Studies on the Relationship between Infection with Bovine C ......phocytosis (PL). This latter condition is considered a preclinical stage of cattle leukemia by some investigators

[CANCER RESEARCH 34, 893-900, April 1974]

SUMMARY

Leukemic cows and adult cattle in several herds underlong-term surveillance were examined for the presence ofantibodies to bovine C-type viruses (BLV) in order toinvestigate further the possible relationships between infection with this virus, bovine leukemia, and persistent lymphocytosis (PL). This latter condition is considered apreclinical stage of cattle leukemia by some investigators.Fluorescent antibodies reacting specifically with a vinionantigen in the cytoplasm of BLV-infected cells were found in90% ofleukemic cows and in 80 to 100% ofclinically normalcows with PL in multiple-case herds. The antibody titers ofanimals with PL were often lower than those of theleukemic cows. Clinically normal cattle with consistentlynormal lymphocyte counts in the multiple-case herds alsoshowed fluorescent antibodies to BLV; however, in each oftheseherdsthe incidence(25 to 76%)and, in most cases,thetiters of antibodies found in the nonlymphocytotic animalgroups were lower than those of the corresponding PLgroups. The antibody was also present in one-third of thecattle in a single-case herd in which the PL rate was notsignificant. In contrast, antibody was found in only 4 out of214 animals from leukemia-free herds. In two multiple-caseherds in which pedigree data are available, it was observedthat animals from high-risk families had fluorescent antibodies to BLV at a significantly higher frequency, and oftenat higher titers, than animals from minimal-risk families.These data are consistent with the hypothesis that BLV maybe the etiological agent of bovine leukemia and perhaps ofPL but that host and virus factors may play significant rolesin the host's responseto BLV infection.

Virus was detected electron microscopically in 19 out of20 antibody-positive cattle and in none of 12 antibody-negative animals examined. This shows that the fluorescentantibody test is as sensitive as electron microscopy for thedemonstration of current BLV infection.

The distribution of precipitating antibodies reacting inimmunodiffusion tests with the intraspecies group-specific&s-I) BLV antigen among our various cattle populationscorrelated closely with the distribution of the fluorescent

1This investigation was supported by USPHS Cancer Center Grant 1P01 CA 14193-01 ofthe National Cancer Institute, USPHS Contract PH43-65-1013 within the Virus Cancer Program of the National CancerInstitute, USPHS Grant 5 P06 RR 00182, and a Cooperative Agreementwith the U. S. Department of Agriculture.

Received September 17, 1973; accepted January 15, 1974.

antibodies. This and the observation that semipunified BLVgs-1 antigen completely removes the specific fluorescentactivity of our standard BLV reference serum indicates thatthe fluorescent and precipitating antibodies react with thesame antigen. A comparison ofthe fluorescent and precipitating activities in the same bovine sera showed that thefluorescent antibody test is substantially more sensitive thanthe immunodiffusion test for the demonstration of BLVinfection.

The significance of these data is discussed in terms of thevalue of the bovine system as a model for studies on thenatural history of leukemia.

INTRODUCTION

Particles similar in morphology and size to the matureforms of the C-type leukemia viruses of other species wereoriginally detected by Miller et al. (28) in short-termcultures of BC2 cells from leukemic cattle. Studies in ourlaboratory confirmed and extended this observation, demonstrating that the particles do in fact representviruses(33).A C-type virus wasalso found in permanentcultures (NBCcell lines) established from peripheral lymphocytes ofleukemic cows (10, 12, 13). We have shown that the virusesfrom the BC cultures and NBC cell lines share a vinionantigen as demonstrated by the immunofluorescence technique in the cytoplasm of acetone-fixed cells as well as anantigen detected by gel diffusion experiments in ethertreated virus preparations or cell extracts of virus-infectedcells (8, 9, 11). The presence of a precipitin antigen in theC-type virus isolated from the BC cultures was confirmedby Miller and Olson (29). Studies in our laboratory havealso indicated that the precipitin antigen of the bovine virusmost probably represents the intraspecies bovine gs-Iantigenic determinant. While this antigen is ether resistant,soluble, and apparently located within the vinion, its specificity differs from that of the munineand feline gs antigens(7, 8, 11).

The morphological and antigenic similarities of theviruses found in the short-term BC cultures and in the NBCcell lines indicate that these agents represent different

2 The abbreviations used are: BC, buffy coat; NBC, New Bolton Center;

gs, group specific; BLV, bovine C-type leukemia virus; PL, persistentlymphocytosis; PHA, phytohemagglutinin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; BSV, bovine syncytia-inducing virus.

APRIL 1974 893

Studies on the Relationship between Infection with Bovine C-typeVirus, Leukemia, and Persistent Lymphocytosis in Cattle1

Jorge F. Ferrer, Donald A. Abt, Diane M. Bhatt, and Robert R. Marshak

Section of Viral Oncology, Comparative Leukemia Studies Unit, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center,Kennett Square, Pennsylvania 19348

on April 4, 2021. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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J. F. Ferrer, D. A. Abt, D. M. Bhatt, and R. R. Marshak

isolates of the same bovine C-type virus, hereafter referredto as BLV.3

Epidemiological and pedigree studies have shown that theadult form of bovine leukemia, the most frequent form ofthe disease, occurs in striking herd and familial aggregations (2, 6, 26). On the basisof the observation that manymultiple-case herds contain some clinically normal animalswith PL, Bendixen (4) proposed that PL is due to the sameagent responsible for bovine leukemia and that it representsa subclinical form of the disease. On the other hand,long-term herd studies in the Eastern United States haveshown that, although in many herds leukemia and PLaggregates in the same families, in other herds theseaggregations do not coincide (1, 2, 26).

During the past I 5 years we have intensively studied alarge number of multiple-case, single-case, contact, andleukemia-free herds by means of periodic blood counts,pedigree analyses, clinical surveillance, and detailed postmortem examination. These cattle populations have provided us with an extraordinary opportunity for large-scaleseroepidemiological studies on the distribution of BLVinfection and its possible relationship to bovine leukemiaand PL. In a previous report we showed that fluorescent andprecipitating antibodies to BLV occur in high incidence in 2multiple-case herds, particularly in animals with leukemiaand PL, whereas, in leukemia-free herds, these antibodieswere either absent or found in a much lower percentage ofanimals ( 11). Subsequently, Olson et a!. (30) reportedsimilar results on the distribution ofprecipitating antibodiesto BLV among various herds. Since then our seroepidemiological studies have been extended to include more animalsfrom our various herd categories. Owing to extensiveknowledge of the pedignees and the hematological andclinical characteristics of these cattle populations, they areparticularly well suited for studieson the possiblerelationship between BLV infection and PL.

MATERIALS AND METHODS

Herds

The hematological, clinical, and pedigree data werecollected according to methods described previously (2, 6,25â€”27). Serum samples used in this study were collectedfrom adult animals, aliquoted, and stored at â€”70Â°.

The pertinent characteristics of the herds used in thisstudy at the time of serumcollection may besummarizedasfollows:

Herd BA. A purebred Jersey herd of approximately 60animals in which 14 cases of leukemia have been documented during a 10-year period. Approximately 26% of thecattle have PL.

3 Final proof for the etiological role of BLV in cattle leukemia has yet to

be obtained. The designation BLV is appropriate; however, on the basis ofstrong indirect evidence, i.e.: (a) morphologically, as in the case of the otherknown leukemia viruses, BLV is a C-type virus; (b) as shown in this paperand in previous studies ( 11, 28, 33), BLV infection and bovine leukemia areclosely associated; and (c) transmission studies indicate that BLV isleukemogenic (31).

Herd BB. A purebred Guernsey herd of approximately 35animals in which 4 cases of leukemia have been documentedduring a 14-year period. Approximately 33% of the cattlehave PL.

Herd BF. A purebred Jersey herd of approximately 120animals in which 47 cases of leukemia have been documented during a 15-year period. Approximately 40% of thecattle have PL.

Herd BJ. A purebred Jersey herd of approximately 225animals in which 2 cases of leukemia have been documentedduring the past 3 years. This herd had been under oursurveillance as a contact herd (2, 25) for 8 years prior tooccurrence ofthe 1st leukemia case. Approximately 18% ofthe cattle have PL.

Herd BG. A purebred Holstein herd of approximately 220animals that had been under our surveillance for 11 years asa leukemia-free control herd. Owing to the occurrence of 1leukemia case in the summer of 1973, this herd is nowclassified as a single-case herd. Approximately 1.8% of thecattlehave PL.

All leukemia cases in the above herds were adult femalecattle.

Herd BH. A leukemia-free purebred Guernsey herd ofapproximately 35 animals that has beenunder our surveillance for 8 years. None of these cattle has ever had PL.

Herd BI. A leukemia-free purebred Guernsey herd ofapproximately 175 animals that has been under our surveillance for 8 years. None of these cattle has ever had PL.

Herd GB. A leukemia-free purebred Guernsey herd ofapproximately 145 animals that has been under our surveillance for S years. None of these cattle has even had PL.

Cell Cultures

Cell line NBC-l3 was established from peripheral lymphocytes ofa cow with leukemia and is routinely maintainedas a suspension culture in McCoy's SA modified mediumsupplemented with 10 or 20% heat-inactivated fetal calfserum. Details on the derivation, maintenance, and charactenistics of BLV production of this cell line have beenreported earlier (13, 16).

The procedures for the short-term cultures of bovine BCcells have also been described in detail (33). PHA was usedat the concentration ofO.01 or 0.02 ml/ml ofmedium. Virusand cells for antigen preparation or for electron microscopicexamination were collected from these cultures 48 to 72 hrafter they were set up.

Preparation of Cells for Immunofluorescent Tests

Target cells were obtained from NBC-13 cultures showing 15 to 20% of the cells with viral BLV antigen. Thepresence of this antigen was determined by immunofluorescent tests with our standard BLV reference serum obtainedfrom a cow (27â€”125) in which leukemia had regressedspontaneously. The specificity and characteristics of thisserum have been fully described in previous papers (7, 9).NBC-13 cells were washed twice in PBS at pH 7.2 bylow-speed centnifugation (500 x g for IS mm), adjusted to aconcentration of approximately 2 x 106 cells/ml of PBS,

894 CANCER RESEARCH VOL. 34



BL V, Leukemia, and Lymphocytosis

and added to special slides containing 8 wells and preparedas described by Hinshaut et al. ( 19). After the addition of thecells, the slides were air dried, fixed for 10 mm in acetone atroom temperature, and stored at â€”70Â°until used.

Fluorescent Staining

The indirect technique was used in this study. Approximately 0.05 ml of serum was placed in each well andincubated for 45 mm at 37Â°in a humidified incubator. Theslides were then washed 3 times in PBS (5-mm intervals foreach wash), dipped in distilled water, and air dried. Next,FITC-conjugated goat anti-bovine 7S globulin diluted I : 10with a solution of bovine serum albumin : rhodamine ( 1:30in PBS), was added to each well, and the slides wereincubated at 37Â° for 40 mm. The procedures for thepreparation and adsorption of the FITC-conjugate havebeen described previously (9). After incubation with theconjugate, the slides were again washed twice in PBS,dipped in distilled water, and air dried. Then, a small dropof glycerin, diluted 1 : 1 in PBS, was placed next to eachwell, and the slides were covered with a 24- x 50-mmcovenslip.

As controls the cells were incubated with either a knownpositive serum (Reference Serum 27â€”125),a known negative serum (from cows in leukemia-free Herd BH), or PBSand then with the FITC-conjugate.

Slides were examined with a Zeiss fluorescent microscopewith a dark-field condenser, FITC filter, barrier filters (65and 50), and a quartz halogen bulb as the light source.

Antigen for Immunodiffusion and for ImmunofluorescenceAbsorption Tests

Viral pellets were obtained from clarified (centnifugationat 2,000 x g for 15 mm) culture fluids by centnifugation at56,000 x g for 2 hr. The pellets were dispensed in a smallvolume of PBS and mixed with 5 ml of peroxide-free ether(anesthetic grade). The mixture was continuously agitatedat room temperature until most of the ether evaporated.This procedure was repeated once more and then nitrogengas was bubbled through the mixture until the ethercompletely evaporated. Ether-treated cell extracts wereprepared as follows. Approximately I x l0@ cells werepacked by centnifugation at 2,000 x g for 20 mm, resuspended in 15 ml ofdistilled water, and then homogenized ina Polytron (Bnonwill Scientific, Rochester, N. Y.) homogenizer in the cold at speed 30 for S mm. This homogenate wasthen concentrated by lyophilization at about 3 ml andtreated twice with IS ml of ether as described for the viralpellets. The homogenate was then clarified by 2 cycles ofcentrifugation at 15,000 x g for 30 mm and concentrated bylyophilization to about 1 ml.

Semipurified BLV gs antigen, kindly provided by ourassociate,Dr. Hugh McDonald, waspreparedby placing anether-treated and clarified BLV preparation on a G-l0OSephadex (bead form) (Pharmacia, Uppsala, Sweden) column (1.5 x 100 cm) equilibrated in 0.02 M Tnis-HC1:O.2 MNaCI:0.02 M NaN3 , pH 7.8. The column flow rate was 6 to8 ml/hn, and I-mI fractions were collected; the void volume

of the column was 53 ml. Antigen reactivity of the fractionswas determined by immunodiffusion with Reference Serum27â€”125. The antigen was eluted at approximately 7 1 ml. Thefinal preparation used in the immunofluorescence absorption tests was a pool of the most active fractions.

Immunofluorescence Absorption Tests. Reference Serum27â€”125was diluted 1:64 (approximately two 2-fold dilutions below its end point) and mixed with an equal volumeof the antigen preparation. After incubation at 37Â°for 1 hrwith occasional agitation and remaining overnight in thecold, the mixture was centrifuged at 5000 x g for 15 mm.The supernatant fluid was removed and a further 2-folddilution was made in PBS. Samples of these undiluted anddiluted supernatant fluids were tested by the indirect immunofluorescence technique on acetone-fixed N BC- 13.cells.

Immunodiffusion Ouchterlony Tests. These were carriedout in 2% Noble agar (Immunoplate, Pattern C, HylandLaboratories, Los Angeles, Calif). The wells were filledtwice with the reagents, and the plates were incubated atroom temperature in a humidified chamber. The slides wereexamined at regular intervals with magnifying lenses underindirect illumination over a 4-day period. Optimal precipitationoccurredby 18or 24 hr and remainedunchangeduntilat least the 4th day. Subsequently, the plates were immersedin sodium barbital buffer (pH 7.4) for 2 days and in distilledwater for I day and then air dried at room temperature. Thedried agar plates were stained for 10 mm in 0.1% Amidoblack lOB (1.0 g Amido black lOB in a solution of 100 mlglacial acetic acid, 700 ml methanol, and 200 ml distilledwater) and then destained in the same solution withoutAmido black lOB for 2 to 3 mm. After this stainingprocedure, bands that were weak on the unstained platesusually became more noticeable.

For the seroepidemiological survey, the standard antigenpreparation (ether-treated BLV obtained from cell lineNBC-l3) was placed in the center well and, as a positivecontrol, Reference Serum 27â€”125was added to alternateperipheral wells. All the positive sera found in this studyformed precipitating lines showing reaction of completeidentity with the line formed by Serum 27â€”125.

Electron Microscopy. Methods for the preparation of cellcultures for electron microscopy have been described elsewhere (9, 33). In order to ensure that each cell sectionviewed represented a different cell, only a single section oneach block was examined.

RESULTS

Fluorescent Antibodies. All sera tested except the controlswere coded in order to avoid bias in scoring the results. Allpositive sera gave a fluorescent reaction pattern similar tothat observed with Reference Serum 27â€”125, i.e., brightstaining which was either diffuse or granular and confinedsolely to the cytoplasm. Extensive studies have demonstrated that, in the NBC cell lines, the presence of the fluorescent antigen and the presence of the virus, as determinedby electron microscopic examination, are closely correlated.Furthermore, in the present study, many of the sera thatwere found to react with BLV-producing NBC-13 cellswere tested against cells from NBC-13 cultures maintained

APRIL 1974 895



Cattle categoryNo.

antibody positive/total no.

testedTite?

distribution of positivesera2â€”48â€”3264-256Leukemic'62/69

(90)C18 (29)â€•24 (39Y'20(32)â€•Multiple-caseBAherdPLe15/16

(94)1 (7)14(93)0(0)NonL24/48 (50)14 (58)10 (42)0(0)Multiple-case

BBherdPL8/10(80)6 (75)2(25)0(0)Non

L2/8 (25)2 (100)0 (0)0(0)Multiple-caseBFherdPL32/35

(91)17 (53)15 (47)0(0)NonL16/21 (76)14 (88)2 (12)0(0)Multiple-case

BJherdPL24/24(100)8 (33)14 (59)2(8)Non

L16/34 (47)13 (81)3 (19)0(0)Single-caseBG herd18/54 (33)12 (67)6 (33)0(0)Leukemia-free

BH herd0/32 (0)0 (0)0 (0)0(0)Leukemia-freeBI herd2/40 (5)2 (100)0 (0)0(0)Leukemia-freeGB herd2/142 (1)2 (100)0 (0)0 (0)


under conditions in which neither the virus nor its antigenare detected (9, 13). Negative results were obtained in allcases. Fluorescent cells were not observed in the controlwells in which a known negative bovine serum and the conjugate or PBS and the conjugate were tested against BLVproducing N BC-l 3 cells.

As shown in Table 1, the majority (90%) of leukemiccattle referred to our veterinary hospital or occurring in ourmultiple-case study herds had fluorescent antibodiesto BLVwith titers that in most cases were relatively high. Clinicallynormal cattle with PL in the multiple-case herds had asimilar incidence (80 to 100%) of antibody, but the titerswere generally lower than in the leukemic group. Cattle withconsistently normal lymphocyte counts in the multiple-caseherds also had BLV antibody; however, in each of theseherds the groups of nonlymphocytotic animals had lowerincidences and usually lower titers than the correspondinggroups with PL. In single-case Herd BG, which had a PLrate of only I .8%, sera were collected for 3 years before theleukemia case occurred. Of the 54 Herd BG cattle tested, 18(33%) had antibodies and most of these were in low titer.The reactors in Herd BG included the cow that subsequentlydeveloped leukemia (titer, I : 16) and 2 animals (titers, I :4and 1: 8) which had lymphocyte counts above expectationbut below the level required for classification as PL. Theoverall incidence of antibody in the animals from themultiple- and single-case herds studied was I 15 out of 250(46%). In contrast, antibodies were present in only 4 of the214 (2%) animals tested in the 3 herds in which no leukemiaor PL has ever been detected.

Correlation between Fluorescent Antibodies and VirusInfection. In order to determine whether or not the presenceof fluorescent antibodies to BLV is associated with the

actual presence of virus, a group of 32 cattle (25 from themultiple-case BF herd, 3 from the leukemia-free BI herd,and 4 leukemia referral cases) were examined for both BLVantibody and C-type virus particles in PHA-treated, shortterm BC cultures. We have shown previously that the invitro maintenance of these cells is essential for expression ofBLV and that this is enhanced by the addition of PHA tothe culture medium (33). Cells for electron microscopicexamination were collected 48 to 72 hr after the cultureswere initiated and in some cases at both intervals. A culturewas not considered negative for virus particles until at least300 cell sections, each representing a different cell, wereexamined. The results (Table 2) show that there is indeed avery close and direct correlation between the presence orabsence of BLV antibody and the presence or absence of thevirus; only I out of 20 antibody-positive animals testedwasnegative for C-type virus particles, and no virus particleswere present in any of the cultures derived from antibodynegative animals.

Relationship between Leukemia Risk and BLV Infection.The availability of 2 multiple-case herds (BA and BF), inwhich previous studies demonstrated that the disease aggregates along family lines (2, 6), provided us with anopportunity further to examine the relationship betweenleukemia risk and virus infection. Table 3 shows that theincidence of fluorescent antibody (86%) among cattle infamilies with maximal leukemia risk is significantly higherthan that (63%) found among animals from families withminimal risk (x2 = 8.513, d.f. = I,p < 0.01); moreover, theantibody titers of the animals with maximal risk were inmost cases higher than the titers of the animals withminimal risk.

Precipitating Antibody. Many ofthe bovine sera that were

Table 1

Distribution offluorescent antibodies to BL V among cattle

a Histopathologically confirmed cases referred to our clinic or occurring in the multiple-casestudy herds.

b Reciprocal of the highest serum dilution showing fluorescent activity.

CNumbers in parentheses,% of total.d Numbers in parentheses, % of positive sera.

e PL, clinically normal animals with PL; Non L, clinically normal, nonlymphocytotic animals.




Cattle categoryFluorescentantibodiesa

(positive/total)Precipitatingantibodiesâ€•

(positive/total)Leukemicc

.55/60 (92)â€•44/60(73)Multipleand single

caseherdsPL49/51(96)38/51(74)Non

L67/148 (45)23/148(15)Leukemia-freeherd4/120 (3)2/120 (2)

Fluorescentantibody titerâ€•Precipitating

antibodiesâ€•(positive/total)

J. F: Ferrer, D. A . A bi, D. M. Bhatt, and R. R . Marshak

DISCUSSION

Confirming previously reported results from this laboratory (9, 11), the present seroepidemiological survey showsthat BLV infection, as evaluated by the presence offluorescent antiviral antibodies, is highly prevalent in cattlewith leukemia and in clinically normal animals frommultiple-case and single-case herds, whereas it is infrequenton absent in leukemia-free herds. That the presence offluorescent antibody to BLV does represent actual ratherthan past infection, at least in adult animals, is demonstrated by the fact that C-type virus particles were found inshort-term BC cultures of l9 out of 20 animals examined. Apossible exception to this relationship has emerged fromunpublished studies on newborn calves in the multiple-caseBF herd. These calves possess fluorescent antibodies to BLVwhich most likely have been acquired passively throughcolostrum.

The observation that all animals which yielded C-typevirus-positive BC cultures had fluorescent antibodies mdicates that, for the detection of BLV infection, the fluorescent antibody test is at least as sensitive as electronmicroscopic examination of PHA-treated BC cultures. Inaddition, the fluorescent antibody technique is simpler,fasten, and less expensive than electron microscopic examination.

The pattern ofdistnibution of BLV, as determined by thepresent seroepidemiological survey, among herds with different incidences of leukemia (Table I ) as well as withinHerds BA and BF in which pedigree data demonstratefamilial aggregations of the disease (Table 3), cleanlyestablishes that there is a close association between leukemia or leukemia risk and BLV infection. This pattern andrecently reported transmission studies suggesting that BLVis leukemogenic (31) strongly supports the view that BLV isthe etiological agent of bovine leukemia. On the other hand,the incidence of leukemia in the multiple- and single-caseherds studied (Ref. 2; â€œMaterials and Methodsâ€•) is muchlower than the incidence of BLV infection. In addition, inmultiple-case Herds BA and BF we found that more thanone-half of the animals with minimum risk of the disease areinfected with the virus. These observations are consistentwith the idea that, if BLV is in fact the causative agent ofbovine leukemia, the development of the disease is stronglyinfluenced by host genetic factors.

It is conceivable that, in general, the titer of fluorescentantibodies reflects the degree of BLV infection. If this is thecase, the finding that the antibody titers of leukemic cattleare usually higher than those of clinically normal animals inmultiple-case herds could be taken to indicate that the levelof virus infection is also an important determinant indevelopment of the disease. Also, in multiple-case HerdsBA and BF, there seems to be a positive correlation betweenantibody titers and leukemia risk (Table 3).

It is also possible that the immune mechanism in somecattle may be impaired (leukemic infiltration, geneticfactors, etc.) and that such animals would be incapable ofproducing antibody on would produce only low levels ofantibody to BLV. This could explain the absence on low

titers of antibody found in some of the leukemic andhigh-leukemia-risk animals in our study.

Our data showing that the incidence of fluorescentantibodies to BLV in clinically normal animals with PL inmultiple-case herds is quite similar to that observed amongleukemic cows are compatible with the view of Bendixen (4)that PL is due to the same agent that is responsible forleukemia. The fact that in most cases cattle with PL hadlower titers of antibody than did leukemic animals mayindicate that animals with PL are either genetically moreresistant to BLV or have been exposed to a lower virus dose.The same reasoning could explain why cattle without PL inthe multiple-case herds studied here have even a lowerincidence as well as lower titers of BLV antibody. Thus,depending on host genetic susceptibility and/or virus dose,response to BLV infection could be the production ofantibody alone, the production of antibody with PL, or theproduction of antibody with leukemia with or without PL.In any case, the fact that cattle without PL in multiple-caseand single-case herds have antibodies to BLV clearly showsthat PL cannot be used as a reliable indicator of BLVinfection.

On the other hand, previous studies have shown that PLand leukemia may occur in different families within thesame herd and, moreover, that 35% of leukemic cattle donot have histories of PL (2, 26). Thus, it is possible thatbovine leukemia and PL may not have a common etiology.From a cow with lymphocytosis, Van Der Maaten et al. (34)have recently isolated a virus (R-29) that, upon injectioninto newborn calves, causes PL. Morphologically, R-29 isnot a C-type virus and preliminary experiments in ourlaboratory show that it is antigenically different from BLV(D. M. Bhatt, C. Diglio, and J. F. Ferrer, unpublishedexperiments). Thus, it is possible that PL may be dueto R-29 and that this virus may occur simultaneouslywith BLV in many herds as a result of a particularlyfavorable ecology.

The present data showing that precipitating antibodies tothe BLV gs-I antigen are frequent in multiple- and singlecase herds and very infrequent or absent in leukemia-freeherds also confirm and extend results of a previous study(11). It should be clarified that in that communication wehad reported a 17% incidence of precipitating antibody in aleukemia-free herd. This was Herd BG which, as mentionedin â€œMaterialsand Methods,â€• subsequently developed I caseof leukemia and has been therefore reclassified as asingle-case herd. The observation that the fluorescentactivity of Reference Serum 27â€”125 on BLV-infected cellscan be completely removed by incubation with semipunifiedand ether-treated BLV precipitin antigen indicates that thefluorescent antibodies found in this study are also reactingwith BLV gs-1. It is important that the gs antigen of otherC-type viruses can also induce both fluorescent and precipitating antibodies and that, of the known antigens of, orassociated with, other leukemia viruses, only the gs antigenis ether resistant and can be detected in the cytoplasm ofacetone-fixed cells (5, 14, 15, 18, 20, 23).

Studies on the occurrence of fluorescent and precipitatingantibodies in the same sera showed that, of the sera with




BL V. Leukemia, and Lymphocytosis

fluorescent antibody titers between I : 2 and I : 4, only 24%had precipitating activity (Table 5). Thus, it is clean thatfluorescent antibodies are a much more sensitive marker forBLV infection than precipitating antibodies.

Our previous results (1 1) on the frequent occurrence ofprecipitating antibody to BLV in clinically normal animalsfrom multiple-case herds has been recently confirmed byOlson et al. (30), using ether-extracted virus pellets fromshort-term BC cultures as antigen. These authors did notfind any precipitating sera in a leukemia-free herd kept inisolation, an observation that is also in agreement with ourresults (Table 4) showing that the sera of only 2 out of 120animals in leukemia-free herds had precipitating activity.On the other hand, Olson et a!. found a significantly higherincidence (up to 16%) of precipitating antibody in nonisolated herds that have been free of the disease for at least 13years. However, since 3 cases did occur in one ofthese herdsprior to that time, it is perhaps not surprising that BLVinfection, as indicated by the presence of antibodies, is stillpresent in this herd. It is also possible that such herds,containing considerable numbers of infected animals, maydevelop the disease at some future time. This possibility isillustrated by our Herd BG in which no leukemia cases hadbeen detected during 15 years of close observation. Asdiscussed above, Herd BG, unlike our other leukemia-freeherds, had a relatively high incidence of both fluorescent(33%) and precipitating (17%) antibodies and has recentlydeveloped a case of leukemia. This emphasizes the value ofthe BLV antibody test, particularly the detection of fluorescent antibodies, as an indicator of leukemia risk for a givenherd.

In considering results on the distribution of antibodies toBLV in the cattle population, it is important to take intoaccount that, as shown by Malmquist et al. (24), animalsfrom multiple-case on leukemia-free herds frequently possess both fluorescent and precipitating antibodies against aubiquitous BSV which can be isolated from BC cells. It isextremely unlikely that in our seroepidemiological surveyantibodies to BSV could have been mistaken for BLVantibodies since the target cells for the immunofluorescencetests and the viral antigen preparations for the immunodiffusion experiments were obtained from cultures of cell lineNBC-13 rather than from short-term bovine BC cultures.NBC-13 has been extensively examined by electron microscopy and by immunofluorescence, and in no instance havewe observed particles resembling BSV or cells with nuclearfluorescent staining which predominate in cultures infectedwith this agent (7, 9, 10, 12, 13). By periodic testing we havealso found that NBC-13 cells are free of other commonindigenous bovine viruses, i.e., parainfluenza-3 (P13), bovine viral diarrhea (BVD) and infectious bovine rhinotracheitis (IBR) viruses.

It has been well established that mice are immunologically tolerant to the gs antigen of their indigenous leukemiavirus, most likely as a consequence of vertical transmissionof the viral genome or of the whole virus early in intrauterme life (20, 2 1). The observation that most cattle withleukemia and those in multiple-case herds possess naturallyoccurring antibodies to the gs antigen of the putative

etiological agent of the disease indicates that the virus-host,and perhaps the tumor-host, immunological interactions inbovine leukemia may be different from those of munineleukemia and that, unlike the munine leukemia virus, BLVis naturally transmitted to already immunologically competent hosts. Although bovine fetuses are capable of producingcirculating antibodies (22, 32), we have failed to detectfluorescent antibody to BLV in most calves from multiplecase Herd BF before the ingestion of colostrum (unpub.lished observations). Thus, it appears likely that naturalinfection with BLV usually takes place either through themilk on by horizontal transmission later in life.

In view of these fundamental differences, it is necessarynow to consider the possibility that the bovine system is amore relevant model than the murine system for studies onthe natural history of human leukemia. At present, thebovine system represents the only available model of aC-type virus-associated leukemia occurring spontaneouslyand frequently in a large, relatively outbred population ofgenetically well-defined animals living in close contact withman and serving as a major source of human food. In thisregard, 2 out of 6 chimpanzees fed since birth on milk fromBF cows infected with BLV died with leukemia at 34 and 45weeks of age, respectively (H. McClure, R. R. Marshak,and R. P. Custer, manuscript in preparation). Leukemia hasnever before been reported in this primate. If the suspicionthat BLV is leukemogenic for chimpanzees is confirmed infuture studies, it will then be important to consider seriouslythe possible public health significance of bovine leukemia.

The availability of multiple-case herds in which it ispossible, on the basis of pedigree studies, to identify with ahigh degree of accuracy those cattle that are likely todevelop leukemia and those that will not develop thedisease, despite natural infection with BLV, offers a uniquesystem for studies on the participation of immunologicaland genetic factors in the etiology and pathogenesis of anaturally occurring leukemia as well as an opportunity todevelop immunoprophylactic approaches to the disease.

ACKNOWLEDGMENTS

We are indebted to Dr. Neale D. Stock, Vicki Baliga, and David Johnson for their contributions in the electron microscopic studies. We alsothank Betty Thompson and Marian Lewis for help in the preparation ofthe manuscript.

R

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1974;34:893-900. Cancer Res Jorge F. Ferrer, Donald A. Abt, Diane M. Bhatt, et al. C-type Virus, Leukemia, and Persistent Lymphocytosis in CattleStudies on the Relationship between Infection with Bovine

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