The transfer of copper from ewes with Border Disease to their lambs

j. COMP.

PATH. 1979.

THE

Vot.

361

89.

TRANSFER BORDER

OF COPPER DISEASE TO

FROM THEIR

EWES LAMBS

WITH

B)

B. F. SANSOM,

H. W.

Agricultural

Council, .Newbury.

Research Compton,

SYMOISDS

and P. J.

Institute for Research on Animal Berkshire RG16 O.,VJV~, U.h-.

TAYLOR Diseases,

INTRODUCTION

Earlier studies on Border Disease in sheep have described its pathological effects on the central nervous system of neonatal lambs, and changes in copper metabolism. The copper concentration in the cerebrum of affected lambs was reduced, while that in the spinal cordwas increased (Patterson, Brush, Foulkes and Sweasey, 1974), and affected lambs were hypocupraemic (Patterson and Sweasey, 1973; Patterson et al., 1974). It was therefore decided to measure the transfer of copper across the placenta of sheep affected by Border Disease and its uptake into the tissues of the affected lambs, by means of radioactive copper-64. The work was undertaken as part of a longer term study of those lambs which survived with Border Disease, the results of which will be published separately. MATERIALS

AND

METHODS

Sheep. Sixty Dorset Horn gimmers were mated in late August with two Dorset Horn rams; at approximately 30 days gestation they were moved from the Central Veterinary Laboratory, Weybridge to Worcester Veterinary Investigation Centre and at 40 days gestation 30 were injected intramuscularly with 5 ml of a Border Disease virus tissue culture suspension. Subsequently 6 injected and 6 control ewes were selected so that their expected parturition dates (for a gestation period of 145 days) lay between January 20th and 24th 1977 and on January 3rd they were moved to the Institute for Research on Animal Diseases at Compton where they were housed in separate loose boxes. At all three laboratories they were fed hay and concentrates which were each derived from a single source. One of the control ewes lambed before it could be injected with 64Cu. &p/~-64. 64CuC1, was obtained from the Radiochemical Centre, Amersham. Three control and 3 infected sheep were dosed on January 18th when the specific activity was 20 mCi per mg copper, and 2 control and 3 infected sheepwere dosedon January 19th when the specific activity was 5.4 mCi per mg copper. Experimental procedure. Each sheep received an injection into a jugular vein of approximately 850 uCi 64Cu in 3 ml sterile saline. Blood samples were taken from the other jugular vein approximately 5, 30 and 90 min, and 3, 6, 10 and 24 h after the injection and placed in heparinized bottles. The plasma was separated and 64Cu and Cu determined. Immediately after the last blood sample had been taken each sheep was killed with sodium pentobarbitone (Trinaven) and its uterus removed as rapidly as possible. The lambs were removed alive and their radioactivity measured in a whole-body counter. The uterus and its fluid contents were also retained for determination of 64Cu by whole-body counting. A blood sample was taken from the 0021-9975/79/030361+06

$02.00/O

10 1979 Academic

Press Inc.

(London)

Limited

362

B. F. SANSOM

et

cd.

lambs which were then killed by i.v. injection of Trinaven and liver, brain and spinal cord removed. Whole-body cciunting. The 64Cu in the lambs, the uteri and contents and in the lambs’ livers was determined in the Compton whole-body counter (Sansom, Taylor, Wheelock and Vagg, 1971) by comparison with known fractions of the dose solution prepared in “phantoms” of suitable size. Variations in the efficiency of counting with variations in weight (from approximately 50 g for the liver, to nearly 10 kg in the largest lambs and uteri) were less than 10 per cent. Other measurements. The brains and spinal cords were dried on filter paper and weighed. Representative samples of the cerebrum, weighing approximately 15 g, and of the spinal cord, weighing approximately 4 g, were placed in polyethylene vials and their 64Cu content was measured. Subsequently the dry matter content of these samples was determined by taking 5 g of the cerebrum and the whole sample of spinal cord and drying them at 100 “C overnight before re-weighing. Tests on a few samples showed that longer periods of drying did not cause further losses of weight. Blood plasma and tissue copper concentrations were determined by atomic absorption spectrophotometry. Radioactivity determinations and calculations. The samples of plasma, cerebrum and spinal cord were counted in an NE831 1 y-spectrometer. Corrections were made for radioactive decay by interposiqg a standard solution of 64Cu between each group of four samples, and all counts were performed with a probable error of less than 1 per cent. For each ewe the specific radioactivities of blood plasma Cu were calculated as per cent dose per mg Cu in the seven samples taken between 5 min and 24 h after intravenous injection. The mean specific activities of the blood plasma Cu during the 24 h of the experiment were then calculated by the method described by Twardock, Symonds, Sansom and Rowlands ( 1973) except for the following modification which was necessary in the calculations for 4 (2 control and 2 infected) ewes. In these individuals there was a small increase in the specific radioactivity of plasma Cu between 10 and 24 h, instead of a small decrease, and it was assumed that the specific radioactivity of unbound plasma Cu decreased between 10 and 24 h at the mean rate of decline of plasma copper specific radioactivity in the remaining ewes (see discussion). Cu transfer from the dam to the foetus or to foetal tissues was calculated as:

mean

specific

per cent of dose of 64Cu in foetus or tissue activity of maternal plasma 64Cu (per cent dose per mg). RESULTS

The mean specific radioactivities of copper in the ewes’ plasma were mean&s.e.m.) and 1*801&0.188 per cent dose per mg Cu for 2.035 10.239 ( the 5 control ewes and the 6 infected ewes respectively. The difference is not significant. Table 1 shows the mean percentages of the dose of 64Cu transferred to the lambs, to the lambs’ livers and to the uteri and their contents together with the calculated transfers of Cu. None of the differences is significant. Table 2 shows the mean fresh weights, dry matter contents, copper concentrations and total copper contents of the cerebrums and spinal cords of the 8 lambs from control ewes and 9 lambs from infected ewes. The cerebrums and spinal cords of lambs from control ewes were heavier (P < O-05; P < 0.01 respectively) than those of lambs from infected ewes, and they contained a higher proportion of dry matter (P < 0.001). However, the dry matter of

COPPER

TRANSPORT

IN BORDER

363

DISEASE

tissues from control lambs contained lower concentrations of copper than tissues from infected lambs (I’ < O-01) with the result that there was no difference between the total Cu content of the tissues of control lambs and lambs infected with Border Disease. The cerebrums of both control and infected lambs contained higher concentrations of copper than the spinal cords. TABLE 1 THE

MEAN

(f

SEM) @%U AND INFECTED WITH

THE CALCULATED CU TRANSFERRED FROM 5 CONTROL BORDER DISEASE T O THE LAMBS, T O THE LAMBS’ LIVERS T O THE UTERI AND THEIR CONTENTS

Tissue

LambsWh (per cent dose) cu (I%) Lambs’ liversYh (per cent dose) 1I !+y? .-.. “Tu (per cent dose) al (I%) No significant

Controls (8)

Infected (9)

1.73kO.15 895k 151

1.64kO.21 968+ 177

-, 0.88kO.14 467 f 53 3.51 + 0.47 1737* 173

SHEEP AND

AND

6

SHEEP

0.92+0.13 549*114 2.94 * 0.09 1698,132

differences.

TABLE 2 THE

MEAN (+SEM) CEREBRUM

FRESH WEIGHT, DRY MATTER, COPPER CONCENTRATION AND SPINAL CORD TISSUE OF 8 LAMBS FROM CONTROL SHEEP SHEEP INFECTED WITH BORDER DISEASE

Tissue

corltrols

Cerebrumweight (g) dry matter (mg per g) cu (I*!+ per g) total cu (pg) Spinal cordweight (g) dry matter (mg per g) Cu (w per g) total Cu (Kg)

* P i

0.05.

t P i: 0.01.

AND TOTAL AND 9 LAMBS

(8)

Infected (9)

+ 1.13 +2.0 + 0.65 k4.3

37.4 +0.55* 149.1 + 2.0: 18.26+ 1.85t 103.5 +9.1

9.8 kO.33 238.0 k3.5 8.21+ 1.07 18.9 +2.3

7.9 *0.34f 186.8 *3.5: 11.69+ 1.09t 16.73 + 1.02

40.6 158.0 14.40 88.9

COPPER FROM

OF

:: P < 0.001.

Table 3 shows the mean total 64Cu in the cerebrums and spinal cords of the control and infected iambs, and also the calculated total transfer of Cu to these tissues. There were no differences between infected and control sheep in .either of these measurements. Table 4 shows the percentage of the total copper in cerebrum and spinal cord which was transferred to these tissues during the 24 h of the experiment. These

364

B.

F. SANSOM

et al.

values have been calculated from the ratio of the Cu transferred to the total Cu in the tissues. They show that there was no difference between control and infected lambs, but that a higher proportion of spinal cord copper was deposited during 24 h than of cerebrum copper. TAI3I.E 3 THE MEAN ( + SEM) TOTAL 9*c:u AND THE MEAN CALCLmATED SHEEP

AND

SHEEP

INFECTEP

\VITM

DURING TIIE 24

Cerebrum Total “Yh (per Total Ch (I*g) Spinal cordTotal Wh (per Total Ch (fog!

BORDER H AFTER

TOTAL COPPER TRANSFERRED

DISEASE T O THE CEREBRUMS AND A SINGLE INTRAVENOUS INJECTION

SPINAL

CORDS

FROM CONTROL OF THEIR

LAMBS

OF 64~uc1,

ContYols (8)

Infected (9)

cent

dose

Y 103)

4.19+0.23 2.15+0.30

3.80 + 0.39 2.22 + 0.29

cent

dose

7 103)

1.39+0.10 0.72+0.12

0.73 + 0.076

1.28kO.13

4

TABLE

THE MEAN (f SEM) COPPER TRANSFERRED FROM CONTROL SHEEP AND SHEEP INFECTED WITH BORDER DISEASE T O THE CEREBRUMS AND SPINAL CORDS OF THEIR LAMBS DURING 24 H AFTER A SINGLE INTRAVENOUS INJECTION OF “‘&cl, EXPRESSE” AS A PERCENTAGE OF THE TOTAL COPPER IN THE TISSUES

Tis’isJur

Controls

Cerebrum (per cent) Spinal cord (per cent!

* P <

0.001

between

2.41

(8)

2.39 + 0.25 4.40 + 0.40*

kO.31

4.ok%-&o.77*

cerebrum

and

spinal

cord.

DISCUSSION

In order to measure the transfer of Cu from the ewe to her lamb, it has been assumed that Cu was transferred across the placenta in the same form as the 64Cu in the ewe’s plasma. However, there may have been other components of 64Cu which could not be transferred to the foetus and there may have plasma been components of plasma Cu which were transferred to the foetus but which did not become labelled with 64Cu. The significance of any differences observed in this experiment between the transfer rates of 64Cu or Cu to the foetuses of control ewes or ewes infected with Border Disease depends upon the assumption either that these components were present in negligible quantities or that their relative quantities were not affected by Border Disease. The 64Cu was injected as 64CuC1,. Most of the 64Cu probably remained in the ionic form, and its rapid disappearance from the plasma was due partly to its uptake into maternal tissues and partly to its transfer to the foetus. However, in four of the ewes (2 controls and 2 infected) between 10 and 24 h after

COPPER

TRANSPORT

IN BORDER

DISEASE

365

injection, the plasma 64Cu concentration increased slightly. This increase was presumably due to the secretion into the plasma of 64Cu incorporated previously, at a higher specific radioactivity, into an organic constituent of plasmaprobably ceruloplasmin-and this protein was unlikely to have been transferred across the placenta. Thus in the later stages of the experiment there was WIu in the plasma of at least 4 ewes in a form which was not transferable to the foetus and its tissues. An approximate correction for the resulting over-estimate of the mean specific radioactivity of these 4 ewes’ plasma Cu has been made by applying to them the mean rate of decline in 64Cu specific radioactivity between 10 and 24 h derived from the other 7 ewes. Evidence that the calculated values of Cu transferred to tissues (Tables 1 and 3) are reasonable is provided by Table 4 in which the amount of copper transferred during the 24 h of the experiment has been expressed as a percentage of the total copper in the tissues. Between 2 and 3 per cent of cerebrum copper and between 3 and 5 per cent of spinal cord copper was laid down in these tissues irrespective of whether the ewes were normal or infected with Border Disease. Approximately 80 pey cent of spinal cord copper is located in myelin and according to Patterson, Sweasey and Hebert (1971) the first phase of myelination begins in the CNS about 20 days before birth. The apparent daily rate of deposition of approximately 4 per cent of spinal cord copper is in good agreement with these data. There was a significantly lower rate of copper deposition in the cerebrum, but the reason for this is unknown. The copper concentration in the dry matter of the spinal cords of infected lambs was significantly greater (P < 0.01) than in control lambs, in agreement with earlier observations (Patterson and Sweasey, 1975; Patterson et al., 1974), but the total Cu contents of the spinal cords were similar. However, in contrast with the data obtained from our experimentally infected lambs in which the cerebrum copper concentrations were greater, Patterson et al. (1974) observed lower concentrations of Cu in the cerebrums of field cases of Border Disease. The concentrations of ‘j4Cu in the dry matter of spinal cord and cerebrums of infected lambs were also greater than in controls (P < 0.05 for spinal cord) and there was a greater calculated transfer of copper per unit dry matter (P < 0.01 for spinal cord) (Table 3). However, the total copper contents of spinal cords and cerebrums of infected lambs were not different from the controls, and the derived data in Table 4 suggest that the proportional rates of deposition of copper were similar in both groups. Table 4 shows that a greater proportion of total spinal cord Cu than of cerebrum Cu was transferred during the 24 h of the experiment. The transfer of Cu to the spinal cord is in accord with earlier data, as mentioned above, and it is therefore probable that the pattern of Cu deposition in the cerebrum is quite different from that in the spinal cord. The results of these experiments suggest that infection with Border Disease has no effect on either the rate of transfer of Cu from the ewe to her lamb, or on the distribution of Cu in the central nervous system of infected lambs. Pifferences between control and infected lambs in the quantities of Cu, 64Cu and Cu transferred during 24 h, in the cerebrums and spinal cords, were due to the smaller weights and dry matter contents of the tissues of infected lambs. I3

366

B. F. SANSOM

et al.

SUMMARY

The transfer of ‘j4Cu and copper to their lambs was measured in late pregnancy in normal ewes and in ewes infected with Border Disease. The differences previously observed between copper concentrations in the central nervous

systems

of normal

and infected

lambs

were

confirmed,

but

there

were

no differences between the rates of transfer of 64Cu or copper from ewes to lambs.

The

different

copper

concentrations

were

due

to the smaller

weights

and dry matter contents of the tissuesof infected lambs. Evidence was obtained that the time-course of deposition of copper in the cerebrum was different from that in the spinal

cord. ACKNOWLEDGMENTS

We thank D. Sweasey and D. S. P. Patterson of the Central Veterinary Laboratory, Weybridge, and staff of the Worcester Veterinary Investigation Centre, for assistance during the organisation of the experiment and for valuable discussions. REFERENCES

Patterson, D. S. P., Brush, P. J., Foulkes, J. A., and Sweasey, D. (1974). Copper metabolism and the composition of wool in Border Disease. Veterinary Record, 95, 214-215. Patterson, D. S. P., and Sweasey, D. (1973). Hypocupraemia in experimental Border Disease. Veterinary Record, 93, 484-485. Patterson, D. S. P., and Sweasey, D. (1975). Possible annular distribution of copper in the myelin of spinal cord nerves with special reference to the sheep. Biochemical Society Transactions, 3, 118-l 19. Patterson, D. S. P., Sweasey, D., and Hebert, C. N. (1971). Changes occurring in the chemical composition of the central nervous system during foetal and postnatal development of the sheep. Journal of Neurochemistry, 18, 2027-2040. Sansom, B. F., Taylor, P. J., Wheelock, D., and Vagg, M. J. (1971). A scanning whole-body counter for cattle. In Mineral Studies with Isotopes in Domestic Animals, I.A.E.A., Vienna, pp. 125-133. Twardock, A. R., Symonds, H. W., Sansom, B. F., and Rowlands, G. J. (1973). The effect of litter size upon foetal growth rate and the placental transfer of calcium and phosphorus in superovulated Scottish half-bred ewes. British Journal of Nutrition, 29, 437-446. [Received for publication,

September 14/h, 19781