Studies on passive immunity in the foal

J.

C:ohw.

155

PATH. 1974. VOL. 84.

STUDIES III.

ON PASSIVE

THE

IMMUNITY

CHARACTERIZATION NEONATAL

IN THE

AND SIGNIFICANCE PROTEINURIA

FOAL OF

BY

L. B. JEFFCOTT Equine Research Station,

and TISZA The Animal

Health

J. JEFFCOTT Trust.

dVezcmarket, Suffolk

INTRODUCTION

A transient proteinuria has been recorded in newly born colostrum fed calves (Smith and Little, 1924; Howe, 1924). Further study by Pierce ( 1960) has shown the main constituent to be low molecular weight maternal S-lactoglobulin. Proteinuria has also been noted in young lambs (McCarthy and McDougall, 1953) kids, puppies and human infants (Pierce, 1961a) and piglets (Martinsson, 1972). Apart from a preliminary report of this study (Jeffcott, 1971, 1973) no previous reference to proteinuria in newly born foals has been found in the literature. The absorption and fate of the macromolecules, y-globulin and the synthetic polymer PVP (mol. wt. 160 000) has been considered in the two preceding papers. This article discussesthe fate of the low molecular weight proteins of milk and colostrum and their ultimate excretion in the urine. The investigation was divided into two parts; firstly the detection and estimation of duration of proteinuria in colostrum fed foals was determined and secondly, characterization of the main protein components in the urine and an assessmentof their molecular size was carried out. MATERIALS

AND

METHODS

Detection and renal clearance rate of urinary protein. Total urine collection for the first 20 to 48 h. was carried out on 21 pony foals. Urinary protein was estimated by a modified sulphosalicylic acid turbidity test (Wooton, 1964). The foals were divided into the following groups according to the amount of colostrum they received. Group 1: foals 1 to 9 suckled normally. They sucked their first colostrum at a mean 65 min. of life. Group 2: foals 10 to 18 were fed supplementary colostrum in the first 30 h. of life. These foals were muzzled for the first 2 to 3 h. Group 3: foal 19 was fed only pooled equine colostrum of a total protein content 20.5 g./lOO ml. for the first 30 h. of life. The foal was kept muzzled throughout this period and was fed by bottle or stomach tube every hour. Group 4: foals 20 and 21 were deprived of colostrurn for the first 18 and 28 h. of life. They were kept muzzled and fed a commercial milk substitute (Equilac, Pegus Ireland Ltd., Spencer Avenue, Dublin, Ireland) by bottle every hour. Molecular weight determination and characterization of urinary proteins by gel jiltration and CAM electrophoresis. It has been shown that a standard relationship exists between the molecular weight of most proteins and their elution volume in Sephadex G-200

(Andrews, 1965). For the determination of approximate molecular size of the urinary

456

L. B. JEFFCOTT

AND

T. J. JEFFCOTT

protein a number of suitable proteins of known molecular weight were first eluted through G-200 at pH 7.2. Gel filtration of pre- and post-colostrum foal sera, colostrum and milk was carried out for comparison of their elution patterns with that of urinary protein. The preparation and use of gel columns was based on the method of Andrews (1964, 1965) using Sephadex G-200 (Pharmacia, Uppsala, Sweden). The buffer used was 0.1 M Tris/O*05 M NaCl solution containing O-065 sodium azide/l and adjusted to pH 7.2. The runs were performed at room temperature, 20f3 “C. at a mean flow rate of 23.0 ml./h. and column effluents were collected in 3.0 to 6.0 ml. fractions. The method of zone electrophoresis of serum, milk, foal urine and protein fractions after gel filtration was based on that of Kohn (1960) and was carried out on cellulose acetate membrane (CAM) strips.

RESULTS

Detection and Renal Clearance of Urinary Proteins

A significant transient proteinuria was detected in the 19 foals that received colostrum. The levels of proteinuria expressed in mg. protein/100 ml. showed considerable individual variation. The smaller foals tended to void smaller volumes of a more concentrated urine than did the larger foals, although the total amount of protein excreted appeared fairly comparable. In order to minimize this variation, the proteinuria was measured as the renal clearance of urinary protein, the amount of protein excreted in mg./min. The results of the 3 groups of foals fed colostrum are summarized in Fig. 1. The levels rose from birth to peak values at 9 to 11 h. The peak level in group 2 which were fed extra colostrum was the highest of all the groups and the lowest was in group 1

20

Age (h.1

Fig.

1, Renal clearance of urinary protein expressed as mg. excreted/min. in 3 groups foals. (......) Foals 1-9, suckled normally; (--)Foals 10-18 fed extra colostrum; fed only colostrum for the first 30 h. of life.

of colostrum fed (- - -) Foal 19

PASSIVE

IMMUNITY

IN

THE

FOAL:

NEONATAL

457

PROTEINURIA

allowed to suckle normally. In the 2 foals which were deprived of colostrum no evidence of a proteinuria was detected. The highest concentration of protein in the urine of these 2 foals was 2.5 mg./lOO ml. and 2-O mg./lOO ml. protein respectively. Molecular

Weight Determinations

Calibration of sephadex column. A number of proteins of known molecular weight were used and their elution volumes determined in a column of G-200 Sephadex, 30 x 3-O cm. A linear relationship was obtained when the logarithm of the molecular weight was plotted against the elution volume, Ve (Fig. 2). The void volume of the column was estimated using Blue Dextran 2000 on a number of occasions; the mean result was 70.0 ml. Most of the proteins tested conformed fairly closely to the smooth curve relationship of log. mol. wt. against Ve. The equine y-globulin was somewhat atypical in this respect possibly due to its higher carbohydrate content. This has been shown to affect the gel filtration behaviour of some other glycoproteins (Andrews, 1964; Whitaker, 1963). Andrews (1965) has demonstrated that human and bovine y-globulin give an apparent molecular weight by gel filtration of 205 000 instead of their true value of about 160 000. Assuming equine y-globulin to be of similar molecular size and carbohydrate content, the correction in molecular weight to 205 000 brings it onto the straight line graph with the other proteins examined (Fig. 2). The amount of protein applied to the column on each run was 10 to 15 mg. except in the case of bovine p-lactoglobulin, when 30 mg. was used. Andrews (1964) showed that unless a concentration of more than 30 mg. was applied there was dissociation of the molecule into sub-units which resulted in a reduction in molecular weight. 5x105r-

t -

Alkaline

Bovine

Corrected value for Equine y globulin (205 000) /-a phosphatase

albumln

Bowne p lactoglobulln (37 000) \ Trypsin (23 800)

---he

Myoglobln-• (I8 000)

/

/eA

4

i Cytochrome (I2 400)

C

I

I

I

I

I

!

150

Elutlon Fig.

2. Gel filtration logarithmic

volume-

of proteins through G-200 Sephadex. molecular weight of the proteins.

Ve (ml.1

Graph

of elution

volume,

?‘e, against

the

458

L.

B. JEFFCOTT

AND

T. J. JEFFCOTT

Foal serum. Samples of fresh and frozen foal serum collected before colostrum ingestion (0 h.) and after 24 h. were subjected to gel filtration. The samples were dialysed for 48 h. against Tris/NaCl buffer, pH 7.2, and then a volume of dialysate of 1.0 ml. applied to the column. The elution diagram of serum from 1 foal (ex Suna ‘70) before and after suckling is given in Fig. 3. This pattern was representative of all samples examined. There was no difference detected in fresh or frozen samples of serum, and this was also true for colostrum, milk and urine. Three peaks were detected in the post-colostrum serum sample. Peak 1 was a small peak just behind the void volume, 70 to 85 ml. Peak II was the largest of the 3 peaks with a maximum protein concentration in the column effluent at 120 ml. and this was followed by Peak III which had its highest point at 150 ml. There was a marked difference in the pre-colostrum serum pattern in that Peak II was virtually absent, the other 2 peaks being very comparable to those of the postcolostrum serum. I

I

I

I

I

I

200

250

300

II 4” 0 m

I’ 0

50

100

I

150 Eff bent

Fig. 3. El&ion strum.

pattern of foal serum through G-ZOO (@- - -0) Foal serum, oh.; (0-O)

I volume

350

(m1.l

Sephadex before and after Foal serum, 24 h.

the ingestion

of colo-

Colostrum and milk. Samples of colostrum (0 h.) and milk (24 h.) were centriwas harvested and fuged at 20 000 g for 1 h. at 4 “C., the clear supernatant dialysed against Tris buffer, pH 7.2, for 48 h. The resultant colostrum plasma was diluted to approximately 6 g. protein/100 ml. and 1.0 to 1.5 ml. applied to the column. The milk plasma had a lower total protein content, and 3.0 to 3.5

PASSIVE

IMMUNITY

IN THE

FOAL:

NEONATAL

459

PROTEINURIA

ml. were applied to the column on each run. The elution diagrams of colostrum and milk plasma from the mare whose foal was examined in the previous section are given in Fig. 4. In both the colostrum and milk the same peaks I and II were seen as in the serum elution patterns. Peak I was a little larger than that of the serum. In the colostrum Peak II at about 120 ml. was very largg;t and almost identical to the post-colostrum serum Peak II. However, by 24 11. this peak had greatly diminished in size. The Peak III of serum was absent from both the milk and colostrum, but there was an additional peak, Peak IV’, present. The inflection point of this peak occurred at about 200 ml. of the effluent volume and it appeared much larger in the 24 11. milk than in the colostrum sample.

0

50

100

150 Effluent

Fig. 4. El&on pattern of mare’s colostrum Colostrum; ( l - - -0) Milk.

200 volume

(0 h.) and milk

250

1

300

3E IO

(ml.)

(24 h.) through

G-ZOO

Sephades.

(a~-~-~)

Foal urine. Urine samples with high protein content were first centrifuged, concentrated in carbowax to a protein concentration of about 1.5 to 2.0 g./lOO ml. and dialysed against frequent changes of Tris/NaCl buffer, pH 7.2, for 48 11. at 4 “C. Volumes of dialysate of I.0 to 2.0 ml. were applied to the column at each run. The elution diagram of a 12-h. urine sample from foal ex Suna ‘70 is given in Fig. 5 together with the pattern of colostrum. Only 1 significant peak was seen in this and other urine samples examined, which corresponded exactly to Peak IV. There was a little evidence of Peak III from 150 to 170 ml., but virtually none of Peaks I and II. K

460

L. B. JEFFCOTT

2

AND

T. J. JEFFCOTT

“r

I

i i 0 w d 0

’

0

I 150 Effluent

Fig.

5. Elution pattern of foal urine ( l - - -0) Foal urine; (0-O)

200 volume

(12 h.) and mare’s Colostrum.

i0 (ml)

colostrum

(0 h.) through

G-200

Sephadex.

Estimation of molecular size of the various peaks. The approximate molecular weights of the 4 peaks by extrapolation from the graph of log. mol. wt. of the standard proteins against Ve (Fig. 2) has been calculated in Table 1. TABLE APPROXIMATE

Peak I 1:: IV

MOLECULAR WEIGHTS ELUTION VOLUMES IN

Efluent volume Volume (ml.)

70-85 110-140 140-170 180-230

of main Mol.

peak wt. range

600000-1000000 100000-280000 40000-100000 5000-26 000

1 OF PEAKS I-IV BASED G-200 SEPHADEX

ON THEIR

Elution Ve (ml.)

volume Mol.

wt.

75

c. 900 000

120 150 200

200 70 000 000 13 000

Characterization of Urinary Proteins Electrophoresisof serum, colostrum, milk and foal urine. The following fractions were differentiated in serum according to their mobilities at pH 8.6. Albumin ran the farthest distance of all the fractions from the site of application towards the anode as a heavy band. Behind this came the u1 and a2 globulins followed by the PI globulin which did not move significantly from the point of application.

PASSIVE Dam serum

IMMUNITY Foal serum (Oh)

IN THE

FOAL:

Foal serum (24 h.)

Fig. 6. CAM main

Milk (24 h.l

Foal urme (12h.j

Peak II

Peak I!z

in serum,

foal urine

Fig. 7. CAM bovine

strips of colostrum, milk and foal urine in comparison with P-lactoglobulin and equine y globulin. (A-E as in Fig 6)

Fig. 8. CAM

strips

n*

of Peaks I to IV

from

gel filtration

Coloetrum (0 h.)

Mdk (24 h.)

Peak Ill

strips showing the different protein fractions protein fractions of milk and colostrum).

461

PROTEINURIA

Foal urine (I2 h.)

Colostrum (Oh.)

Peak I

NEONATAL

of serum,

milk

and milk. bovine

and urine.

(A-E

(A-E

serum

the live albumin

as in Fig 6)

462

L. B. JEFFCOTT

AND

T. J. JEFFCOTT

The Bz peak was located a short distance towards the cathode and behind this as a wide and rather diffuse band was the y-globulin (Fig. 6). The foal serum taken before the ingestion of colostrum was seen to be completely devoid of any y-globulin, but 24 h. after birth a distinct y-globulin band was demonstrated. In the colostrum (0 h.) a total of 5 main protein fractions were distinguishable and these have been designated A to E according to their electrophoretic mobility. The protein of band D was the predominant fraction with the most intense staining and this corresponded to the y-globulin of serum. This band D also contained some 8-globulin as well. By 24 h. after foaling, the milk had a very different appearance to that of the colostrum. The protein concentration had greatly diminished and the large y-globulin band was almost non-existent (Fig. 6). The electrophoretic appearance of dialysed foal urine, containing a high concentration of protein, was similar to that of the 24-h. milk just described. It showed the presence of fractions, A, B and C, was devoid of D (y-globulin) and showed a faint trace of band E. TABLE PROTEIN

FRACTIONS

DETECTED MILK,

BY CAM NEONATAL

2

ELECTROPHORESIS FOAL SERUM AND

IN MARE URINE

Serum components Albumin Sample

SERUM,

COLOSTRUM,

Milk

comfionents

Globulins Kt

u1

p

y

:l

B

C

D yglob

E

Mare serum Foal serum (0 h.) Foal serum (24 h.) Mare colostrum (0 h.) Mare milk (24 h.) Foal urine (12 h.)

The differences in the electrophoretic pattern of serum, colostrum, milk and foal urine have been summarized in Table 2. The only bands of protein detectable in both serum and colostrum were y-globulin, P-globulin, and possibly some ~1sglobulin. The exact identification of bands, A, B, C and E was not undertaken in this investigation; however, fraction B had a similar mobility to that of bovine 8-lactoglobulin (Fig. 7). Electrophoresis of samples from gel jiltration Peaks I to IV. After gel filtration of colostrum, milk, foal serum and urine, fractions of the effluent volume containing the protein peaks were concentrated and subjected to CAM electrophoresis (Fig. 8). The gel filtration of Peak I obtained from serum, colostrum and milk showed only 1 band which migrated towards the anode into the c(~ globulin region. Peak II from colostrum, milk and post-colostrum serum was composed mainly of B and a very high concentration of y-globulin. In Peak III from foal serum, there was a high content of albumin, but it also contained some CQ, ~1~ and 8i globulin. Peak IV eluted from colostrum, milk and foal urine did not contain any of the serum components and was comprised of fractions, B, C and E.

PASSIVE

IMMUNITY

IN THE

FOAL:

NEONATAL

PROTEINURIA

463

DISCUSSION

The urinary proteins were examined in some detail to determine as far as possible their molecular size and character. It was shown that the bulk of the proteinuria in these foals was composed of low molecular weight proteins (5000 to 26 000) derived from the colostrum, probably j3-lactoglobulin and cc-lactalbumin. In the newborn calf proof that most of the urinary protein was low molecular weight maternal 8-lactoglobulin was provided by the immunological, electrophoretic and ultracentrifuge data of Pierce (1959, 1960) and Pierce and Johnson (1960). They showed that 8-lactoglobulin was absorbed from the gut along with the immune lactoglobulin, but that it was not detrctable in the blood as it is selectively cleared from the circulation, probably by glomerular filtration due to its small molecular size of 35 400 to 40 000. There appears to be a considerable disparity in the molecular size of the urinary proteins in the foal and the calf. However this may be explained by the fact that in the bovine 8-lactoglobulin occurs in the dimeric form (37 000 mol. wt. / although its dissociation into half molecules occurs easily (Andrews, 1964). Kessler and Brew (1970) have shown in porcine milk that 8-lactoglobulin occurs in the monomeric form (18 500 mol. wt.) and possibly this is also the case in thr. horse. In the neonatal piglet immunoelectrophoretic analysis of urine (Martinsson, 1972) revealed three different proteins. Two of these were found to be IgG fragments although no intact IgG was detected. It has been reported by Hardy (1969a,b) that in calves the amount of proteolysis during y-globulin absorption is very small relative to that seen in the piglet. This would also appear to be tht cast for the foal (Jeffcott, 197413). The greater the amount of colostral protein fed in the first 12 h. or so of lifi: the more marked the proteinuria. However the pattern of decline of urinary protein levels was not altered by feeding high concentrations of protein after the cessation of uptake of macromolecules by the small intestine. This has also been noted to occur in the calf (Pierce, 1961b). The absorption of macromolecules by the neonatal intestine of the foal, like other ungulates, is considered to be nonselective, although there may be two routes of uptake according to the molecular size (Jeffcott, 1972). Molecules over 70 000 are taken up by specialized epithelial cells while smaller molecules may pass directly into the intestinal capillaries or by the duodenal cells and hence to the portal circulation. There was no evidence of the smaller molecular size milk protein (5000 to 26 000) detectable in the foal’s serum before or after ingestion of colostrum. Tt is assumed that these milk proteins are absorbed, but are very rapidly cleared from the circulation and excreted in the urine by virtue of their small size. They are well below the renal threshold for proteins which is about 70 000 (Bayliss, Kerridge and Russell, 1933). The large molecules such as IgG antibodies (mol. wt. 160 000) are retained in the foal’s circulation only gradually declining over the ensuing 5 months (Jeffcott, 1974a). It was concluded that the presence of neonatal proteinuria in colostrum fed foals merely reflected the absorption and subsequent excretion of low molecular weight proteins of no immunological \-alue and was not due to a transient permeability or malfunction of the kidney.

464

L.

B. JEFFCOTT

AND

T. J. JEFFCOTT

SUMMARY

A marked but transient proteinuria was detected in all foals that received colostrum in the first 24 h. of life. Initially there were rising levels of urinary protein which reached a peak by 6 to 12 h., followed by sharply declining levels to 24 to 28 h. of life. Feeding high protein colostrum during and after the time of intestinal closure did not prolong the period of proteinuria. Analysis of the urinary protein by CAM electrophoresis showed that it was devoid of obvious serum components. It more closely resembled the protein composition of milk and colostrum, but contained no evidence of y-globulin. On gel filtration it was found to consist almost exclusively of low molecular weight protein. It was concluded that the small molecular weight milk proteins were absorbed by the intestine along with the larger molecules, but were selectively excreted in the urine by virtue of their small size. The cessation of proteinuria was judged therefore to be a reflection of cessation of absorption of macromolecules by the foal’s intestine.

ACKNOWLEDGMENTS

The results presented in this paper were incorporated by one of us (LBJ) into a thesis for the degree of Ph.D. in the University of London. The financial assistance of the Horserace Betting Levy Board is gratefully acknowledged.

REFERENCES

Andrews, P. (1964). Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochemical Journal, 91, 222-233. Andrews, P. (1965). The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochemical Journal, 96, 595-606. Bayliss, L. E., Kerridge, P. M. T., and Russell, D. S. (1933). The excretion of protein by the mammalian kidney. Journal of Physiology, 77, 386-398. Hardy, R. N. (1969a). The breakdown of [ 1311] y globulin in the digestive tract of the newborn pig. Journal of Physiology, 205, 435-45 1. Hardy, R. N. (1969b). Proteolytic activity during the absorption of [1311] y globulin in the newborn calf. Journal of Physiology, 205, 453-470. Howe, P. E. (1924). The relation between the ingestion of colostrum or blood serum and the appearance of globulin and albumin in the blood and urine of the newborn calf. Journal of Experimental Medicine, 39, 3 13-320. Jeffcott, L. B. (1971). Duration of permeability of the intestine to macromolecules in the newly-born foal. Veterinary Record, 88, 340-341. Jeffcott, L. B. (1972). Passive immunity and its transfer with special reference to the horse. Biological Reviews, 47, 439-464. Jeffcott, L. B. (1973). The Mechanism of Transfer of Maternal Immunity to the Foal. In Equine Infectious Diseases III Proceedings of Third International Conference, Paris (1972), 419-435, J. T. Bryans and H. Gerber, Eds. S. Karger, Basel. Jeffcott, L. B. (1974a). Studies on passive immunity in the foal: 1. y globulin and antibody variations associated with the maternal transfer of immunity and the onset of active immunity. Journal of Comparative Pathology, 84, 93-101. Jeffcott, L. B. (1974b). Studies on passive immunity in the foal : II. The absorption of 1251-labelled PVP (polyvinyl pyrrolidone) by the neonatal intestine. Journal of Comparative Pathology, 84, 279-289.

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Kessler, E., and Brew, K. (1970). The whey proteins of pig’s milk isolation and characterization of a B-lactoglobulin. Biochimica et biophysics acta, 200, 449-458. In Chromatographic and Electrophoretic TechKohn, J. (1960). Zone Electrophoresis. niques. Volume II, I. Smith, Ed., William Heinemann, London. Martinsson, K. (1972). Studies on the proteinuria of newborn piglets with special reference to IgG fragments. Acta ueterinaria scandinavica, 13, 87-95. McCarthy, E. F., and McDougall, E. I. (1953). Absorption of immune globulin by the young lamb after ingestion of colostrum. Biochemical Journal, 55, 177-182. Pierce, A. E. (1959). Studies on the proteinuria of the newborn calf. Journal of Physiology, 148, 469-488. Pierce, A. E. (1960). S-lactoglobulins in the urine of the newborn suckled calf Nature, 188, 940-941. Pierce, A. E. (1961a). Proteinuria in the newly born. Proceedings of the Royal SocieLv of Medicine, 54, 996-999. Pierce, A. E. (1961b). Further studies on the proteinuria in the newborn calf. Journal of Physiology, 156, 136-149. Pierce, -4. E. and Johnson, P. (1960). Ultra centrifuge and electrophoretic studies on the proteinuria of the newborn calf. Journal of Hygiene, Cambridge, 58, 247-260. Smith, T. and Little, R. B. (1924). Proteinuria in newborn calves following feeding of colostrum. Journal of Experimental Medicine, 39, 303-312. Whitaker, J. R. (1963). Determination of molecular weights of proteins by gel filtration on Sephadex. Analytical Chemistry, 35, 1950-l 953. Wooton, I. D. P. (1964). Micromethods in Medical Biochemistry. 4th Edit. J. A. Churchill Ltd., London. [Receivedfor publication,

August lst, 19731