[CANCER RESEARCH 45, 3225-3230, July 1985]

Characterization of the Blood Lymphocyte Population in Cattle Infected withthe Bovine Leukemia Virus1

E. N. Esteban,2 R. M. Thorn,3 and J. F. Ferrer4

Comparative Leukemia Studies Unit, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennet! Square, Pennsylvania 19348

ABSTRACT

Blood leukocytes of cattle characterized in terms of bovineleukemia virus (BLV) infection and persistent lymphocytosis (PL)were examined for the presence of lymphocyte subpopulationmarkers and viral antigens. The percentages of cells with surfaceand intracytoplasmic immunoglobulin M (IgM) and erythrocyte-antibody-rosetting cells agreed closely in all infected cattle. This

correlation and the results of double labeling experiments indicate that virtually all the surface IgM-positive B-lymphocytes in

the blood of these animals carry Fc receptors. In PL cattle, thepercentages of surface IgM-positive cells were more than twice

those of normal cells and accounted for all the increase inperipheral blood lymphocytes. B-cells accounted for most of the

increase in peripheral blood lymphocytes seen in cattle with PL.In contrast, most BLV-infected, nonlymphocytotic cattle hadnormal percentages of B-cells. Thus, the expansion of the B-cell

population in blood, while being a conspicuous characteristic ofPL, is not necessarily a consequence of BLV infection per se.

Comparisons of the percentages of IgM-positive and erythro-cyte-antibody complement-rosetting cells, together with the results of double labeling experiments, indicate that about one-halfthe B-cells in the blood of cattle with PL lacked C-3 receptors.The proportion of these cells (most likely immature B-lymphocytes) was smaller in the blood of BLV-infected nonlymphocytotic

cattle.Direct comparison showed that, in BLV-infected cattle with or

without PL, and in BLV-free cattle, virtually all erythrocyte-

rosetting blood cells had peanut agglutinin receptors. With onlyone exception, the numbers of erythrocyte-positive cells in theblood of BLV-infected cattle with or without PL were within

normal values.The "null" blood cell population, estimated as the difference

between the IgM-positive and erythrocyte-positive populations,was essentially unaffected in BLV-infected cattle without PL, but

it was absent in PL cattle.The large majority of the B-lymphocytes present in the blood

of cattle with PL were infected with BLV. The proportion ofinfected B-lymphocytes in the blood of BLV-positive nonlympho

cytotic cattle was much lower. Even in cattle with low or moderate levels of BLV-infected blood lymphocytes, the percentagesof these cells were remarkably constant during the 12-monthperiod of the study. The data indicate that most of the BLV-infected B-lymphocytes of cattle with PL lack C-3 receptors.

1This work was supported by a grant from the Kleberg Foundation.2Present address: Cátedra de Virologia, Facultad de Ciencias Veterinarias,

U. N. C. P. B. A., Pinto 399, Tandil, Buenos Aires, Argentina.3Present address: Cambridge BioScience Corporation, 35 South Street, Hop-

kinton, MA 01748.4To whom requests for reprints should be addressed.

Received 10/15/84; revised 3/25/85; accepted 3/26/85.

INTRODUCTION

BLV5 infection in cattle is, as a rule, persistent even though

virus-neutralizing antibodies are continuously present, usually in

high titers, in all infected animals. Only a small fraction (probablyless than 10%) of the BLV-infected cattle develop leukemia

(lymphosarcoma). The rest of these animals either remain asasymptomatic carriers or develop a condition known as PL (forreview, see Ref. 1). It has been assumed that PL represents apreleukemic stage. However, most cattle with PL are otherwiseclinically normal and do not develop leukemia even when keptuntil advanced ages. Moreover, there is no evidence that theseanimals harbor neoplastic cells (2). Thus, PL in cattle should beregarded as an essentially benign response to BLV infection.The available evidence strongly suggests that the developmentof lymphosarcoma and PL in cattle depends to a large extentupon the host genetic constitution (3, 4).

Several workers (5-11) have found increased percentages of

PBL bearing slg in cattle with PL, but they did not distinguishwhether these cells had synthesized or adsorbed the immunoglobulin. DeLima and Mitsherlich (12) showed that PL-positivecattle have elevated percentages of I SL bearing C-3 receptors,

as determined by EAC rosetting. Kjmar ef al. (6) reportedincreases in the percentages of slg-positive cells and cells bearing C-3 receptors (as determined by binding of aggregates of

human IgG) in the blood of 5 cows with PL. These 2 studies,however, do not clarify the relation of BLV infection to theobserved increases in PBL bearing B-cell markers, becauseneither the control nor the PL-positive animals were examined

for BLV.Takashima ef al. (11) showed that the concentration of slg-

positive or EAC-positive PBL was higher in BLV-infected cattlewith PL than in BLV-free cattle. Since BLV-infected cattle without

PL were not included, this study also fails to clarify whether ornot BLV infection per se was responsible for the changes in theconcentration of PBL bearing B-cell markers. More conclusive

information on this question was reported by Kenyon and Piper(5) who studied animals that had been examined for both BLVinfection and PL. This study showed that, as compared withBLV-free cattle, about 65% of the BLV-infected cattle withoutPL had normal concentrations of slg-positive or EAC-positive

PBL. In contrast, the percentages of these cells were greatlyincreased in all cattle with PL.

The available information on the changes in the concentrationof T-cells in the blood of BLV-infected cattle is scant. In 3 PL

cattle examined by Paul ef al. (8), the percentages of PBL formingerythrocyte rosettes were about one-half of that of nonlympho-

5The abbreviations used are: BLV, bovine leukemia virus; AIP, amplified immu-noperoxidase assay; EA, erythrocyte-antibody rosetting; EAC, erythrocyte-anti-body complement rosetting; PBL, peripheral blood leukocytes; PBS, phosphate-buffered solution; PL. persistent lymphocytosis; PNA, peanut agglutinin; slg, surface immunoglobulin; slgM, surface immunoglobulin M.

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BLOOD POPULATION OF BLV-INFECTED CATTLE

cytotic cattle of unknown BLV status. On the other hand, Tak-ashima ef al. (11) found no significant differences between BLV-

infected cattle with PL and noninfected cattle in regard to theconcentration of erythrocyte-positive PBL.

From the studies of Kenyon and Piper (13) and Paul ef al. (14),it appears that the BLV-infected PBL in cattle are predominantly,if not exclusively, B-cells. However, no information is availableon the contribution of BLV-infected PBL to the PL status.

The purpose of the present study was to obtain more conclusive information on the changes in PBL populations associatedwith BLV infection and PL. With this purpose, the blood of arelatively large number of animals of the same breed, rigorouslycharacterized in terms of BLV infection and PL and matchedwith regard to age, was examined for the presence of cellsbearing IgM on the surface and cytoplasm; cells forming EA,EAC, or erythrocyte rosettes; and cells with receptors for PNA.Using a highly sensitive and specific assay developed recently(15), we have also compared the concentration of BLV-infectedPBL in BLV-infected cattle with or without PL.

MATERIALS AND METHODS

Animals. Twenty-one adult cattle naturally infected with BLV, 10 ofwhich had PL, and 5 BLV-free control animals of approximately the sameages were obtained from a high-incidence study herd (herd BF) main

tained at the University of Pennsylvania. The characteristics of this herdhave been described (3, 4). None of the experimental animals developedleukemia or any other clinically detectable abnormality during the observation period. The BLV-infected animals had antibodies against the virion

glycoprotein antigen, as determined by radioimmunoassay (16), and werepositive in an in vitro BLV infectivity assay (17). Animals were classifiedin regard to PL according to criteria previously established (4).

Preparation of Peripheral Blood Lymphocytes. The isolation, culture,and harvest of blood lymphocytes were done as described in the accompanying paper (15).

Immunofluorescence Assay for slg. PBL were washed 3 times andresuspended to a final concentration of 10 x 106 viable cells/ml of PBS.

One hundred pi of this suspension were mixed with 400 n\ of normalgoat serum to prevent nonspecific reactions. A monospecific rabbit anti-

bovine IgM serum (Miles Laboratories, Inc.) was added to the mixture toa final concentration of 1:400. After incubation for 1 h at 4°C,the cells

were washed 3 times and resuspended in 300 n\ of PBS, pH 7.4.Fluoresceinated goat anti-rabbit IgG (heavy and light) (Miles Laboratories,

Inc.) was then added to the cell suspension to a final concentration of1:300. Following incubation for 45 min at 4°C, the cells were washed

again 3 times with cold PBS and resuspended in a 25% (v/v) glycerol in0.15 M NaCI. Each test included controls in which the rabbit antiserumwas replaced with normal rabbit serum or PBS. At least 200 cells werecounted using a Zeiss fluorescence microscope to determine the percentage of cells with slg.

Immunofluorescence Assay for Intracellular Immunoglobulin. Thepreparation of the cell smears and the performance of the tests wereessentially the same as that of the AIP test described in the accompanying paper (15). The first incubation was done with monospecific rabbitantisera against bovine IgM, lgG1, lgG2, or IgA (Miles Laboratories, Inc.),which were previously diluted 1:300 in normal goat serum to preventnonspecific reactions. For counterstaining, we used 0.02% Basic Blue24 (Sigma Chemical Co.) for 10 min. In the control, the monospecificrabbit antisera were replaced with normal rabbit sera. As positive controlsfor lgG1 and lgG2, we used smears of cell suspensions prepared fromthe spleen of a BLV-free animal.

Erythrocyte, EA, and EAC Rosette Test Procedures. These weredone as described in the accompanying paper (15).

Determination of PNA Receptors. The lymphocytes were isolated as

described above and adjusted to 10 x I06/ml. To 100 pi of cell suspen

sion, fluorescein isothiocyanate:PNA (E.Y. Laboratories, Inc.) was addedat the final concentration of 1:100, incubated at 4°Cfor 30 min, and

washed 3 times with PBS (pH 7.4), and the cells were scored using afluorescent microscope.

Simultaneous slg Assay and EA or EAC Resetting. PBL wereassayed for slg as described above, washed, and divided into 2 aliquots.One of these aliquots was used for EA resetting, and the other wasused for EAC resetting following the procedures described earlier, exceptthat the incubations were done at 4°Cto prevent slg capping. Also, the

ethidium bromide staining solution was not used. At least 200 cells werescored for slg and rosette formation using a fluorescent microscope.The numbers of cells forming EA or EAC rosettes were not influencedby prereacting them with the rabbit anti-IgM serum and correspondingconjugate or by incubating them at 4°Cinstead of 37°C.

AIP Assay. This test has been described in detail in the accompanyingpaper (15).

RESULTS

Table 1 shows the percentages of PBL carrying surface orintracytoplasmic IgM, as well as the percentages of PBL formingEA, EAC, and erythrocyte rosettes in 3 categories of cattle: BLVfree; BLV infected without PL; and BLV infected with PL. Thenumbers given in the table are the average of 2 (cells with slg)or 3 (cells forming EA, EAC, or erythrocyte rosettes) measurements performed at monthly intervals. These replicate measurements were remarkably constant for each animal. The resultsshow that the percentages of PBL carrying slg were withinnormal values in 8 of 11 infected cattle without PL, and it waselevated significantly (3 or more SDs above the mean) in theremaining 3 animals. In contrast, in all cattle with PL, the bloodconcentration of these cells was markedly increased.

In the 2 groups of infected cattle, the percentages of PBL withintracellular IgM were in good agreement with those of slgM-bearing cells. Furthermore, less than 9% of the cells with intracellular immunoglobulin reacted with monospecific antiseraagainst lgG1, lgG2, or IgA (results not shown). Thus, the largemajority of the slg-bearing PBL were B-lymphocytes.

Table 1 also shows that, whereas all but one (BF-301 ) of theinfected nonlymphocytotic cattle had normal levels of EA-roset-ting PBL, the concentration of these cells was greatly increasedin all cattle with PL. In nearly all infected cattle, the percentagesof EA-rosetting and slg-positive PBL were in close agreement,thus suggesting that they represent the same B-cell population.This was confirmed in double labeling tests on 4 infected cattle,2 of which had PL; 99% of the slg-bearing PBL of these animalsformed EA rosettes. Thus, slg-positive EA-positive B-cells ac

count for most of the increase in lymphocytes circulating in theblood of cattle with PL.

The percentages of PBL cells forming EAC rosettes werewithin normal values in infected cattle, even in those with PL(Table 1). It is clear, therefore, that a large proportion of the B-lymphocytes in the blood of cattle with PL were EAC negative.This was also shown directly in double labeling experiments inwhich about 50% of the slg-bearing PBL of the 2 BLV-infectedcattle with PL examined formed EAC rosettes. In 2 BLV-infectednonlymphocytotic cattle, the proportion of EAC-rosetting slg-positive cells was about 80%.

In most of the cattle with PL and in one of the infectednonlymphocytotic cattle, the percentages of PBL forming eryth-

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BLOOD POPULATION OF BLV-INFECTED CATTLE

Table 1Percentagesof PBL with variousmarkers in BLV-freeand BLV-infectedcattle with and without PL

CattleBLVfree

BF-366BF-360BF-359BF-358BF-352Total

PBL/cu mmofblood4,300

5,1004,0007,2005,200igMOn

thesurface24

37322823IntracellularND"

NONONONOEA47

38434646RosettesEAC31

32354120Erythrocyte29

363026

33

BLV infected, PL negativeBF-362 3,600BF-349 5,000BF-338 3,500BF-335 3,700BF-339 4,200BF-309 4,600BF-304 4,200BF-301 5,500BF-290 1,700BF-284 2,700BF-281 2,700

BLV infected, PL positiveBF-340 14,500BF-337 10,600BF-334 11,000BF-331 12,200BF-314 10,400BF-308 10,900BF-295 11,700BF-286 10,800BF-238 20,200BF-177 11,400

28.8 ±5.1°

3942442135545573143138

40.4 ±15

828186868288859083

85±2.8

5149612740475265314049

46.5±10.9

8589868884918790NO82

86.8±2.7

44±3.2

4544512639454868263544

42.8±11.2

73747480848171727783

76.9±4.5

31.8+ 6.8

4331431529353744262932

33.0±8

434234444233422945

39.8±5.3

30.8±3.4

2629243730343716293426

29 ± 6

1011161211101716439

13.5+ 4.1'ND,notdone.6Mean±SE.

rocyte rosettes were significantly lower than in the BLV-free

controls. It is important to note, however, that the absolutenumber of erythrocyte-positive cells was within normal values inall but one of the PL animals. The exception was a cow (BF-238)

whose PBL counts were by far the highest.PBL samples of 3 cattle with PL, 3 BLV-infected cattle without

PL, and 6 noninfected cows were examined in parallel in theerythrocyte rosette and PNA tests. The percentages of erythrocyte-positive and PNA-positive cells were virtually identical for

each animal.Using the AIP test (15), we also examined the proportion of

BLV-infected cells in PBL samples obtained at approximatelymonthly intervals during a 12-month period from all the infected

animals. The results are summarized in Table 2. Cattle with PLconsistently showed greater percentages of AlP-positive PBL

than nonlymphocytotic cattle. In both groups of animals, thepercentages of infected PBL were remarkably constant duringthe entire observation period. Only 3 cattle (BF-362, BF-301,BF-304) showed deviations greater than the 95% confidenceinterval of the values obtained during the 12-month observation

period. In 2 of them, the deviations were seen at parturition.In both PL and non-PL cattle, the number of BLV-infected PBL

correlated well with the number of slg-positive (Chart ÃŒA)or EA-positive (Chart 1B) cells. The number of BLV-infected PBL alsocorrelated well with the number of EAC-positive cells in non-PL

cattle. However, in PL cattle, there was little or no correlation(Chart 1C). Thus, most of the BLV-infected B-lymphocytes in

these cattle are EAC negative.

DISCUSSION

The presence of nonlabile, monomeric surface IgM is a characteristic shared by all the B-cells of the species examined. B-

cells also have Fc and EAC receptors, but these are neitherunique to B-cells nor are they present in all B-cells. There arealso non-B-cells with Fc receptors that can bind serum immu-noglobulin of all classes. Thus, to identify true B-cells, it is

necessary to use antibody specific for subclass immunoglobulinor to show that the slg is not labile. There are also cells believedto be in the B-cell lineage that lack slgM but have intracellular

IgM. Erythrocyte receptors appear to be a unique characteristicof T-cells, although it is not yet clear whether all T-cells of cattle

have erythrocyte receptors (see Ref. 18).Several investigators (5-11 ) have reported on the presence of

slg on bovine PBL. However, they did not use antisera specificfor IgM and made no attempt to demonstrate that the immunoglobulin was endogenous rather than cytophilic. In the presentstudy, we have used class-specific antisera to show that the slgpresent on the PBL of BLV-infected cattle with or without PL,and in BLV-free cattle, is predominantly, if not exclusively, IgM.

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Table 2Percentagesof BLV-infected cells in cattle with or without PL

BLV-infectedcattlePL

negativeBF-362BF-349BF-338BF-335BF-329BF-309BF-304BF-301BF-209BF-284BF-281Date

tested9/82433048236NDCND632263610/82402945236NDND602274211/8265*33361.9193551541.9133012/823426301.8a22407879a2.118191/833443220.921506145111272/83362940126454637220263/83272537a1.927483137118345/83363135225483731123287/8325394403826a4744021408/833740501.8383147501.42243%of BLV-infectedcells throughout

periodtested37.7

±0.4"32.5

±5.838.7±8.11.5

±0.628.8±7.040.3±8.349.7±3.550±3.71.4

±0.619.9±4.832.5

±7.4

a Parturition, the Wood sample was always taken before.6 Mean ±SE.c ND, not done.d Average.

30.2 ±5.8"

PLpositiveBF-340BF-337BF-334BF-331BF-314BF-308BF-295BF-286BF-238BF-17779868584NDND7280917871798382NDND698189707268587867748168858082817785808678828387857768808385a697567848182848384868577858271859288838770677073868984868585897987818281808580787568ND738583828280788478ND7779.4±5.681.1

±5.479.3±9.183.3±2.780.2±5.382.3±4.677.2±6.975.5±5.482.1±8.278.5±5.079.8±2.3d

20-

m

oCL

I2-

8-

4-

A IOo

20-

m

1k«&

¿ff

5

4 8 12 16

AIP-Positive Cells per mm3 Blood x IO3

16-

12-

8-

4-

B

4 8 12 16

AIP-Positive Cells per mm3 Blood x IO3

ioQ

•ooo

O

•

f

o£

20-

16-

12-

8-

4-

4 8 12 16

AIP-Positive Cells per mm3 Blood x IO3

Chart 1. Correlation between BLV-infected (AlP-positive)lymphocytes and slg-positive W, EA-positive(B),and EAC-positive(C) lymphocytes. O, results from non-PLanimals;•,results from PL animals.

The large majority of the slgM-positive PBL from infected cattlealso had cytoplasmic IgM, thus confirming that they were B-lymphocytes. Consistent with this conclusion are also the resultsshowing that most of these cells had Fc receptors, as determinedby their ability to form EA rosettes. This latter observation is atvariance with the results of Kumar ef al. (6), who found that asignificant proportion of the slg-bearing PBL of cattle with PLdid not react with fluoresceinated heat-aggregated human im-munoglobulin. It is likely that this discrepancy reflects a highersensitivity of the EA-rosetting technique for the detection ofbovine cells with Fc receptors.

Our results confirm the conclusion (5-12) that PL is associated

with an increase in the percentage of circulating B-cells. Indeed,our data show that the expansion of the B-cell population accounts for virtually all of the increase in PBL associated with PL.A possible exception was Cow BF-238 which also showed amarked increase in the number of erythrocyte-positive PBL. BF-238 had, by far, the highest blood lymphocyte counts among theanimals examined. Thus, the possibility should be consideredthat, in animals such as this, with exceptionally high numbers ofPBL, the increase in lymphocyte subpopulations in blood is moregeneralized. It should be noted that the sum of slgM-positiveand erythrocyte-positive PBL in BF-238 was 133%. Thus, it isalso possible that some of the PBL of BF-238 had both slg and

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BLOOD POPULATION OF BLV-INFECTED CATTLE

erythrocyte receptors. Usinger and Splitter (19) have demonstrated a very small (less than 1%) subpopulation of cells bearingboth slg (class unknown) and PNA receptors in undefined cattle.BF-238 was one of the cattle tested for both erythrocyte recep

tors and PNA receptors, and the percentages of cells with thesemarkers were almost identical. Therefore, this cow could conceivably have had a substantial increase in the PNA-positive B-

cell subpopulation described by Usinger and Splitter (19).Other studies (5-11 ) have not shown as high percentages of

slg-positive PBL in PL cattle as those found by us. A likelyexplanation is that anti-IgM antibody is more efficient in detecting

slgM than the pouvaient antisera used in the other studies.It is important to stress that most of the BLV-infected non-PL

cattle examined by us, as well as those examined by Kenyonand Piper (5), had normal percentages of B-cells in blood. Thus,

contrary to a commonly stated conclusion, BLV infection per sedoes not necessarily involve an expansion of the B-cell popula

tion in blood. A corollary of this finding is that the developmentof PL involves, in addition to BLV infection, other factors. Thereare data indicating that these factors are largely associated withthe host's genetic constitution (3, 4).

Approximately one-half of the circulating B-cells which wedetected in cattle with PL lacked C-3 receptor, as shown by their

inability to form EAC rosettes. Such cells, which most likely arerelatively immature B-lymphocytes, were also present, but in alower proportion, in the blood of BLV-infected nonlymphocytotic

cattle. Kenyon and Piper (5) and Takashima ef al. (11) did notfind evidence for the presence of the slg-bearing lymphocyteslacking C-3 receptors in the blood of BLV-infected cattle. This

may have been due to an overestimation of the percentage ofEAC-positive cells in PL cattle. In the case of Kenyon and Piper

(5), this overestimation was probably due to the use of trypanblue in counting rosettes. Since this dye, unlike the acridine-

orange:ethidium bromide dye used by us, does not stain viablecells, the nonrosetting viable lymphocytes must be distinguishedby size and morphology. In our experience, such identification ishighly equivocal; it always leads to undercounting of nonrosettedlymphocytes, particularly in PL cattle, and hence to overestimation of the percentage of rosetting cells. The differences of ourresults with those of Takashima ef al. (11) could be ascribed tothe fact that we used a different complement in the EAC-rosetting

technique.The percentages of erythrocyte-rosetting cells detected by us

in BLV-free and BLV-infected cattle are in good agreement with

those reported by Takashima ef al. (11), but they are considerablylower than those found by Paul ef al. (8). It should be noted,however, that the non-PL cattle examined by Paul ef al. (8) were

only 1 to 2 years old, an age at which the PBL counts in cattlebegin to gradually decrease. Also, these workers used trypanblue staining in counting rosettes and might have thereforeoverestimated the percentages of erythrocyte-rosetting cells be

cause of the reasons mentioned earlier.With the exception of Cow BF-238, the absolute numbers of

erythrocyte-rosetting PBL were within normal values in BLV-infected cattle with or without PL. Thus, the erythrocyte-positive,presumably T-, cell population of cattle does not seem to be

affected by either BLV infection per se or by the development ofPL.

The sum of the percentages of IgM-bearing cells and erythrocyte-positive cells was about 100% (except in BF-238) in PL

cattle, 70% in BLV-infected non-PL cattle, and 60% in BLV-free

cattle. By this calculation, it would appear that PL, but notnecessarily BLV infection per se, is associated with the loss of a"null" lymphocyte population.

The close correlation between the numbers of BLV-infectedcells and the number of slg-positive or EA-positive cells in theblood of animals with or without PL implies that B-lymphocytesare the main, if not the only, target for BLV infection in cattle.From our results, it is clear that most of the PBL of cattle withPL were infected with BLV. The proportion of BLV-infected

lymphocytes in the blood of nonlymphocytotic cattle was muchlower and more variable. The data in Chart 1C indicate that themajority of the BLV-infected PBL in cattle with PL belong to theEAC-negative B-cell subpopulation. In contrast, all, or nearly all,BLV-infected PBL of non-PL cattle were EAC positive. This

indicates that the development of PL is associated with therelease into the blood stream of cells which do not usuallycirculate in significant numbers.

Previous studies (20) have shown that, as the consequenceof a specific plasma-blocking protein, BLV particles are not

synthesized or are seldom synthesized in vivo. If synthesized,BLV particles are most likely prevented from infecting other cellsby the virus-neutralizing antibodies that are present, usually inhigh titers, in all BLV-infected cattle (10, 21). Thus, it is reasonable to assume that the BLV-infected PBL in cattle are derivedfrom precursor B-lymphoid cells which are infected during the

first phase of the infectious process. This assumption providesthe most likely explanation for our observation that the concentration of BLV-infected PBL was remarkably constant for as

many as 12 months, even in cattle with low or moderate levelsof these cells. An alternative explanation is that new lymphocytesbecome infected periodically in vivo and are released into theblood stream at about the same rate at which previously infectedcells are removed from circulation. Lymphocyte recirculationstudies are needed to resolve this issue.

It is conceivable that the number of B-cell precursors that

become infected with BLV and therefore the percentage ofinfected PBL depend to a large extent on the levels of the BLVplasma blocking factor that are present during the initial stagesof infection. Thus, the animals that develop PL could be thosethat, following infection, synthesize the blocking factor less rapidly and efficiently.

ACKNOWLEDGMENTS

We thank Dr. Donald Abt, who has played an essential role in the developmentof the BF herd. We also thank Betty Thompson for secretarial help.

REFERENCES

1. Ferrer, J. F. Bovine lymphosarcoma. Adv. Vet. Sci. Comp. Med., 24: 1-68,1980.

2. Ferrer, J. F., Marshak, R. R., Abt, D. A., and Kenyon, S. J. Persistentlymphocytosis in cattle: its cause, nature, and relation to lymphosarcoma. Ann.Rech. Vet., 9:851-857, 1978.

3. Abt, D. A., Marshak, R. R., Ferrer, J. F., Piper, C. E., and Bhatt, D. M. Studieson the development of persistent lymphocytosis and infection with the bovineC-type leukemia virus (BLV) in cattle. Vet. Microbiol., 1: 287-300,1976.

4. Abt, D. A., Marshak, R. R., Kulp, H. W., and Pollock, R. J. Studies on therelationship between lymphocytosis and bovine leukosis. Proceedings of theFourth International Symposium on Comparative Leukemia Research. Bibl.Haematol., 36: 527-536, 1970.

5. Kenyon, S. J., and Piper, C. E. Cellular basis of persistent lymphocytosis incattle infected with bovine leukemia virus. Infect. Immun., 76: 891-897,1977.

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10,

11

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1985;45:3225-3230. Cancer Res   E. N. Esteban, R. M. Thorn and J. F. Ferrer  Infected with the Bovine Leukemia VirusCharacterization of the Blood Lymphocyte Population in Cattle

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