVERV ET AL.
significant in p r o t e c t i n g t h e calf during t h e s e difficult 3 wk a f t e r birth.
REFERENCES
1 Brambell, F.W.R. 1970. The transmission of passive immunity from mother to young. Page 201 in Frontiers of biology. Vol. 18. A. Neuberger and E. L. Tatem, ed. North Holland Publishing Co., Amsterdam. 2 Brandon, M. R., and A. K. Lascelles. 1971. Relative efficiency of absorption of IgG1, lgG2, IgA, and lgM in the newborn calf. Australian J. Exp. Biol. Med. Sci. 49:629. 3 Butler, J. E. 1974. lmmunoglobulins of the mammary secretions. Page 217 in Lactation: A comprehensive treatise. Vol. III. B. L. Larson and V. R. Smith, ed. Academic Press, NY. 4 Comline, R. S., H. E. Roberts, and D. A. Titchen. 1951. Route of absorption of colostrum in the newborn animal. Nature 167:561. 5 Comline, R. S., H. E. Roberts, and D. A. Titchen. 1951. Histological changes in the epithelium of the small intestine during protein absorption in the newborn. Nature 168: 84. 6 Fahey, J. L., and E. M. McKelvey. 1965. Quantitative determination of serum immunoglobulins in antibody-agar plates. J. Immunol. 94: 84. 7 Hunter, W. M., and F. C. Greenwood. 1962. Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature 194:495. 8 Logan, E. F. 1974. Quantitative studies on serum immunogiobulin levels in suckled calves from birth
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to five weeks. Vet. Rec. 94: 367. 9 MacDougall, D. F., and W. Mulligan. 1969. The distribution and metabolism of fast lgG immunoglobulins in neonatal calf. J. Physiol. 201:77P. 10 McEwan, A. D., E. W. Fisher, and I. E. Selman. 1968. The effect of colostrum on the volume and composition of the plasma of calves. Res. Vet. Sci. 9:284. 11 Milstein, C. P., and A. Feinstein. 1968. Comparative studies of two types of bovine immunoglobulin G heavy chains. Biochem. J. 107: 559. 12 Olson, D. P., and G. L. Waxier. 1976. Immune response of the bovine fetus and neonate to Escbericbia coli: quantitation and qualitation of the humoral immune response. Amer. J. Vet. Res. 37:639. 13 Sasaki, M., C. L. Davis, and B. L. Larson. 1976. Production and turnover of lgG1 and IgG2 immunoglobulins in the bovine around parturition. J. Dairy Sci. 59:2896. 14 Sasaki, M., C. L. Davis, and B. L. Larson. 1977. lmmunoglobulin IgG1 metabolism in newborn calves. J. Dairy Sci. 60:623. 15 Sehultz, R. D. 1973. Development aspects of the fetal bovine immune response: a review. Cornell Vet. 63:507. 16 Smith, E. L., and A. Holm. 1948. Transference of immunity to the newborn from colostrum. J. Biol. Chem. 175: 349. 17 Sterling, K. 1951. The turnover rate of serum albumin in man as measured by 131 I-tagged albumin. J. Clin. Invest. 30:1228. 18 Utsumi, S., and F. Karush. 1964. The subunits of purified rabbit antibody. Biochemistry 3:1329.