A simple method for the demonstration of factors in bovine colostrum capable of causing anaemia in lambs reared free from maedi on bovine colostrum

Veterinary Immunology and Immunopathology, 10 (1985) 297--303 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

297

A SIMPLE METHOD FOR THE DEMONSTRATION OF FACTORS IN BOVINE COLOSTRUM CAPABLE OF CAUSING ANAEMIA IN LAMBS REARED FREE FROM MAEDI ON BOVINE COLOSTRUM

W.E. BERNADINA 1 and P. FRANKEN 2 1 Faculty of Veterinary Medicine, Department of Immunology, Yalelaan i, 3584 CL Utrecht 2

(The Netherlands)

Veterinary Health Service, Gelderland (The Netherlands)

(Accepted 20 June 1985)

ABSTRACT Bernadina, W.E. and Franken P., 1985. A simple method for the demonstration of factors in bovine colostrum capable of causing anaemia in lambs reared free from maedi on bovine colostrum. Vet. Immunol. Immunopathol., i0: 297-303. Previous studies demonstrated that the anaemia encountered in lambs reared on bovine colostrum and a milk substitute was associated with the presence of immune complexes on lamb erythrocytes. In the present study the usefulness of a panel of 20 sheep sera for the detection of "anti-sheep" factors in bovine colostrum by double im~unodiffusion in agarose was investigated. Utilising this method, 353 batches of bovine colostrum have been examined, 132 of which were declared safe for use in the rearing of lambs. When fed to lambs, only 2 samples (1.5%) caused anaemia as compared with up to 20% before this test was introduced. Experiments designed to determine whether the bovine colostra, declared anaemia-prone, would indeed cause anaemia when fed to lambs, showed our method to fully discriminate between safe and unsafe colostra for the rearing of lambs. In a follow-up collaborative study, set up to cover most of the Netherlands, the general validity of the test system described was demonstrated, using 114 batches of safe colostrum to feed 723 lambs. Further experiments are needed to determine the exact nature of the factor(s) involved in this phenomenon.

INTRODUCTION

"Zwoegerziekte", Netherlands,

a

progressive

interstitial

pneumonia

of

sheep

in

the

accounts for substantial financial losses for our sheep-farmers.

The disease is caused by a persistent infection with a maedi/visna virus

(De

Boer, 1975). Studies on the epidemiology of the "zwoegerziekte" (= maedi) have disclosed that transmission of the virus via the ingestion of colostrum/milk from infected ewes is the most

0165-2427/85/$03.30

important way of

infection

© 1985 Elsevier Science Publishers B.V.

(De Boer et al.,

298 1979;

Houwers,

1980).

"zwoegerziekte"

is

Hence,

the

advocated

following

in

the

eradication

Netherlands.

programme

Ewes

with

a

for

persistent

infection with a maedi/visna virus are traced by serological methods 1970;

Houwers

separated

et

al.,

immediately

1982). from

The

the

lambs

dams

at

born

to

birth

these

and

(De Boer

infected

reared

the

ewes

artificially

are in

isolation on bovine colostrum and a milk substitute. Bovine colostrum appears to be a good alternative source of immunoglobulins for lambs

(Logan et al.,

1978; Kessels, 1980; Franken and Elving, 1982). However, in some cases severe anaemia

(mostly non-haemolytic)

(Franken et

al.,

1982).

We

developed

have

in

shown that

lambs

fed

anaemia

on bovine

is

colostrum

associated with

the

presence of immune complexes (IC) on lamb erythrocytes (unpublished data). The question

arose

whether

a

simple

method

could

be developed to distinguish ! between safe and unsafe colostra for rearing lambs free from maedi. A suitable test has in fact been developed and this has already been used routinely in the field. The test which has been described briefly 1983)

is

bovine

based

colostrum

details of

on

the

and

presence

a panel

the procedure

or

of

absence

20

sheep

of

(Bernadina and Franken,

immunoprecipitation

sera.

The

present

and also presents results of

between

paper

reports

field trials

carried

out with colostra declared safe.

MATERIALS AND METHODS

Sera

Serum random

samples were

taken

from five different

from

20

flocks by

adult the

sheep

(Texel breed)

Regional Animal

selected

at

Health Service

of

Gelderland and sent to our laboratory. The sera were stored in aliquots at -20 °C and specimens were thawed only once before testing and discarded.

Bovine colostra Colostrum samples

(two samples of

ca

i0 ml

per

cow)

milking post-partem were collected by the participants

taken

at

the

first

(i.e. Regional Animal

Health Services of Gelderland, West-Nederland and Limburg

and veterinarians

practising in the northern provinces of the Netherlands and in Belgium)

and

sent to our laboratory. All samples not used immediately for whey production and subsequent use in the assay to be described below were stored at -20 °C until required. The colostra used in this study were collected in two periods. At

the

test)

first

collection

(1981/1982;

colostrum samples were

taken

introduction

of

the

gel

from 353 cows.

At

the

second

precipitation collection

299 (1983/1984;

evaluation of the gel precipitation

test)

colostrum

samples were

taken from 234 cows.

Preparation of cow colostral whey For each cow one sample of colostrum was thawed and whey was prepared using a

slightly

modified

casein were

method

of

Reyero

and

removed by centrifugation

Stoekl

(1977).

In brief,

at 45000 g. for 30 minutes

fat

and

at 4 °C. A

solution of 10% (w/v) dextran sulphate 500 (sodium salt, Pharmacia, Sweden) was added

(0.02 ml/ml sample) and stirred at room temperature for 30 minutes. Then

3 M CaCl 2 (0.i ml/ml sample) room temperature The supernatant liter)

was added and stirred for another 30 minutes at

followed by centrifugation (whey) was dialysed

first against 0.9% NaCI/EDTA

for 24 h and overnight against PBS

at 4 °C. After

dialysation

(4 °C) at 15000 g for I0 minutes.

the whey

was

(phosphate-buffered concentrated

to

(2 g EDTA/-

saline, pH 7.2)

starting volume

to

perform the precipitation test.

Gel precipitation tests Analysis

by

(Indubiose A37,

the

(GPT)

double

diffusion

technique

was

l'Industrie Biologique Francaise)

performed

in

1%

agarose

in barbitone acetate buffer

pH 8.2. Eleven ml gel was poured over an 8 x 8 cm glass plate. Troughs long)

flanked by two rows of ten wells

(6 cm

(~ 3 mm) at a distance of 4-6 mm from

the trough were cut. One hundred and fifty ~i

(150 91)

of undiluted whey

(or

an equivalent volume of diluted whey) and 8-10 ~i of each of the 20 sheep sera were

placed

in the

troughs

and wells

respectively.

The

precipitation

bands

were observed after incubation at 22 °C in a moist chamber for 1-2 days. After washing in PBS the plates

were

(24 h) and distilled water stained with

a Coomassie

(2 h) to remove excess of protein, Blue/methanol

solution.

Destaining

was achieved with an acetic acid/methanol solution.

RESULTS/DISCUSSION

In the tested

first

against

capacity

series a panel

of bovine

of experiments, of

20

sheep

colostral whey

353 samples of bovine

sera.

As

can be

to precipitate

seen

colostrum were

in Table

i,

the

the 20 sheep sera differed

remakably. Since we had no idea as to where to draw the line

300 TABLE 1 Proportions

of

various

bovine

colostral

whey

samples

precipitating

various

c a t e g o r i e s of sheep sera.

no. of c o l o s t r a l w h e y

Total

no. of sheep sera p r e c i p i t a t e d

70

(19.8) a

20

46

(13.0)

15 - 19

36 (10.2)

i0 - 14

35

(9.9)

5 -

9

34

(9.6)

1 -

4

132

(37.4)

0

353

a

Values

in p a r e n t h e s e s

denote

the p e r c e n t a g e

of the

tested bovine

colostra

that p r e c i p i t a t e d the given amount o f sheep sera.

between

safe

and

unsafe

colostra

for

the

rearing

of

lambs,

we

decided

release only the 132 n o n - p r e c i p i t a t i n g b a t c h e s of bovine c o l o s t r u m i) for use in the e r a d i c a t i o n - p r o g r a m m e

to

(cf. Table

for m a e d i in sheep, w h i l e c h e c k i n g on

their c a p a c i t y to induce a n a e m i a in n e w b o r n lambs. All 132 samples w e r e fed to lambs

and

lambs

fed on these colostra.

two

batches

were

lowered

concentrations

of

causing

samples of bovine

reported Assuming

reacting

to have

caused

anaemia

in

some

of

the

that maybe p r e c i p i t a t i o n was m i s s e d by factors,

c o l o s t r u m was

the

test

with

the

repeated with varying

2

anaemia-

concentrations

of b o t h the w h e y and the sheep sera. N o p r e c i p i t a t i o n was o b s e r v e d w i t h any of the 20 sheep sera used. The next q u e s t i o n

to be a n s w e r e d was w h e t h e r

the colostra,

w h i c h had b e e n

d e c l a r e d n o n - s a f e by our test procedure, w e r e capable of causing a n a e m i a w h e n fed to n e w b o r n

lambs.

We already had reasons to suspect that this w a s indeed

the case. Some s h e e p - f a r m e r s h a d fed 3 b a t c h e s of b o v i n e c o l o s t r u m to ii lambs not

awaiting

the

results

d e v e l o p e d severe a n a e m i a Service,

Gelderland,

of

test.

All

lambs

(personal communication,

1982).

sera in our p r e c i p i t a t i o n

our

Two

of

test w h i l e

these

colostra

fed

C.D.W.

on

these

K6nig,

precipitated

the o t h e r one p r e c i p i t a t e d

colostra

A n i m a l Health all

20

sheep

16 of the

20

sheep sera used. M o r e c o n c l u s i v e e v i d e n c e was o b t a i n e d from an e x p e r i m e n t set

301 up by the Department of Large Animal Medicine

(Faculty of Veterinary Medicine,

Utrecht),

"unsafe" by our procedure,

in which bovine

colostra,

declared

were

used with great success to mimic the anaemia encountered in lambs, reared free from maedi on bovine colostrum.

TABLE 2 Proportions

of

bovine

colostral

whey

reactive

non-reactive

and

with

the panel of 20 sheep sera in the lambing season 1983-1984.

no. of samples

no. of samples preci-

no. of samples not pre-

tested

pitating sheep sera

cipitating sheep sera

234

72

162

(144)

Value in parentheses denotes the number of colostra released to check on the general validity of the GPT in preventing IC-associated anaemia in lambs.

The results reported above permitted an evaluation of our GPT in terms of its reliability anaemia which

in reducing

regions of the Netherlands was

set

scale,

up

in the

immune

season

feeding

1983/1984

lambs with

to

register,

colostra

on

declared

a country-wide safe by

mediated

(non-haemolytic)

form

of

bovine

colostrum-induced

the colostra causing haemolytic anaemia in lambs had to be excluded.

A test procedure developed

to detect these haemolysis-

(Bernadina et al., submitted)

prone bovine

colostra had been

and this test was used in the lambing

season 1983/1984 to exclude the colostrum samples, before mentioned, field trial bovine

the GPT

Since the gel precipitation test was supposed to be a check on the

complex

anaemia,

colostrum-induced

(unpublished data). Therefore a collaborative study

lambing

the results of

procedure.

the number of cases of bovine

till then was reported to amount to Up to 20% for the various

(cf. Table

colostrum

laborative

study,

developed anaemia.

which 114

2). An inquiry revealed had had

been been

released fed

to

723

for

from the

that from the 144 batches of use

lambs,

(Table 16

of

2)

in

which

the

col-

(2.2%)

had

It is of note that six sheep-farmers participating

in the

inquiry had also used 12 batches of bovine colostrum which had not been subjected to our precipitation test to feed their lambs. These colostra were fed

302 to 77 lambs, 16 of which

(20.8%) developed anaemia, underscoring unequivocally

the general validity of our test. Although this study has demonstrated in

lambs

could

to

detect

be

reduced

anaemia-causing

explained.

Several

factors

possibilities

immune-complex-mediated

anaemia;

in our panel

sera,

of sheep

that bovine colostrum-induced

substantially, in

the

some

exist,

failure

colostrum

including

of our test samples

(I)

the

anaemia

system(s)

remains

presence

to be

of

non-

(2) the absence of certain genetic variation

and/or

(3)

feeding

of non-safe

colostra

to the

lambs by the sheep-farmers. for

the

anaemia in the lambs reared on these colostra, more elaborate experiments

As

for

the

factor(s)

present

in bovine

colostra

and

responsible

are

needed to determine their exact nature.

CONCLUSIONS

Despite

its

anaemia-prone we

were

able

simplicity

our

test

and non-anaemia-prone to

reduce

the

discriminates

surprisingly

bovine colostra.

anaemia-inducing

well

between

Utilising this technique

bovine

colostra

to

about

2%

compared to more than 20% before the introduction of this test. It

should

essential Methods.

to

be

stressed

prepare

Colostral

however,

bovine

whey

that

colostral

prepared

for

whey

otherwise,

optimal as

test

decribed

e.g. by

results, in

addition

it

is

Materials

and

of rennet,

was

found to be much less effective. More

sheep

sera may be

added to the pool

to include most of the genetic

variation present in the part of the country under investigation.

ACKNOWLEDGEMENTS

The

authors would

by the Editorial

like

to acknowledge

Secretariat-AGRI

the

linguistic

assistance

rendered

section of Elsevier Science Publishers

BV.

Miss Aletta de Beaufort is thanked for her excellent secretarial assistance.

REFERENCES

Bernadina, W., Stam, D., v. Zandwijk, L., Holzhauer, M. Optimizing the test procedure for the demonstration of factors in bovine colostrum causing anaemia in lambs reared on bovine colostrum (submitted).

303 Bernadina, W.E., Franken, P., 1983. Feeding cows colostrum to lambs. Vet. Rec. 113 (2): July 9, 47. De Boer, G.F., 1970. Zwoegerziekte. A persistent virus infection in sheep. Thesis. University of Utrecht, The Netherlands. De Boer, G.F., 1975. Zwoegerziekte virus, the causative agent for progressive interstitial pneumonia (maedi) and meningo-leucoencephalitis (visna) in sheep. Res. Vet. Sci. 18: 15-25. De Boer, G.F., Terpstra, C., Houwers, D.J., Hendriks, J., 1979. Studies in epidemiology of maedi/visna in sheep. Res. Vet. Sci. 26: 202-208. Franken, P., Elving, L., 1982. Een vergelijking van een aantal parameters in het bloed van normaal opgefokte lammeren en van lammeren, die zwoegervrij met rundercolostrum zijn opgefokt. Tijdschr. v. Diergeneesk.: 107, 315-324. Franken, P. Bernadina, W.E., K6nig, C.D.W., Elving, L., van den Ingh, T.S.G.A.M., van Dijk, S., 1982. Anemie bij zwoegervrij opgefokte lammeren. Tijdschr. v. Diergeneesk. 107: 583-585. Houwers, D.J., 1980. Maedi and maedi control. Tijdschr. v. Diergeneesk. 105: 661-664. Houwers, D.J., Gielkens, A.L.J., Schaake jr., J., 1982. An indirect enzymelinked immunosorbent assay (ELISA) for the detection of antibodies to maedivisna virus. Vet. Microbiol., 7: 209-219. Kessels, F., 1980. Het moederloos opfokken van lammeren, in het kader van de zwoegerziektebestrijding. Ed. Stichting Gezondheidszorg voor Dieren, 's Gravenhage. Logan, E.F., Forster, W.H., Irwin, D., 1978. A note on bovine colostrum as an alternative source of immunoglobulin for lambs. Anim. Prod., 26: 93-96. Reyero, C., Stockl, W. 1977. Isolation of porcine colostral immunoglobulins and preparation of monospecific anti- d , anti-y and anti-~ chain antibodies using agarose-linked immunosorbents. J. Imp. Meth., 15: 211-221.