Isolation of a p15 polypeptide from bovine leukemia virus and detection of specific antibodies in leukemic cattle

VIROLOGY

77,

501-509

(1977)

Isolation of a ~15 Polypeptide from Bovine Leukemia Virus and Detection of Specific Antibodies in Leukemic Cattle’ OSKAR

Federal

R. KAADEN, BERND FRANK WEILAND,

Research

Institute

for

Animal

Virus Accepted

FRENZEL, BERNHARD DIETZSCHOLD, AND MANFRED MUSSGAY Diseases,

D-74

November

Tiibingen,

Federal

Republic

of Germany

11,1976

Bovine leukemia virus (BLV) was isolated and purified from cultivated leukocytes of leukemic cattle and from long-term cultures of BLV-infected fetal lamb kidney cells. Because of their morphological characteristics and the detection of virion-associated reverse transcriptase activity, the particles were considered to be oncornaviruses. From purified BLV preparations, a precipitating basic protein designated BLV ~15 with an apparent molecular weight of 14,800 was isolated. The antigen was heterogeneous in charge with an isoelectric point of 8.3 (8.2-8.5). The antigen cross-reacted with antisera from leukotic cattle and sheep in both agar-gel immunodiffusion and complement fixation tests. No serological relationship was detected, however, either to murine and feline leukemia viruses or to bovine syncytial and Maedi-Visna viruses by either technique. A total of 564 sera from different cattle farms with a history of enzootic bovine leukosis was investigated in the double immunodiffusion test using the purified BLV ~15 antigen. In addition, serologically positive reagents were detected among hematologitally positive (51.5%), suspect (14.5%), and negative (0.9%) animals. INTRODUCTION

The detection and isolation of C-type virus particles from blood leukocytes of highly leukemic cattle or from long-term cultures cocultivated with leukocytes from leukotic cattle provided experimental evidence that the etiological agent of enzootic bovine leukosis (EBL) is an oncornavirus. The morphological and hydrodynamic properties of the virus particles now commonly referred to as bovine leukemia virus (BLV) are similar to those of other known mammalian oncornaviruses (Weiland et al., 1974; Weiland et al., 1976). Furthermore, BLV contains an RNA-directed DNA polymerase (Dietzschold et al., 1974; Gilden et al., 1975) specific for oncornaviruses and a high molecular weight 60-70 S RNA, demonstrated by the simultaneous detection of 70 S RNA and reverse transcriptase activity using the viral RNA as endogenous template (Kettmann et al., ’ Supported by Deutsche Forschungsgemeinschaft. * Address reprint requests to Dr. Mussgay. Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

1975).

Recently, two precipitating antigens were isolated from BLV preparations: an antigen (~24) with a molecular weight of 24,000 (Gilden et al., 1975) and an ethersensitive antigen considered to be a membrane component of BLV (Onuma et al., 1975). The present study deals with the isolation and characterization of another BLV protein which also reacts with sera from leukotic cattle and sheep in the agargel immunodiffision test. MATERIALS

AND METHODS

Cultivation of cells and purification of virus particles. The isolation and cultiva-

tion of peripheral blood leukocytes from 1eu k emit cattle with white blood cell counts ranging from 20,000 to 600,000 leukocyteslpl and the purification of the virus particles from the culture supernatant by ultracentrifugation and rate zonal and/or buoyant density centrifugation in sucrose gradients have been described (Weiland et al., 1974). More recently, long-term cul501 ISSN

0042-6822

502

KAADEN

tures of fetal lamb kidney cells (FLK) infected with BLV were used as the source of the virus (Van der Maaten et al., 1974). Maedi-Visna virus (MVV) was propagated in sheep choroid plexus cells and isolated from the supernatant of infected cells between 7 and 10 days p.i. Electron microscopy. Details of the methods used have been described (Weiland et al., 1974). Assay for DNA polymerase (reverse trunscriptase). Purified preparations of BLV, Friend mouse leukemia (FLV), and avian myeloblastosis virus (AMV) were assayed for DNA polymerase activity using the synthetic templates poly(dA):poly(dT) and poly(rA):(dT),, (Collaborative Research, Inc., Boston, Massachusetts). The conditions of the assay were identical to those already described (Dietzschold et al., 1974). Purified AMV polymerase was purchased from Collaborative Research, Inc. Purified FLV was provided by Dr. V. Moennig, Hannover. Labeling of virus and antigen. For labeling of BLV with 5 &i/ml of [YSlmethionine (Amersham Buchler, Braunschweig, West Germany; specific activity, 500 mCi/ mmol), cultivated cells were incubated for 36 hr in Eagle’s medium reduced in methionine (10%). Iodination with lz51of isolated antigen by the chloramine-T method was performed according to the standard procedure (Greenwood et al., 1963). The specific activity of the iodinated antigen ranged from 5 X lo3 to 2 X lo4 cpm/ng of protein. BSA as a marker protein was methylated (Kiehn and Holland, 1970) with 13Hldimethylsulfate (Amersham Buchler, Braunschweig; specific activity, 150 mCi/mmol). Protein determination. Protein content was determined by the method of Lowry et aL. (1951), with BSA as reference. Antigen preparation. Virus purified by rate zonal and density gradient centrifugation was treated with 0.5% (w/v) Nonidet P-40 (NP40) dissolved in Tris-HCl buffer, pH 7.4 (0.01 M Tris-HCl, 0.05 M NaCl), or treated with Tween 80-ether (O’Connor et al., 1966). Unbroken virus was sedimented by ultracentrifugation at 100,000 g for 2 hr in an SW 50.1 Spinco rotor. Purification procedures. Soluble anti-

ET

AL.

gen from high-speed centrifugation was applied to a CNBr-activated Sepharose 4B column (15 x 1 cm) containing the immobilized immunoglobulin fraction of a cattle serum with BLV-specific antibodies. Activation of Sepharose 4B by CNBr, coupling of immunoglobulins, and binding of the antigen were done according to the method of Axen and Porath (1966). BLV antigen was eluted from the column by washing with glycine-HCl buffer, pH 2.0. After intensive dialysis against 1% glycerol, 1% glycine, and 0.5% NP40, the antigen was subjected to isoelectric focusing for 48 hr in a O-4070 sucrose gradient containing 1% ampholytes, pH 3.5-10.0, and 2 M urea. After electrophoresis, the gradient was fractionated, and aliquots of each fraction were taken for pH measurement and serological tests. Antigen-containing fractions were dialyzed against Tris-HCl buffer, pH 7.4, and layered on top of a 5-25% (w/v) sucrose gradient containing 0.5% NP40 and 2 M urea. The gradient was centrifuged at 230,000 g for 16 hr at 5” in an SW 65 Spinco rotor. 13Hlmethylated BSA served as a marker protein. Technical details of the purification procedures are shown in Table 1 and in the figure legends. Serological tests. The following antisera were used: sera from cattle and sheep with a positive hematological record of enzootic leukosis. The antisera formed precipitin lines with BLV antigens and showed immunofluorescence reactions if examined in the anticomplement immunofluorescence test (Frenzel et al., 1975). Bovine syncytial virus (BSV) and a homologous antiserum were provided by Dr. M. J. Van der Maaten, Ames, Iowa. The antiserum against MVV was produced in experimentally infected sheep. Antisera against murine leukemia virus, strain Rauscher (MLV-R) from rabbit, and feline leukemia virus, strain Theilen (FeLV-Theilen) from guinea pig, were products of Electro-Nucleonics Laboratory, Inc., Bethesda, Maryland. The hyperimmune serum against BLV p15 was produced in a rabbit. The immunization schedule entailed four injections of purified BLV ~15 applied at weekly intervals, one intravenously fol-

ISOLATION

lowed by three intramuscular injections of antigen with complete Freund’s adjuvant. Double immunodiffusion tests were conducted in 0.8% agarose gels prepared in 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl. Complement fixation (CF) tests were performed using two units of each C’ and antiserum (Schmidt and Lennette, 1965). The antigens used in the CF tests were prepared by Tween BO-ether treatment from the following virus preparations: BSV (obtained from Dr. Van der Maaten); ovine leukemia virus (OLV, obtained from Dr. Paulsen, Giessen); FeLVl Theilen and MLV-R (both purchased from Electra-Nucleonics Laboratory). The procedures for the anticomplement immunofluorescence and absorption tests have been described before (Frenzel et al., 1975). Sera to be tested for precipitating antibodies were obtained from several cattle farms with known hematological status of EBL according to the Gottingen leukosis key (Tolle, 1965).

Polyacrylamide-gel (PAGE). Electrophoresis

electrophoresis

was performed in slab gels prepared from a 6-14% (w/v) acrylamide gradient. Diallyltartamide as a cross-linker was used at a ratio of acrylamide:cross-linker of 3O:l. Proteins were dissociated in the presence of 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol by heating at 100” for 3 min. The buffer system (Laemmli, 1970) and procedures employed in the preparation of PAGE and processing of gels for microdensitometry were according to the method described by Gibson and Roizman (1974).

OF

BLV

503

~15

size, were similar to those of other known mammalian oncornaviruses. Negatively stained virions occasionally displayed knob-like projections on the viral envelope (results not shown). Biochemical characterization of the BLV preparations was carried out by assay of reverse transcriptase activity using poly(rA):(dt),, and poly(dA):poly(dT) as template primers. Friend leukemia virus and purified AMV-DNA polymerase were included as controls. The results represented in Table 1 showed that BLV preparations, like those of FLV, contained a DNA polymerase capable of response to poly(rA): (dT)12, whereas poly(dA):poly(dT) exhibited a poor template activity for the oncornavirus-specific enzymes. Both the morphological characteristics and the detection of an oncornavirus-specific DNA polymerase suggested that the isolated virus particles were indeed oncornaviruses.

Isolation

and Purification

TABLE COMPARISON

of the Isolated

1

OF TEMPLATE

RESPONSE

OF DNA

POLYMERASES FROM BOVINE AND FRIEND MOUSE LEUKEMIA AND AVIAN MYELOBLASTOSIS VIRUSES TO END~CENOUS RNA, PoLv(dA):PoLv(dTl, AND

PoLv(rAl:(dT),,fi

RESULTS

Characterization Particles

of BLV pl5

Purified BLV particles were degraded by NP40 dissolved in hypotonic buffer and subjected to the purification procedures summarized in Table 2. The use of NP40 was found to give more reproducible results than treatment with Tween-ether. After high-speed centrifugation at 100,000 g for 2 hr and affinity chromatography on a Sepharose 4B-immunoglobulin column,

Virus

C-type particles revealing the typical hydrodynamic properties of oncornaviruses were isolated from the tissue culture supernatant of cultivated lymphocytes both from leukemic cattle and from infected fetal lamb kidney cells. Electron microscopic examination of the purified BLV fractions showed pleomorphic particles which, on the basis of their shape and

Source of polymerase

Endogenous template

[3H]TTP PolytdA): poly(dT)

BLV FLV Purified AMV polymerase

0.12 0.45 0.68

incorporated

(pmol)

0.75 1.05 0.98

Pqyly;Al: 12 94 143 785

a The enzyme activity is expressed as picomoles tritiated TTP incorporated into a TCA-insoluble product. The zero-time value of 0.05 pmol was subtracted from each determination.

504

KAADEN

the antigen preparations were electrofocused in a pH 3.5-10 Ampholine gradient. Because of the strong tendency of the antigen to aggregate at low salt concentrations, the electrophoresis was performed in the presence of 2 M urea. The distribution of precipitating and [35S]methionine-labeled antigen activities is shown in Fig. 1. When tested with a BLV antiserum from cattle, the precipitating antigen was detected in the pH range from 8.2 to 6.5 with maximum activity at pH 8.3. ElectrophoTABLE ISOLATION Antigen

PROCEDURE prepared

from

CF activity per milligram of protein”

BLV purified by rate zonal and buoyant density centrifugation BLV disrupted by NP40 Supernatant after highspeed centrifugation (2 hr at 100,000 g) Affinity chromatography column eluted with glytine-HCl buffer, pH 2.0 Isoelectric focusing, pH range 8.2 to 8.5 Rate zonal centrifugation in a 5-25% sucrose gradient BLV

(’ CF tests were p15 serum. b nt, Not tested.

2 FOR BLV

carried

~15

ANTIGEN Total protein recovered

(mg)

nt*

15

1,700 5,120

15

2.4

9,210

1.8

24,400

0.4

with

a rabbit

retie analysis at this stage of purification revealed one major and two minor proteins (results not shown). Final purification of the antigen was achieved by rate zonal centrifugation in a 5-25% sucrose gradient containing 2 M urea. Sedimentation analysis of iodinated BLV antigen together with 13H]methylated BSA marker protein is shown in Fig. 2. Under the sedimentation conditions described, the precipitating activity of the BLV antigen was detected in fractions 14-16 clearly separated from the faster-migrating BSA. By the methods indicated in Table 2, about 30% of the total viral BLV p15 activity was recovered in the final isolate as determined by CF tests. The efficiency of the isolation methods is also demonstrated by a 14-fold increase of the specific complement-fixing activity per milligram of protein. Electrophoretic

3.2

8,050

out

ET AL.

anti-

Analysis

Prior to electrophoretic analysis in SDSPAGE, the antigen-containing fractions obtained after isoelectric focusing and rate zonal sedimentation were dialyzed against 0.01 m Tris-HCl buffer, pH 7.4, and then lyophilized. The freeze-dried antigen was subsequently treated with 2% SDS and 5% mercaptoethanol and electrophoresed in a 6-14% acrylamide gradient using a 3% stacker gel and a discontinuous buffer system. Marker proteins were run under identical conditions. In repeated experiments, the protein, purified as indicated

15

0 0 x

Fraction

number

FIG. 1. Isoelectric focusing of BLV ~15 antigen in an Ampholine distribution of the [3SSImethionine (0-O) and of the precipitating

gradient, (hatched

pH 3.5-10.0 area) activities

(O-----O). The is indicated.

ISOLATION

OF

above and subjected to PAGE, was homogeneous in its electrophoretic behavior and identifiable as a single polypeptide with a relative molecular weight of 14,800 (Figs. 3 and 4). Because of its electrophoretic migration in SDS-PAGE, the isolated precip-

BLV

505

~15

itating antigen was designated as BLV ~15. If compared with purified FLV, however, the BLV antigen migrated to a relative position corresponding to p12 of Friend murine leukemia virus (Fig. 3). A polypeptide migrating to a position identi-

‘Ii-BSA

Fraclions

FIG. 2. Rate zonal sedimentation of [1Z51]iodinated BLV ~15 antigen (O-----O) together with 13H1methylated BSA (0-e) in a 5-25% sucrose gradient containing 0.5% NP40. The gradient was run for 16 hr at 5” and 230,000 g in an SW 65 Spinco rotor. The sedimentation is from right to left.

FIG. focusing proteins.

3. SDS slab-gel electrophoresis and rate zonal sedimentation; The electrophoresis was run

in a 6-141 acrylamide sucrose gradient-purified for 6 hr at 15 mA.

gradient BLV

of BLV ~15 antigen after and purified FLV served

isoelectric as marker

506

KAADEN

Relotlve

ET AL.

mlgratlon

distance

FIG. 4. Determination of the relative molecular weight of BLV ~15 protein by SDS-polyacrylamide-gel electronhoresis. The nositions of the marker proteins are indicated. Conditions of the electrophoresis were identical to those of Fig. 3.

cal to that of the isolated BLV antigen was also detected in sucrose gradient-purified BLV preparations if subjected to SDSPAGE (Fig. 3). Besides ~15, at least two other major viral polypeptides designated as p24 and ~50 were identified in the BLV preparation. Serological Analysis and Detection of BLV ~15 Antibodies in Leukemic Cattle

The serological analysis of the purified BLV p15 in the double immunodiffision test is shown in Fig. 5. BLV ~15 (Fig. 5, Ag& formed a single precipitin line with antisera from leukotic cattle (Fig. 5, s1 and sJ. A line of identity was observed between antisera from animals suffering from bovine (sl and SJ and ovine (sJ leukosis. A negative control (sJ was always included in each test. The purified BLV ~15 also failed to react with bovine syncytial virus antisera (results not shown). The immunofluorescence test using antisera from leukotic cattle was shown to detect intracytoplasmic antigens in BLV-infected cells. Therefore, absorption tests were carried out to study the possible role of the BLV p15 antigen in the BLV-specific immunofluorescence reaction. If preincubated with BLV antisera known to be positive in the anticomplement immunofluorescence test, the purified BLV ~15 reduced the BLV-specific intracytoplasmic immunofluorescence but did not completely block the fluorescent activity of the

FIG. 5. Serological analysis of BLV p15 antigen in the double immunodiffision test performed in 0.8% agarose gels. Purified ~15 antigen (Ag,), cytoplasmic extract of cultivated lymphocytes (Ag,), and NF40-treated BLV particles (Ag,) were allowed to react with sera from leukemic cattle (s, and sg) or leukemic sheep (s2) or with bovine serum (sq) proved to be negative in the anticomplement fluorescence test.

antisera. These findings thus revealed that several antigens, among which is BLV ~15, are involved in the immunofluorescence reaction. In another series of experiments, the monocomponent antiserum against BLV ~15 was tested in the CF test using block titrations of different sera and antigens as shown in Table 3. BLV ~15 reacted only in the homologous system and with antiserum from leukotic sheep, thereby indicat-

ISOLATION

OF

BLV

TABLE

507

~15

3

COMPLEMENT FIXATION TESTS USING A RABBIT HYPERIMMUNE AGAINST OVINE LEUKEMIA VIRUS (OLV); MURINE LEUKEMIA

SERUM AGAINST BLV p15 AND ANTISERA VIRUS, STRAIN RAUSCHER (MLV-R); FELINE

LEUKEMIA

SYNCYTIAL

VIRUS,

STRAIN

THEILEN

(FeLV-THEILEN); VIRUS

BOVINE

Antigens

Antisera BLV

BLV ~15 OLV MLV-R FeLV-Theilen BSV MVV

~15

12814” 128/8 neg. neg. neg. neg.

OLV

MLV-R

6414 25618 neg. neg. neg. neg.

o The results of checkerboard experiments antigen titer at 50% inhibition of hemolysis. * neg., s10/2 (antiserum 1:lO over antigen

VIRUS

(BSV);

AND

MAEDI-VISNA

(MVV) against FeLV-Theilen

neg. B neg. 6414 1614 neg. neg.

are expressed

neg. neg.

812 3214 neg. neg.

as the reciprocal

BSV

MVV

neg. neg. neg. neg. 32/16 neg.

neg. neg. neg. neg. neg. 3218

of the antiserum

titer

over

the

1:2).

ing a lack of serological reactivity with the other tested virus antigens. Table 4 presents the results of the double immunodifiion tests using the BLV p15 antigen and a total of 564 cattle sera. The sera were obtained from farms with a known hematological status of EBL according to the Giittingen leukosis key. All of the positive sera were obtained from cattle farms with a known positive or at least suspect background for EBL. Furthermore, it is evident that a few positive sera (0.9%) were also found among hematologically negative animals which were kept in an EBL-positive environment. DISCUSSION

Despite some minor differences in morphology and ion requirement of the reverse transcriptase, BLV particles are considered to be possible members of subgenus C of mammalian oncornaviruses (Dalton et al., 1975). The conclusion that BLV is indeed the etiological agent of enzootic bovine leukosis but not an induced endogenous bovine or ovine oncornavirus mainly rests on the direct correlation between the incidence of BLV-specific antibodies and the clinical expression of the disease. Furthermore, nucleic acid hybridization experiments performed with BLVRNA and complementary DNA showed that BLV is not endogenous to the cellular genome of normal cattle or sheep tissues (Callaghan et al., 1976). Contrary to murine, feline, and rat leukemia viruses,

TABLE PRECIPITATING DIFFERENT HEMATOLOGICAL

Hematological status (“Gottingen leukosis key”) Positive Suspect Negative

4

ANTIBODIES CATTLE

AGAINST

FARMS

BLV

WITH

p15 IN

KNOWN

STATUS OF ENZOOTIC LEUKOSIS

BOVINE

Number of sera tested

Positive reagents with BLV ~15 (%)

68 55 441

35 (51.5) 8 (14.5) 4 (0.9)

however, there are no experimental findings at present indicating a serological relationship of BLV to any other oncornaviruses of subgenus C. Hitherto, two proteins have been isolated from BLV: BLV p24 does not share any inter-species crossreactivity with antisera against p30 from other mammalian oncornaviruses in immunodiffision, complement fixation tests, and radioimmunoassays (Gilden et al., 1975), and it has been suggested by Onuma et al. (1975) that a second (ether-sensitive) antigen is a membrane protein of BLV. Precipitating antibodies to both BLV p24 (ether-resistant) and ether-sensitive (envelope) antigens have been demonstrated in cattle, naturally infected or experimentally exposed to BLV infections. The ~15 antigen isolated from sucrose gradient-purified BLV and purified by affinity chromatography, isoelectric focusing, and rate zonal centrifugation showed precipitin lines of identity if allowed to

KAADEN

508

react with sera from cattle or sheep suffering from enzootic leukosis but did not show any serological reactivity in CF tests with antisera against MLV, FeLV, BSV, and MVV (Table 3). The purified p15 antigen partially inhibited the anticomplement fluorescence reaction in the cytoplasm of BLV-infected cells, thus indicating that more than one antigen may be involved in the immunofluorescence reaction. The polypeptide analysis of BLV demonstrated remarkable differences from the known polypeptide pattern of other mammalian oncornaviruses if FLV was used as a reference. The main polypeptides of BLV were found to be in the molecular weight range of 15,000, 24,000 and 50,000. A detailed electrophoretic analysis of BLV polypeptides is under study. It is interesting to note the good correlation between the hematological status of EBL and the presence of BLV-specific precipitating antibodies against BLV p15 as shown in Table 3. Although of less sensitivity than blood cell counting, the double immunodiffision test with the isolated p15 fully supports the epizootiological observations and hematological data of the EBL eradication program. Serological findings using sera from a larger cattle population including anticomplement fluorescence, complement fixation, and double immunodiffusion tests are being evaluated at present in greater detail, and studies are also under way to determine the localization of BLV p15 within the virus particle. The results presented here may be seen as a contribution to the classification of BLV within the family Retraviridae. Note added in proof. Recently, a BLV glycoprotein with a relative molecular weight of 69,000 daltons has been detected and isolated (Frenzel et al., manuscript in preparation). ACKNOWLEDGMENTS We are very grateful to Dr. J. M. Van der Maaten, Ames, Iowa, for the generous giR of FLK cells and BSV, Dr. V. Moennig, Hannover, for providing purified FLV, and to Dr. J. Paulsen, Giessen, for supplying us with sheep antisera and homologous antigens. We also would like to thank Miss R. Neth and Mr. Dieter Sautter for their valuable technical assistance during part of this work.

ET

AL.

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(London)

210,367-369.

CALLAGHAN, R., LIEBER, M. M., GRAVES, D. C., and FERRER, J. F. (1976). Bovine leukemia virus genes in the DNA of leukemic cattle. Science 192, 1005 1007. DALTON, A. J., HEINE, U. J., and MELNICK, J. L. (1975). Symposium: Characterization of oncornaviruses and related viruses. J. Nut. Cancer Inst. 55, 941-943. DIETZSCHOLD, B., KAADEN, 0. R., UEBERSCH~R, S., WEILAND, F., and STRAUB, 0. C. (1974). Suggestive evidence for an oncornavirus-specific DNA polymerase from C-type particles of bovine leukosis. Z. Naturforsch. 29c, 72-75. FRENZEL, B., MUSSGAY, M., SCHNEIDER, L. G., and STRAUB, 0. C. (1975). Immunofluorescence test for bovine leukosis-associated complement-fixing antibodies. Zentralbl. Vet. Med. B 22, 519-523. GIBSON, W., and ROIZMAN, B. (1974). Proteins specified by herpes simplex virus. IX. Staining and radiolabelling properties of B capsid and virion proteins in polyacrylamide gels. J. Viral. 13, 155165.

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O’CONNOR, T., RAUSCHER, F. J., DETHE, G., FINK, M. A., and GERBER, P. (1966). Murine leukemia viruses: Rupture with ether and detergents to subviral constituents. Nat. Cancer Inst. Monogr. 22, 205-215.

ONUMA, M., OLSON, C., BAUMGARTENER, L. E., and

ISOLATION PEARSON, L. D. (1975). An ether-sensitive antigen associated with bovine leukemia virus infection. J. Not. Cancer Inst. 55, 1155-1158. SCHMIDT, N. J., and LENNEITE, E. M. (1965). Basic techniques of virology. In “Viral and Rickettsial Infections in Man” (F. L. Horsfall and J. Tamm, eds.), pp. 1189-1231. Lippincott, Philadelphia. TOLLE, A. (1965). Zur Beurteiiung quantitativer hamatologischer Befunde im Rahmen der LeukoseDiagnostik beim Rind. Zentralbl. Vet. Med. B 12, 281-290. VAN DER MAATEN, M. J., MILLER, J. M., and

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509

BOOTHE, A. D. (1974). Replicating type-C virus particles in monolayer cell cultures of tissues from cattle with lymphosarcoma. J. Nut. Cancer Inst. 52, 491-497. WEILAND, F., UEBERSCH~, S., STRAUB, 0. C., KAADEN, 0. R., and DIETZSCHOLD, B. (1974). C-type particles in cultured lymphocytes from highly leukemic cattle. Znteruirology 4, 140-149. WEILAND, F., and UEBERSCHAR, S. (1976). Ultrastructural comparison of bovine leukemia virus (BLV) with C-type particles of other species. Arch. Viral. 52, 187-190.