Studies on passive immunity in the foal

1. COMP.

279

PATH. 1974.VoL.84.

STUDIES II.

THE

ON

PASSIVE

ABSORPTION PYRROLIDONE)

OF

IMMUNITY 1251-L~~~~~~~ BY THE NEONATAL

IN PVP

THE

FOAL

(POLYVINYL INTESTINE

BY

L. B. JEFFCOTT Equine Research Station, Animal

Health

Trust, h’ewmarket,

Suffolk

INTRODUCTION There is little published work on the mechanism and uptake of macromolecules by newly born foals. In other ungulates, calves and piglets, uptake by the absorptive cells of the small intestine is apparently nonselective (Bangham, Ingram, Roy, Shillam and Terry, 1958; Lecce, 1966 ; Pierce and Smith, 1967; Hardy, Hockaday and Tapp, 1971). The proteins and macromolecules are absorbed without significant digestion within the intestinal cells and retain their biological activity (Pierce, Risdall and Shaw, 1964; Asplund, Grummer and Phillips, 1962). The macromolecule employed in this study for the assessment of uptake by the neonatal intestine was iodinated polyvinyl pyrrolidone, 1251-PVP K.60. This synthetic polymer has a mean molecular weight of 160 000 which approximates that 1965) and has been shown to be well absorbed by the for y globulin (Andrews, intestine of the calf and piglet (Hardy, 1969a,b). A preliminary account of certain aspects of this work has been previously published (Jeffcott, 1971a). MATERIALS

AND

METHODS

Animals. Newly born crossbred pony foals were used in these experiments. Parturition was attended in all cases and the foals muzzled immediately after birth to prevent access to colostrum. They were kept with their dams at all times and were divided into the following 3 groups:Colostrum fedfoals. Eleven foals were muzzled for the first 2 to 3 h. of life and then allowed to suck at will. They were fed one dose of approximately 0.02 mCi labelled ‘2%PVP K.60 in 50 ml. equine colostrum 3 to 20 h. of life. Foals 1 to 4 were dosed at 3 h.; foals 5, 6, 7, 8, 9, 10 and 11 were dosed at 7, 10, 12, 14, 16, 18 and 20 h. respectively. Colostrum deprived foals. Foals 12 and 13 were completely deprived of colostrum for the first 18 and 28 h. They were fed during this time by bottle at hourly intervals 250 to 300 ml. of a mare’s milk substitute (“Equilac”, Pegus Ireland Ltd., Spencer Avenue, Dublin, Ireland). They were dosed at 3, 9, 20, 36 and 48 h. after birth with is5I-PVP K.60 in 2 per cent PVP K.60 mixed with 50 ml. 3 to 4-d. mares milk in place of colostrum. Foal 14. This foal was kept muzzled for the first 30 h. and fed only high protein (20 g./lOO ml.) equine colostrum. The mare’s udder was stripped every hour and the foal allowed to suck at will after 30 h. The colostrum was obtained by pooling samples of mares’ colostrum obtained from a local Thoroughbred stud and from the mares whose foals were colostrum deprived. The foal was dosed once at 15 h. with 125I-PVP K.60 in 2 per cent. PVP K.60 and 50 ml. colostrum. Collection of samples.Five ml. samples of blood were taken in 2 bottles containing heparin as anti-coagulant. Total urine collection in 13 of the foals during the first 24 to 48 h. was carried out. The urine was collected by natural collection into a metal urine collector or by catheterization. This latter procedure was performed on the filly

280

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JEFFCOTT

foals with a large metal bitch catheter, and on the colts with a plastic Tieman’s catheter (size FG12, Portex Ltd., Hythe, Kent). Measurementof radioactivity. The isotope used to assess quantitative uptake by the intestine was lz51-PVP K.60 (polyvinyl pyrrolidone) supplied by the Radiochemical Centre, Amersham. Doses of approximately 0.2 mCi were fed by stomach tube to foals in 2 per cent. PVP (Fluka A. G., Switzerland) and mixed with 50 ml. mare’s colostrum or milk. Radioactivity was measured in a liquid scintillation counter (Packard-Tricarb machine, model 3320) using 1.0 ml. plasma and 0.1 ml. urine aliquots in 0.4 per cent. (w/v) butyl-PBD in 1 :l toluene/Triton X-100 as scintillant (Chapman and Marcroft, 1971). The efficiency of counting was determined by using an internal standard. Haematocrit readings were carried out on all blood samples taken. Uptake into the plasma was then calculated as a percentage of the total dose introduced into the stomach of each foal. Blood volumeestimations.Iodinated human serum albumin (1251-HSA from the Radiochemical Centre) was provided in I.0 ml. vials containing 2.8 to 5.5 pCi. The contents of each vial were diluted to 5.0 ml. in sterile saline and 2 aliquots of 0.1 ml. mixed with the toluene/Triton scintillant were taken for counting. A volume of 4.0 to 4.5 ml. of the diluted albumin was injected i.v. into the foal. Blood samples were taken at 0, 2, 8, 20, 25, 30, 45, 60, 75 and 90 min. after the injection of labelled material. Plasma samples were counted as previously described and haematocrit readings taken on all samples. The logarithmic concentration of labelled albumin was plotted against the time after injection. Extrapolation of the linear portion of the regression line to zero gave the theoretical initial concentration of labelled material from which the total volume of plasma was determined. The foal was weighed at the end of the experiment and the plasma volume in ml./kg. calculated. Gel jiltration. The preparation and use of gel columns was based on the method described by Andrews (1964, 1965). A column of 38.0 x 3.0 cm. of Sephadex G-100 (Pharmacia, Uppsala, Sweden) was used throughout; occasionally the top 1 to 2 cm. of the gel were replaced to ensure even flow through the column. The buffer used was a O-1 M Tris/O.05 M NaCl solution containing 0.065 g. sodium azide/l., adjusted to pH 7.2 with hydrochloric acid. The runs were performed at room temperature, 20&3 “C., at a mean flow rate of 23.0 ml./h. Aliquots of 2.0 to 3.0 ml. of plasma or urine were applied to the column, the urine having been concentrated 7 to 10 times on a rotary evaporator. Column effluents were collected in 3.0 to 6.0 ml. fractions with the aid of a Locarte fraction collector. Aliquots of 1.0 ml. of the effluent fractions were counted in the scintillation counter. The void volume (Vo) of the column was estimated using 1.5 ml. of a 4 g./lOO ml. solution of Blue Dextran 2000 (Pharmacia, Uppsala, Sweden). The elution volumes of PVP polymers of varying molecular weights (11 000 to 36 000) were determined. PVP of molecular weights of 11 000 and 24 500 was obtained from Koch-Light Laboratories; 17 000 to 20 000 and 40 000 from British Oxygen Chemicals; 30 000 to 40 000 from May & Baker Ltd; 44 000 from British Drug Houses; 160 000 from Fluka, Switzerland and 360 000 from Sigma Chemicals. Each PVP solution (25 mg.) was applied to the column and the column effluent read at 225 nm in a Unicam SP 500. RESULTS

Measurementof Absorption of PVP from the Intestine Serial blood samples were taken at hourly intervals from the foals after dosing with labelled PVP and the plasma radioactivity was determined in a scintillation spectrometer. Gel filtration of plasma on Sephadex G-100 revealed that there was virtually no degradation of the 1251-PVP K.60 molecule during absorption by the small intestine. Since it was not practicable to carry out

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plasma volume estimations on the foals immediately after birth, this was done between 2 and 53 d. All the foals were weighed at birth and again when the plasma volume was determined, and haematocrit readings were made on all blood samples. The plasma volume at the time of dosing with PVP was calculated from these results (Table 1). The results ranged from 46.4 to 73.3 ml./kg. for plasma volume and 76.0 to 123.3 ml./kg. for blood volume. TABLE RESULTS

OF BLOOD

AND

PLASMA

1

VOLUME

DETERMINATIONS

Plasma volume at time of 1”51-PVP dosing mi./kg. (by extrapolation)

IN

14

Body weight at birth (k.1

FOALS

Age at which blood volume determined (dqvJ)

---

106.1 123.3 116.3 76.5 76.0 116.2 85.0 80.0 84.5 95.5 77.1 82-l 99.3 85.7

63.5 72.1 64.8 50.7 46.4 73.3 .54.4 55.8 51.5 60.7 50.3 52.3 62.7

48.1

20.9

22.7 29.5 15.9 23.2 30.0 36.4 19.4 18.6 35.5 25.9 24.5 20.5 28.6

2 2 2 6

6 6 1; 16 31 42 46 48 53

The blood radioactivity for each foal was calculated as a percentage of the total dose given. The results of absorption of PVP by the 11 colostrum fed foals is shown in Fig. 1 and a marked reduction in the uptake of PVP can be seen according to the age of the foal. Maximum absorption in foals 1 to 4 dosed at 3 h. was a mean 22.09 per cent., but the uptake in foal 11 after dosing at 20 h. was only 0.93 per cent. of the dose administered.

Hwrs

Fig.

1. Absorption represents

after

feedIng

‘=I-PVP

of 1z51-PVP K.60 from equine colostrum the mean result of foals 1 to 4).

K 60

in foals 1-l 1 (the line of absorption

at 3 h.

The colostrum deprived foals 12 and 13 fed PVP at 3 h. showed a considerably lower percentage absorption than the foals 1 to 4 fed at the same time with colostrum (Fig. 2). Although there was lower absorption, the duration of

282

L.

B.

JEFFCOTT

permeability appeared to be the same as the colostrum fed foals. The foal fed colostrum continuously for 30 h. and dosed with PVP at 15 h. showed maximum absorption of only 1.87 per cent. (Fig. 2), which was less than the peak values recorded for the 16 and 18-h. dosings of 4.35 per cent. and 2.11 per cent. respectively.

Hours

after

feeding

‘251-PVP

K60

Fig. 2. Effect ofcolostrum deprivation and extra colostrum feeding on the absorption of i’sI-PVP K.60 from the intestine of newly born foals. (0) Foals 1 to 4 fed colostrum and dosed at 3 h. (0) Foals 12 and 13 deprived of colostrum and dosed at 3 h. (A) Foal 14 fed colostrum for first 30 h. of life and dosed at 15 h.

Excretion oj’PVP

in the Urine

The excretion by the kidney of 1251-PVP K-60 was examined in foals 1 to 13 dosed between 3 and 20 h. The percentage excretion of the total dose in the first 36 h. was calculated and has been summarized for foals 1 to 11 in Fig. 3. There was a gradual fall from the high values (15.7 per cent.) recorded for the foals dosed at 3 h. to 5 per cent. or under when dosing took place after 16 h. For foals 12 and 13 a mean of 13 per cent. of the total dose was excreted in the urine.

3

10

12 Age

Fig. 3. Percentage 11 foals.

of the total

The renal clearance excreted per h. for all After dosing, there was peak of 1.7 per cent./h. around 12 h. followed

dose administered

14

15

I6

16

20

at d0s1r-q (h)

of i2sI-PVP

K.60

which

was excreted

in the urine

of

of PVP expressed as the percentage of the total dose the colostrum fed foals except foal 5 is shown in Fig. 4. a sharp rise of radioactivity in the urine which reached a at 7 to 9 h. after birth. The level fell fairly steeply to by a more gradual fall to less than 0.2 per cent./h. after

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28 h. A similar pattern of PVP disappearance from the urine was seen in the colostrum deprived foals 12 and 13. Both these foals were dosed 4 times in the first 48 h.

00’

” 0

5

I

I

I

I

IO

20

30

40

Age (h)

K.60 Fig. 4. Pattern of renal clearance of ‘z51-PVP and 20 h. of life (foals 1 to 4 and 6 to 11).

in 10 foals fed approximately

0.02 mCi

between

3

Estimation of Molecular Size of Urinary PVP Plasma samples taken after feeding rz61-PVP K-60 submitted to gel filtration through Sephadex G-100 exhibited a sharp peak of radioactivity in the column effluent just behind the void volume (Fig. 5). However, when samples of urine were put through the column a larger, more diffuse zone of activity was detected in the column effluent with its peak at 131.8 ml. and a secondary peak at the exclusion limit of the gel.

Effluent

Fig. 5. Elution pattern through K.60 at 3 h. of life. (0)

Sephadex Plasma,

wlune

G-100 of plasma (0) Urine.

(ml)

and urine

from

foal 4 dosed with

12sI-P\‘P

In order to estimate approximately the molecular size of this peak of urinary lz51-PVP K.60, a number of PVP solutions of various molecular weights were eluted through the column. Their elution volumes, Ve, representing the maximum concentration of solute in the effluent volume, (Table 2) were interpolated from an elution diagram to the nearest 0.1 ml. by triangulation. All

284

L.

B. JEFFCOTT

PVP molecules of more than 40 000 were eluted between 85.0 and 87.6 ml., a little behind the column void volume of 80.5 ml. PVP solutions of less than 40 000 molecular weight were eluted at increasing Ve, and the smallest polymer examined, approximate molecular weight 11 000, had a Ve of 194.0 ml. (Fig. 6). At the limit of exclusion of the gel, after 250 ml. had been eluted, a second peak of presumably very low molecular size polymer was usually detected. This peak was particularly large in the case of PVP 11 000. From these results it appears that the peak of molecular size of excreted 1251-PVP K-60 in the urine was much lower than that of the plasma and was between 11 000 and 20 000. TABLE2 GEL

FILTRATION

Approx.

OF PVP

mol. wt.

(POLYVINYL SEPHADEX

of PVP

PYRROLIDONE)

Elution

11000 17-20 000 24 500 30-40 000 40 000 44 000 160 000 360 000 ‘251-PVP ‘z51-PVP Void volume

so

IN G-100

volume Ve (ml.)

194.0 100.5 93.5 87.0 85.7 85.0 85.3 87.6 131.8 84.5 80.5

in urine in plasma of column-Vo

150 Effluent

2Kl

250 volume

3co

:

(ml)

Fig. 6. Elution pattern of foal urine through G-100 Sephadex compared with PVP (mol. wt 11 000 and 24 500). (0-O) PVP mol. wt 11 000. (O--O) mol. wt 24 500. (0) Foal urine. DISCUSSION

The maximum absorption of PVP K-60 by the neonatal very soon after birth. This was followed by a continuous and tion in uptake until at 20 h. of life less than 1 per cent. of the stered reached the systemic circulation. There are few other the duration of transfer of macromolecules to new born foals. and Kinkaid (1950) found that absorption of hyperimmune

intestine occurred progressive reductotal dose adminireports concerning Bruner, Doll, Hull Salmonella abortus

STUDIES

ON

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IMMUNITY

IN

THE

FOAL

285

equi antiserum in 2 foals had ceased by 24 to 36 h. Rossdale and Scarnell (1961)

demonstrated that absorption had ceased by 36 h. in a foal which had been muzzled to prevent haemolytic disease. Jeffcott (1971a) found by feeding various hyperimmune antisera to 13 foals after birth that absorption had ceased by 24 h. The importance of colostrum in assisting the absorption of PVP was demonstrated here. The foals deprived of colostrum exhibited a reduced efficiency of absorption although the duration of permeability was not noticeably altered. It would appear, therefore, that the factors that enhance macromolecular absorption from the intestine present in bovine colostrum (Balfour and Comline, 1962; Hardy, 1969a) are also present in mares colostrum. The exact nature of these factors has not yet been determined. They are only effective during the restricted period after birth and accelerate absorption, but are not entirely responsible for it. This point has a practical application if newly born foals with low or inadequate immunity are to be fed a y-globulin preparation or hyperimmune serum at birth to bolster maternal immunity. The addition of some equine colostrum will considerably improve the uptake of such material. The neonatal foal’s intestine has been shown to take up colostral y-globulin and a variety of specific antibodies (Jeffcott, 1971a) in addition to the synthetic polymer, PVP. It seems probable that uptake by the intestine is nonselective as occurs in the calf (Bangham et al., 1958; Hardy, 1969a) and the piglet (Hardy et al., 1971). Th e selection of suitable immune proteins in ungulates takes place not at the site of absorption but in the mammary gland ( Lascelles, 1963; Pierce and Feinstein, 1965). This would mean that immediately after birth the foals intestinal cells will absorb any large molecule presented to it. If a foal, whose dam has started lactating before parturition and run away ller colostrum, is to be fed supplementary colostrum then this should obviously be given before the foal receives any milk or other feed. The question whethei starvation significantly prolongs the duration of permeability was not investigated here. In the unsuckled piglet absorption continues until death up to 106 1~. (Payne and Marsh, 1962a,b) although the efficiency falls off with timr (Hardy, 1969b), but this is apparently not the case in the calf (Morris, 1968). The efficiency of macromolecular absorption in the foal compares well with figures quoted for the calf (Balfour and Comline, 1962 ; Hardy, 1969a; Kruse, 1970). In the piglet a rather lower percentage of uptake is recorded of about 13 per cent. of the total dose given. McEwan, Fisher and Sclman (1970) point out that for a number of reasons these observed efficiencies are probably considerably lower than the actual values. The most important factor is that only the intravascular component has been measured and no allowance made for the cxtravascular pool of the macromolecules. In the new born calf MacDougall and Mulligan (1969) found an average ratio of 1*2/l .O jextravascular/intra\,ascular) for IgG and in the foal Reilly and MacDougall (1973) using 1?51labelled TgG found the ratio was l*O/l*O (extravascular/intravascular). Extrapolating from these figures would have the effect of doubling the calculated results presented here of absorptive efficiency. The actual mechanism of absorption of macromolecules is very complex, but has been examined in the foal by ,Jeffcott (1971b) and is essentially similar to

286

L.

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JEFFCOTT

that seen in a variety of other species (Clarke and Hardy, 1969a,b, 1970, 1971a,b; Hardy et al., 1971). The uptake of macromolecules from the intestinal lumen occurs by pinocytosis. The resulting microglobules of material move deeper into the cell where they merge with one another to form larger globules. These in turn coalesce to form one or two very large vacuoles at the base of the cells. Finally they discharge their contents into the intercellular space from where they pass to the local lacteals and then on to the systemic circulation. Uptake by these specialized cells appears to follow the all-or-none law of physiological activity in that they absorb protein maximally before passing any into the lymphatics. The process of cessation of macromolecular absorption (closure) is also complex and far from being completely understood. There appears to be a variety of factors involved including the adrenal cortical hormones (Jeffcott, 1972). Clarke and Hardy, (1969a,b, 1970, 1971a,b) have shown that a number of species can take up macromolecules into the intestinal cells after 24 to 36 h., but are no longer able to transfer them out of the cells into the systemic circulation. In foetal lambs (Simpson-Morgan and Smeaton, 1972) it has been shown that there was no sign of cessation of absorption of intact proteins. There is only a very slow turnover of the villous intestinal cells before birth (Smeaton, 1972) whereas immediately after birth the turnover time was as short as 72 h. This is very similar to the epithelial turnover time recorded in young rats by Clarke and Hardy (196913). It would appear that the ability to absorb large molecules after birth is dependent on the existence of specialized epithelial cells lining the intestinal villi. These are rapidly replaced by more mature-looking cells unable to take up macromolecules or discharge them in the intercellular space. This would explain why absorption is maximal immediately after birth and falls off in a linear fashion to complete cessation. In the foal fed colostrum continuously for 30 h. from birth closure occurred earlier than in the other groups. This may have been due to the large amounts of colostral protein absorbed from the intestine before the dosing of PVP at 15 h. leaving relatively few cells capable of taking up PVP. Although PVP K-60 is a synthetic polymer with a mean molecular weight of 160 000, it does contain a fairly wide range of molecular sizes. It was shown by gel filtration that there was no detectable degradation of the molecule during absorption by the gut and that the larger size molecules were retained in the plasma. However a peak of PVP excretion was seen in the urine at 9 h. after birth and this was shown to contain only small molecular weight components, 11 000 to 20 000, and by 28 h. of life very little radioactivity was detectable in the urine at all. The excretion of PVP followed an almost identical pattern to that of proteinuria seen in colostrum fed foals (Jeffcott and Jeffcott, 1974). It was judged to be a reflection of closure of the intestine to macromolecules and to be one further piece of evidence that this process is a gradual one and completed soon after 24 h. of life in the foal. SUMMARY

An iodinated marker, lz51-PVP K-60, (polyvinyl pyrrolidone) of mean molecular weight, 160 000, was shown to be well absorbed from the intestine

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of the newly born foal. The absorption was maximal (22 per cent.) 3 h. after birth, but fell rapidly in a linear decline to less than 1 per cent. by 20 h. of life. There was reduced absorption (10 per cent.) when the foals were deprived of colostrum although the time of cessation of uptake did not alter. The figures for the percentage absorption as only

absorption intravascular

represented

only

the

apparent

efficiency

of

PVP was measured. The real efficiency would doubled taking into account the extravascular

be expected to be approximately pool of PVP. PVP introduced into the stomach of newly born foals reached peak levels ot radioactivity 6 h. after administration. There was apparently no degradation of the PVP molecule during the process of uptake and transfer to the systemic circulation. However high levels of PVP were excreted in the urine up to 9 11. after

birth.

After

this time

the levels

declined

until

by 28 h. very little

radio-

activity was detectable in the urine. The molecular size of the urinary PVP was 11 000 to 20 000. ACKNOWLEDGMENTS

I wish to thank Dr R. N. Hardy for his most valuable advice on this project and foi supplies of lssI-PVP K.60. The results presented in this paper were incorporated in a thesis for the degree of Ph.D., in the University of London. I gratefully acknowledge the help of my colleagues at the Equine Research Station particularly Dr D. I. Chapman for his assistance with the method of measuring radioactivity, to Miss Katherine Whitwell B.V.Sc., M.R.C.V.S., for her help and advice and to Mrs T. J. Jeffcott for her technical assistance. My thanks are also due to the National Stud, Newmarket for supplies of equine colostrum and to British Oxygen Chemicals for supplies of PVP. REFERENCES

Andrews, P. ( 1964). Estimation of the molecular weights of proteins by Sephadex gelfiltration. 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. Asplund, J. M., Grummer, R. H., and Phillips, P. H. (1962). Absorption of colostra1 gamma-globulins and insulin by the newborn pig. Journal of Animal Science. 21. 412-413. R. S. (1962 j. Acceleration of absorption of unchanged Balfilur, W. E., and Comline, globulin in the newborn calf by factors in colostrum. Journal of P/p.riolocg. London, 160, 234-257. Hangham, D. R., Ingram, P. L., Roy, J. H. B., Shillam, K. W. G.. and Terry, R. J. (1958). The absorption of 1311-labelled serum and colostral proteins from the gur of the young calf. Proceedings of the Royal Society, London, B, 149, 184-191. Hruner, D. W., Doll, E. R., Hull, F. E., and Kinkaid, A. S. (1950). Further studies on haemolytic icterus in foals. American Journal of Veterinary Research, 11, 22-25. Chapman. D. I., and Marcroft, J. (1971). The use of Triton X-100 in the liquid scintillation counting of carbon-14 with particular reference to plasma and urine. International Journal of Applied Radiation and Isotopes, 22, 371-377. Clarke, R. M., and Hardy, R. N. (1969a). The use of 1251-polyvinyl pyrrolidont. K-60 in the quantitative assessment of the uptake of macromolecular substance\ by the intestine of the young rat. Journal of Physiology, London, 204, 113-I 25. Clarke, R. M., and Hardy, R. N. (1969b). An analysis of the mechanism of cessation of uptake of macromolecular substances by the intestine of the young rat (‘Closure’). Journal of Physiology, London, 204, 127- 134.

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Clarke, R. M., and Hardy, R. N. (1970). Structural changes in the small intestine associated with the uptake of polyvinyl pyrrolidone by the young ferret, rabbit, guinea-pig, cat and chicken. Journal of Physiology, London, 209, 669-687. Clarke, R. M., and Hardy, R. N. (1971a). Histological changes in the small intestine of the young pig and their relation to macromolecular uptake. Journal of Anatomy,

108,63-77. Clarke, R. M., and Hardy, R. N. ( 1971 b). Structural changes and the uptake of polyvinyl pyrrolidone in the small intestine of the young goat. Journal of Anatomy, 108, 79-87. Hardy, R. N. (1969a). The influence of specific chemical factors in the solvent on the absorption of macromolecular substances from the small intestine of the newborn calf. Journal of Physiology, London, 204, 607-632. Hardy, R. N. ( 196913). The absorption of polyvinyl pyrrolidone in the newborn pig intestine. Journal of Physiology, London, 204, 633-65 1. Hardy, R. N., Hockaday, A. R., and Tapp, R. L. (1971). Observations on the structure of the small intestine in foetal, neonatal and suckling pigs. Philosophical Transactions of the Royal Society, London, B, 259, 5 17-53 1. Jeffcott, L. B. (1971a). Duration of permeability of the intestine to macromolecules in the newly-born foal. Veterinary Record, 88, 340-341. Jeffcott, L. B. (1971 b). Perinatal studies in Equidae with special reference to passive transfer of immunity. Ph.D. Thesis, University of London. Jeffcott, L. B. (1972). Passive immunity and its transfer with special reference to the horse. Biological Reviews, 47, 439-464. Jeffcott, L. B., and Jeffcott, T. J. (1974). Studies on passive immunity in the foal. III. The characterisation and significance of neonatal proteinuria. Journal of Comparative Pathology, 84, in press. Kruse, V. (1970). A note on the estimation by simulation technique of the optimal colostrum dose and feeding time at first feeding after calf’s birth. Animal Production, 12, 661-664. Lascelles, A. K. (1963). A review of the literature on some aspects of immune milk. Dairy Science Abstracts, 25, 359-364. Lecce, J. G. (1966). Absorption of macromolecules by neonatal intestine. Biologia neonatorum, 9, 50-6 1. MacDougall, D. F., and Mulligan, W. ( 1969). The distribution and metabolism of fast IgG immunoglobulin in the neonatal calf. Journal of Physiology, London, 201, 77-78P. McEwan, A. D., Fisher, E. W., and Selman, I. E. (1970). An estimation of the efficiency of absorption of immune globulins from colostrum by newborn calves. Research in Veterinary Science, 11, 239-243. Morris, I. G. (1968). In Handbook of Physiology-Alimentary Canal (C. F. Code, Ed), sect. 6, vol. 3, pp. 1491-1512. Baltimore: American Physiological Society. absorption in the baby Payne, L. C., and Marsh, C. L. (1962a). G amma globulin pig: The nonselective absorption of heterologous globulins and factors influencing absorption time. Journal of .Nutrition, 76, 151-158. Payne, L. C., and Marsh, C. L. (1969b). Absorption of gamma globulin by the small intestine. Federation Proceedings, 21, 909-912. Pierce, A. E., and Feinstein, A. (1965). Biophysical and immunological studies on bovine immune globulins with evidence for selective transport within the mammary gland from maternal plasma to colostrum. Immunology, 8, 106-123. Pierce, A. E., Risdall, P. C., and Shaw, B. (1964). The absorption of orally administered insulin by the newly born calf. Journal of Physiology, London, 171, 203-2 15. Pierce, A. E., and Smith, M. W. (1967). The intestinal absorption of pig and bovine immune lactoglobulin and human serum albumin by the newborn pig. Journal of Physiology, London, 190, 1-18. Reilly, W. J., and MacDougall, D. F. (1973). The metabolism of IgG in the newborn foal, Research in Veterinary Science, 14, 136-137.

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Rossdale, P. D., and Scarnell, J. (1961). Immunisation of the new-born foal against tetanus. Veterinary Record, 73, 184185. Simpson-Morgan, M. W., and Smeaton, T. C. (1972). The transfer of antibodies by neonates and adults. Advances in Veterinary Science and Comparative Medicine, 16, 355-386. Smeaton, T. C. (1972). Quoted by Simpson-Morgan and Smeaton. Advances in Veterinaty Science and Comparative Medicine, 16, 355-386, [Receivedforpublication,

August lst, 19731