[CANCER RESEARCH 39, 1118-1 123, March 1979]
0008-6472/79/0039-0000 $02.00
Humoral Antibody Response to Bovine Leukemia Virus Infection in Cattle
and Sheep1
FrançoiseBex,2 Claudine Bruck,3 Marc Mammerickx, Daniel Portetelle,3 Jacques Ghysdael,4 Yvefte
Cleuter, Madeleine Leclercq, Daniel Dekegel, and ArsèneBurny5
Departmentof MolecularBiology, UniversitOLibre de Bruxelles,67, rue des Chevaux,1640Rhode-St.-Genése,
Belgium(F. B., C. B., D. P., J. G., V. C.,
M. L., A. B.J; Faculty of Agronomy, 5800 Gembloux, Belgium (C. B., D. P., J. G., A. B.J; National Institute for Veterinary Research, 99, Groeselenberg,
1180 Uccle, Brussels, Belgium (M. M.J; and Pasteur Institute 22, rue due Remorqueur, 1140 Brussels, Belgium (0. D.J
ABSTRACT
In this study, 345 cattle from 7 herds with a history of
lymphosarcoma ware tasted for antibody to BLV antigens
by three serological methods, namely immunodiffusion
glycoproteins
and 4 internal,
nonglycosylated
proteins.
The
molecular weights of the glycoproteins have bean estimated
to be 60,000(range,51 000 to 70,000)and 30,000(mange,
30,000 to 48,000). The 4 nonglycosylatad structural proteins
ware p24 and proteins with molecular weights of 15,000
using a bovine leukemiavirus glycoprotain with a molecular 12,000, and 10,000 (3, 4, 6-8, 9, 14, 15, 19, 25, 30, 31, 33,
weight of 60,000 as antigen, and radioimmunoassay using
34). Two lines of evidence indicate that the proteins man
a bovine leukemia virus glycoprotein with a molecular tionad above are viral antigens: (a) BLV-infected animals
weight of 60,000 and a bovine leukemia virus protein with a
molecular weight of 24,000 as antigen. The three tests
undercomparisonagreedfor335 animals,240 beingnaga
tiva in the three tests, and 95 being positive. Results ware
variable in tan cases only. Glycoprotain with a molecular
weight of 60,000 antibody titers ware systematically higher
than ware protein with a molecular weight of 24,000 anti
body titers in bovine sara and milk, as wall as in sara of
experimentally infected sheep. In the latter case, antibodies
to bovine leukemia virus antigens reached maximal values
at the animal death in the tumor phase of the disease.
Ratios of serum antiglycoprotein titer to milk titer varied
between 4 and 117, showing that, if milk pools are to be
used in surveys of bovine leukemia virus infection, use of
vary sensitive techniques of detection is mandatory.
INTRODUCTION
Enzootic bovine baucosis is an infectious disease (for
review, see Rafs. 1 and 28) caused by the ratrovirus BLV.6
The agent has bean identified by morphological (26, 35, 36)
and biochemical (2, 5, 13, 16-18, 39) criteria and has bean
shown to be exogenous to the bovine genus (2, 16, 18).
BLV is now propagated in established call lines of ovine
(38) or bat (10) origin.
It is generally accepted that the BLV proteins consist of 2
, This
work
was
helped
financially
by
the
Fonds
Cancerologique
de
Ia
Caisse Génémale
dEpargne et de Retralte, the European Economic Commu
nity Contract 00000150, and the Belgian Ministry of Agriculture.
2 Held
a
postdoctoral
fellowship
from
the
Institut
pour
l'Encouragement
du Fonds
National
4 Assistant
au
Canceralogique
Fonds
de Ia Recherche
de
Ia
says, based on either gp6Oor p24, with immunodiffusion
performed with gp6O, to apply these radioimmunoassays to
experimental infections of sheep, and to compare antibody
levels
insaraand milkofinfected
dairycows.
Our final aim is to design the most efficient strategy of
BLV detection in field conditions to provide animal sanitary
policy officers with the cheapest, most reliable, and most
practicable method of detection of BLV infection. This must
be done if enzootic bovine laucosis is to be eradicated.
MATERIALSAND METHODS
de Ia Recherche Scientifique dans I'lndustrie et I'Agriculture.
3 Aspirants
synthesize
antibodies
directedagainsttheseproteins;
(b)
BLV-infected calls synthesize protein precursors to the
group of internal viral proteins carrying group-specific
antigen determinants, (p24 and proteins with molecular
weights of 15,000, 12,000, and 10,000), a high-molecular
weight precursor to the gag proteins and the polymerasa
(reverse transcniptase), and a precursor to BLV glycopro
teins. In vitro protein-synthesizing systems programmed
with BLV 385 viral RNA synthesize gag precursors and a
high-molecular-weight precursor to the gag proteins and
the polymerase (reverse transcniptase). Subgenomic frac
tions of BLV RNA code for at least one glycoprotein pnecun
son. In compatant systems, these precursors mature into
the viral structural antigens (6—8).@
The whole arsenal of serological methods has bean used
by epidemiologists to search for BLV antibody carriers in
bovine populations (1, 28). BLV gp6O and p24, the most
abundant viral antigens, are widely used in such surveys.
The purpose of this paper is to compare nadioimmunoas
Scientifique.
Caisse
Générale
d'Epargne
et
Animals, Sera and Milk
de Retraite.
S To
S The
whom
requests
abbreviations
for
reprints
used
are:
should
BLV,
bovine
be
addressed.
leukemia
virus;
gp6O,
glycopro
teins with a molecular weight of 60,000; p24, proteIn with a molecular weight
of 24,000; ID gp6O, immunodiffusion
using a bovine leukemia virus glycopro
tein with a molecular weight of6O,000 as antigen; AlA gp6O, radioimmunoas
say using a bovine leukemia virus glycoprotein with a molecular weight of
60,000 as antigen; RIA p24, radioimmunoassay using a bovine leukemia
virus protein with a molecular weight of 24,000 as antigen; gp, glycoprotein;
PMSF, phenylmethanesulfonyl fluoride; PBS, phosphate-buffered saline
(0.01 N sodium phosphate/0.15 M NaCI).
Received August 2, 1978; accepted December 6, 1978.
1118
In the general survey (345 animals) in which ID gp6O, AlA
gp6O, and AlA p24 were compared, the animals (from
various breads) ware from laukotic herds detected in Bel
gium by the ID gp6O method and submitted to epidemiolog
ical investigations before eradication measures ware ap
plied.
7 J.
Ghysdael,
R.
Ketterman,
and
A.
Bumy,
manuscript
in
preparation.
CANCER RESEARCH VOL. 39
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Humora! Antibody Response to BLV Infection
The reference serum used throughout this study was
from Cow 15 who died of enzootic leucosis in the tumor
phase of the disease. Serum was collected at the animal's
death.
Experimental sheep belong to the experimental hard of
the National Institute for Veterinary Research. None of them
showed traces of BLV antibody before infection. Infection
was achieved by feeding each test animal 20 ml of bovine
leukemic whole blood par day for the first 30 days of his life
(23).
Virus
Na125b,
and 5 @.Lg
ofchboramine-T
were incubatedfor10 mm
at2°.
The iodinatad
proteinwas separatedfrom freeNa'251
by chromatography in a Sephadex G-50 column (1 x 40 cm)
equilibrated in 0.05 M phosphate buffer, pH 7.5/0.1%Tween
80/10k
M
PMSF.
In
the
case
of
gp6O,
the
labeled
antigen
was further purified by affinity chromatography on a 1-mb
bectin/Sephamosa (Lens cu!inaris : Boehningem Mannheim,
Mannheim, Germany) column according to Devare and
Stephenson (4). Purified labeled antigens were at least 95%
pracipitabla by our reference positive serum (Serum 15).
Their specific radioactivities were 2 x 10@cpm/@g for gp6O
and between 1.5and 4.0x 10@'
cpm/@g forp24.
BLV was produced by monolayer cultures of BLV-infactad
fatal lamb kidney calls obtained from Van damMaatan at a!.
Radiolmmunoassays
(38).
medium containing nonessential amino acids and supple
mented with 10% inactivated (0.5 hr) at 56°fetal calf serum.
When the cells reached 100% confluence 4 times, 24-hr
virus fluid harvests were collected. The medium was clan
fied by low-speed cantnifugation (6300 g) for 30 mm at 3°in
the Sorvall HG-4L motor to eliminate cell debris. Virus in
supamnatant fluid was palletad by centrifugation in a SR 35Spinco rotor at 46.600 x g for i.5 hr at 3°.Pellets wane
resuspended in PBS, pH 7.2/i0@ M PMSF. The viral sus
In Sera. Reaction mixtures (200 @I)
were prepared in PBS
and contained 1 mg of bovine serum albumin, 0.5% Triton
x-100, 2 x 10@cpm of 125l-labeled BLV gp6O or p24, and
either 4 .tI of the serum to be tasted or 4 .tl of normal
bovine serum plus 4 @b
of the appropriate dilution of the
serum under investigation. Incubation was carried out for 2
hr at 37°followed by 18 hr at 4°.Two hundred @l
of a 10%
Staphylococcus aureus Cowan I (12) suspension was then
added followedby incubation
for2 hrat4°.
The precipitate
was spun down in a Sorvall HS 4 rotor at 8000 x g for 30
pension was then centrifuged through a 2-mb25% sucrose
mm at 4°.The reaction mixtures were then divided into 2
solutioninPBS ina SW 50.1rotorat46.600x g for1.5hr
at 3°.
equal volume fractions. The bottom fraction (B) contained
the bacterial precipitate with adsorbed antigen-antibody
complexes; the top fraction (T) contained the free antigen.
Both fractions ware counted in a Beckman auto-gamma
counter. The ratio (B —T)/(B + T) was calculated. It
represents the percentage of antigen binding. Serum titers
were expressed as the reciprocal of the dilution capable of
binding 50% of the maximal amount of pmecipitabbe 1251..
labeled antigen.
The
cells
were
grown
in Eagle's
minimum
essential
Viral Antigensp24 and gp6O
Purification of BLV p24 (3, 9, 14, 19, 25, 33) and gp6O (4,
31) and design of nadioimmunoassays have been described
by several groups. The methods we used ware essentially
those outlined by Pontetelle at a!. (33) except for some
details of virus solubilization and gp purification.
In short, the viral pellet from the SW 50.1 centnifugation
was resuspended in PBS, pH 7.2, 1% Triton X-iOO, and 0.4
M KCI at a protein
concentration
of 2 mg/mI
and
incubated
for 10 mm, at 0°.The insoluble viral residue was than spun
down in a SW 50.1 rotor at 100,000 x g for 1 hr at 4°.The
supemnatant was then purified by a Sephadex G-200 filtra
tion in a 2.5- x 100-cm column equilibrated in PBS, pH 7.2/
0.1% Tween 80/0.02% NaNS10@ M PMSF. In these nonma
ducing conditions, most of the gp antigens coma off the
column as a 94,000 molecular weight complex containing
gp6O and glycoprotein with a molecular weight of 30,000
held together by disulfida bonds. The alution profile was
monitored by agar gel immunodiffusion (see below) using
Serum 15 as a reference serum. The protein material from
the ID-positive fractions was then dialyzed against Tnis-HCI
buffer, pH 7.2/0.1% Twean 80/10@ M PMSF and submitted
to DEAE chromatography in a 0.9- x 15-cm column equili
brated with the same buffer. Elution was performed by a
linear gradient of 0.00 to 0.20 M NaCI. BLV gp6O eluted at
0.05 M NaCI in these conditions.
In Milk. Raw milk, 200 @b,
was diluted with 200 @tl
of
Buffer A (PBS, pH = 7.2/0.02% Triton X-100/0.25% bovine
serum albumin 0.02%NaN3/10@M PMSF/0.2M EDTA).Two
hundred @l
of this solution were then used in a radioimmu
noassay test carried out as described here above. For
determinationof titers,
milk dilutions
ware prepared in
Buffer A backing EDTA.
Agar-Gel Immunodiffusion.Theywere performedas de
scnibed by Mammenickx at a!. (24).
Polyacrylamide Gel Electrophoresis and Fluorography.
They were performed as described by Ghysdael at a!. (6, 7).
RESULTS
Fig.1 illustrates
a polyacrylamide
gel profile
of BLV
structural proteins and the migration patterns of purified
gp6O and p24.
In Table 1, we detail the observations we made in herds
where bovine lymphosarcoma occurred. Out of a total of
345, 105 animals reacted positively in at beast one of the 3
tests under comparison. Positive reactors thus amounted
lodinationof BLVp24 and BLVgp6O
to 30.5% of all investigated animals. Such high percentages
lodination of BLV antigens followed the chlomamine-T of BLV-positive animals were previously observed by our
group and others (1, 28) in multiple case herds.
procedure described by Greenwood et a!. (11). Reaction
A comparative analysis of the results obtained shows that
mixtures(100 @l)
containing
5 @tg
ofBLV proteins,
1 mCi of
MARCH 1979
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1979 American Association for Cancer Research.
1119
F.Bex ata!.
Table1
Seroepidemo!ogicalsurveyof 345cattle by 3 methods: ID gp6O,
AlAgp6OandAlAp24
positivein
Results agreed in 335 cases: negative in the 3 tests, 240;
AlAgp6O,
the 3 tests, 95; positive in at least one test, 105; positive
in
Detailsof
104; positive in ID gp6O,101; positive in AlA p24, 99.
resultsare
the 10casesfor whichdifferenttestsshoweddifferent
shown below.AlA
antibody titan), values for this ratio fluctuate between 3.0
and 63.7. The obvious simple conclusion is that themeis no
straight-forward correlation between IgG titans in those
body fluids.
Those milk samples and 9 other samples were analyzed
for theiranti-gp6Oand p24 titers.
The valuesrecorded
correlate (Chart 1) according to the equation
gp6OSema
titers95 IDgp6O
20162
—
270
30242
—
210
8163
—
270
2555
—
where y is anti-gp6O antibody titan, x is anti-p24 antibody
titer, and r is 0.63.
We also investigated potential discrepancies between
titans observed in individual quarters of the udder. Results
of 2 such cases are depicted in Table 3. Animal 2 shows no
variation in anti-gp antibody titan while significant differ
enca in both antibody titans does exist among the 4 quarters
of the udder of Animal 1. The numbers reported in Table 3
<5
<559
+
400
<567
+
480
<5102
+
190
<5118
+
400
<5240
+
660
amaquite reproducible, milk from quarter 2 being the only
+
680
sample reacting
positively
inIDgp6O tests.
The results described so far obviously called for deeper
investigations into the antibody response of BLV-infactad
animals. Accordingly, we turned our attention towards a
flock of sheep of the National Veterinary Institute under
<5a
See
y = 1.86+ 0.34x
titens@' RIAp24
legend
of Chart
1 for definition
of titer.
the 3 methods agreed in 335 cases;240 animalsware
negative in the 3 tests while 95 were positive in the 3 tests.
From the detailed results (Table 1), it is obvious that RIA
gp6O detects the highest number of antibody carriers. In
only one case, RIA gp6O shows a titer lower than 5 while
AlA p24 is clearly
positive
(titer, 25). The 9 other sera of
Table 1 show quite higher gp6O titans than p24 (sea ragras
01
0
sion lines below). In 3 of these cases, RIA p24 titers could
@
be accurately determined ; in 6 cases, p24 titers ware vary
low.
The results displayed in Table 1 call for 2 comments: (a)
surprisingly enough, ID gp6O seams to detect more anti
body carriers than does AlA p24. (b) antibody titers of 300
to 400 seam to be the Iowan limit of detection by ID gp6O.
A
@
general
overview
of
the
situation
encountered
hare
y :1.78+ 0.71x
0
4 I
@1.96+0.62
01
.@
xl•@
.0
C
a3
>
is
-J
2
obtained by calculating the regression line and the corrala
tion coefficient between gp6O and p24 antibody titers.
Thirty sara ware randomly chosen, and antibody titers ware
determined. The following expressions (Chart 1) ware ob
tamed:
0
Cs
U
0
a.
U
4,
I
J
I
I
I
0
1
2
3
4
5
Reciprocal of BLV p24 Antibody titer (log 10)
y = 1.78+0.71x(r=0.72)
Chart1. Comparison
of p24andgp6Oantibodytiters.Titersareexpressed
where y is gp6O antibody titan, x is p24 antibody titer, and r
is correlation coefficient. It follows that both values conra
late quite wall but RIA gp6O systematically gives higher
values (10 to 15 times higher) than does AlA p24.
Survey of milk for presence of BLV antibodies was than
carried out in an attempt to decide if this material would
provide an accurate picture of the animal's status. First, we
compared gp6O and p24 titers in sara and milk of 9 seno
positive animals from a dairy hand. Table 2 illustrates the
nasults obtained.
In sara, gp titers varied from 810 to 15,800. In milk assays,
extreme values varied between 50 and 870. As a consa
quance, the ratio “anti-gp
titer in samum/anti-gp titan in
milk―varied from 3.7 to 117.5. The mange of variation of
anti-p24 titers was also vary wide with values ranging from
“less
than 5―
to 210 being recorded. If we exclude Animal
14 (for which we did not calculate the serum/milk ratio and
1120
as the reciprocals of the highest dilutions capable of binding 50% of 1251..
labeledantigens.+ , bovineserum;•,
bovinemilk; 0, sheepserum;y, anti
gp6Oantibody titer; x, anti-p24 antibody titer.
Table2
Serum/milk ratios for gp6O and p24 antibody
Fomdefinition of titers, see legend to Chart 1
titers
Animalgp6Op245Y
(10,950/290y'
(2,290/40)
7Y
37.6 (15,800/420)
9Y
12.9 (11 200/870)
6.6 (660/100)
14
15Y
16.2 (810/50)
3.7 (1 620/440)
(<50/<5)
3.0 (120/40)
110
21.8 (11,750/540)
20.8 (4,360/210)
140
11 .3 (4,070/360)
19.0 (14,800/780)
160
117.5 (14,100/120)57.3
66037.8
(1,910/30)aNumbersin
parentheses,
10.3 (310/30)
27.0 (540/20)
20.2 (1,820/90)
63.7
actual values recorded
in serum and
milk.
CANCER RESEARCH VOL. 39
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Humoral Antibody Response to BLV Infection
permanent surveillance for several years. These animals
ware infected by bovine leukamic blood. Sara were taken at
intervals to follow the progression of the disease. Table 4
illustrates evolution of antibody titers with time in 6 animals.
From these numbers, constant features emerge: (a) anti
gp6O titers ware always higher than were anti-p24 titans but
varied in parallel with the batters according to the equation
(Chart 1)
y = 1.96+ 0.62x(r= 0.90)
again here y is anti-gp6O antibody titan and x = anti-p24
Table3
gp6O and p24 antibody titers@'in milk from each quarter of the
udder
5021
a See
legend
b Anti-p24
660
3
4220
200
50
3
460
80
60b
to Chart
titems
were
230
70
16080
2
1 for definition
so low
that
of the
milk
Table4
of antibody
titers with time after experimental
sheepClini
infection
of
cal
staSheep
tus@'
fection345
N
N
N
N
702/17/72376
N
N
N
T
455/8/73327
Anti-gp6O
9,570
33,190
9,570
60,4003,990 41,79021
18
10,030
20
63,250120
22,9602
3,990
4,180
4,180
5,51050
360
140
560
N
N
9,570
16,640
2,700
3,030
19,1001,910
2,70023
N
N
702/4/72344
N
N
N
T
402/17/72a
definition
45
the viral invasion. Such an outcome has, indeed, been
58
71
reported
45
58
6,040
11,510
20,9401,210
to adult cattle
(27). If our
highly infected hand (105 reactors out of a total of 345
animals), about 1% of animals (4 of 345) are developing
BLV infection with anti-gp titers lower than 300. This illus
tratas the slow progression of BLV.
Table 1 also exemplifies the fact that the 3 serological
3,480
5,020
7,26017
35
1,740
3,990
45,82013
20
49
7,100
1,820
33,190
109,9101,380
of titer
after BLV inoculation
interpretation is correct, it means that at a given time in a
tests under investigation did agree in the vast majority of
4,580
N, nommal;T, tumom.
b See
population until the last animal would be infected. It might
1,26023
8,150
N
gation only 3 animals were negative by ID gp6O and positive
by AlA gp6O, and one was negative by ID gp6O and positive
by RIA p24. These 4 cases very probably reflect the rate of
vary well be that some animals get infected but overcome
N
N
invasion of BLV in the population examined. We do not
mean, however, that BLV would spread indefinitely in that
10,030
52,600
2,000
T
785/16/71334
to 350. Thus, we observed that in the herds under investi
1,350
N
of in
The results encountered in the general survey including
345 animals are unexpected as far as ID gp6O detects
approximately the same number of reactors as does AlA
gp6O and indeed more reactors than does AlA p24. In that
respect, it must be made clear that agam-gel immunodiffu
sion test was optimized as recommended by the European
Economic Community Working Group in Bovine Laucosis.
It is our experience that ID gp6O run in the optimized
conditions is positive for antibody titers equal on superior
32
45
41,790
N
N
N
785/16/71328
N
postin
titembAnti-p24titem@'Mos.fectionDate
26,370
28,900
328 and 376. In Sheep 376, anti-gp titer varied from 1 ,350 to
63,250 (about 47 times) while, in Sheep 328, the increase
sample
was possible.
Evolution
iad from 120 to 22960 for Sheep 376 within a period of 43
months (increase, 191-fold). By contrast, in Sheep 328,
anti-p24 titer was multiplied only by a factor of 1.4 during a
comparable period of time.
Comparing Animals 345, 327, and 328 and considering
the period of 21 to 23 months after infection, it was found
that antibody titers varied between 50 and 3990, which
amounts to as much as 80-fold; for Anti-gp6O titers, the
same comparison as above could be made between Animals
DISCUSSION
of titer.
no dilution
variations waneobserved. For example: anti-p24 titers var
amounted only to a factor of 2.2 (from 8.150 to 19.100).
Anti-gp titers 45 months after infection in Sheep 345, 376,
and 327 ware 33,190, 63,250, and 3,990, respectively. Only
40 months after infection, Sheep 344 reached an anti-gp
titan of 109.910. It might be informative hare to know that
animals 345, 327, and 334 amastill alive while Sheep 328,
376, and 344 died 78, 45, and 40 months, respectively, after
infection.
ofthe
AnimalQuamtem
uddergp6Op2411
2
antibody titer; (b) ups and downs were rarely observed in
the evolution of antibody titers. As a rule, titers increased
with time until the animal's death; (c) large individual
inlegend
to Chamt1.
35
cases (335). AlA gp6O revealed 3 reactors not detected by
ID gp6O; in turn , the latter test identified 6 reactors unda
tactad by AlA p24. Only in one case did AlA p24 come out
positive while antibodies to gp6O were absent or undetect
able by the methods used. It would be informative to follow
such an animal. Is this the very beginning of BLV infection,
an example of an infection being fought successfully by the
host, or even a case of tolerance versus BLV envelope
glycoprotein? Many interpretations are possible.
MARCH 1979
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1121
F. Bex at a!.
It is also remarkable (Chart 1) that gp titers are systemat
ically higher than are their p24 counterparts, a situation not
observed by Davara and Stephenson (4), although in our
case as in theirs, specific activities of antigens used ware
quite similar. We have no straightforward explanations for
this discrepancy. It might have to do with peculiarities of
the hands observed such as level of infection, time elapsed
since the beginning of infection, breed, or management
practices. It should be pointed out, however,that equations
of regression lines are quite similar in bovine sara and milk
and in sheep sara. We are tempted , therefore, to believe
that the phenomenon holds true in a variety of natural and
experimental situations.
In Table 4, we compared the evolution of antibody titans
in 6 sheep inoculated p.o. with bovine leukemic blood. The
most striking conclusion from this follow-up of 6 animals is
that antibody titers continuously rise up to the animal's
death, but this strong humonal response cleanly does not
protect the infected host even though anti-gp6Oantibodies
exhibit strong cytotoxicity versus BLV-pmoducing fatal Iamb
kidney calls (32). To fight the developing disease success
fully, the recipient animal must obviously react against
other antigens than viral structural proteins. Much attention
should, therefore, be devoted to identification and pumifi
cation of tumor-specific antigens (29) and to much batter
understanding of the cellular immunity reactions of BLV
infected
and -vaccinated
animals.
We finally focused our attention towards the possibility of
detecting BLV infection in milk. Control of milk production
and milk quality is a routine in many, if not all, dairy herds
around the world. In some countries such as the United
Kingdom,8 a milk pool from each dairy herd is analyzed
twice a year. It is, therefore, obvious that if BLV infection
could be detected in milk, surveys on a national scale could
be easily and economically performed. Table 2 summarizes
our findings in 9 milk samples and the 9 corresponding sara
coming from a highly infected herd. Again, gp antibody
titers wane significantly higher than ware p24 titans, but
both titers ware much Iowan in milk than in serum. Ratios of
serum titer to milk titer varied between 4 and 117 for gp6O
and between 3 and 64 for p24. It follows that milk pooling
and sampling will allow detection of BLV infection only if
animals contributing BLV antibodies have sufficiently high
titans. Since our present technique allows clear-cut datac
tion of BLV infection with antibody titans as low as 5, we
had no difficulty in identifying, by milk poo1 analysis, a
leukemic herd in which one cow out of 66 was a BLV
reactor. It is, however, self-evident that such a BLV datac
tion program would miss hands where one or 2 animals with
low titers would be present.
Distribution of BLV antibodies among the 4 quarters of
the udder was also investigated in 2 cows. In Cow 2, BLV
antibodies ware low (p24 antibodies being barely datacta
bla) and evenly distributed while in Cow 1, Quarter 2 had
higher titers for both gp6O and p24 antibodies. This uneven
distribution was confirmed by an agan gal immunodiffusion
test in which only milk from Quarter 2 yielded a positive
result. Low titans encountered in milk most probably reflect
the low concentration of globulins in milk as compared to
8 D.
1122
H.
Roberts,
personal
communication.
serum. The ratio of globulins in serum to globulins in milk
is about 50/1 . Among these globulins, bgG's represent more
than 80% (20). Milk lgG's probably originate from the blood
plasma. Any phenomenon (inflammation, infection, or age)
that would increase filtration of blood globulins into milk
would also increase milk antibody titans to BLV proteins.
Practical conclusions to be derived from our study con
firm the opinion we previously expressed in our comparison
of diagnostic methods of BLV infection (21). Wa are confi
dent that ID gp6O, run in optimal conditions, is the most
practical, the cheapest, and the easiest method. It allows
detection of almost as many animals as much more sophis
ticatad techniques and can make use of sara, even spoiled,
collected in bad field conditions by veterinary practitioners
and reaching the laboratory several days later. Attempts at
eradication of enzootic bovine leucosis using ID gp6O
tasting, according to a wall-designed time schedule taking
into account the rate of BLV propagation, are presently
under way and seam to yield vary promising results (22, 37).
ACKNOWLEDGMENTS
The skillful technical assistance of P. Ridmemont, J. Severs, G. Vanden
dade, and G. Vanheule was greatly appreciated.
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l5OKd.
@-gp60
@
-a,30
@
-p15
g@.
6aKd.
@.p
24
39Kd.
@.....
‘1k;
-p24
21.SKd.
2
3
Fig. 1. Channel 1, fluorograph of a 20% sodium dodecyl sulfate-contain
ing polyacrylamide slab gel of rHlamino acid-labeled BLV proteins. gp6O
and gp3O move as rather broad bands; p12 and p10 are barely separated on
this type of gel. Channel 2, fluomograph of purified ‘21-labeled
BLV gp6O run
on a 15% sodium dodecyl sulfate-containing polyacrylamide gel; Channel 3,
Coomassie blue stain of purified BLV p24 run on a 15% sodium dodecyl
sulfate-containing polyacrylamide gel. Purity of preparations shown in Chan
nels 2 and 3 was estimated to be at least 95%.
MARCH 1979
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1123
Humoral Antibody Response to Bovine Leukemia Virus
Infection in Cattle and Sheep
Françoise Bex, Claudine Bruck, Marc Mammerickx, et al.
Cancer Res 1979;39:1118-1123.
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