0095-1137/78/0008-0233$02.00/0
Copyright ©1978 American SocietyforMicrobiology Printed in U.S.A.
Detection
of Antibody
to
Bovine Syncytial
Virus
and
Respiratory Syncytial
Virus in Bovine
Fetal Serum
E. A. GOULD,t* G. M. ALLAN, E. F. LOGAN,ANDJ. B. McFERRAN
Veterinary Research Laboratories,Stormont,Belfast,BT4 35DNorthern Ireland
Receivedfor publication21March1978
Batches of commercial fetal bovine serum, described by the suppliers as antibody-free, all contained antibodytobovine syncytialvirus (BSV) whentested
byindirect immunofluorescence. Antibodyto bovine respiratorysyncytialvirus
(RSV) was not detected in these sera. Twenty-four percent ofindividual fetal
bovinesera contained antibody to BSV, and 14% contained antibody to RSV
when tested by indirect immunofluorescence. BSV antibody titers infetal sera
fromdamswith high BSV antibody levelswerevariable but alwayshigher than
RSV antibody titers. Radial immunodiffusion studies with BSV-positive sera
revealed thepresenceof immunoglobulinM (IgM), IgG, andIgA, but the quantity
ofthese immunoglobulin was notdirectly related to the BSVantibody titers.
Theevidencesuggeststhat the antibodypresentinfetalsera aroseastheresult
ofinfection rather than from maternaltransferacrossthe placenta.
Atleast 14different viruses have been isolated from bovine fetaltissue (10), including bovine
syncytial virus (BSV) (17), but the isolation of bovinerespiratorysyncytialvirus (RSV) has not beenreported.In mostcases, it was not known how or when the fetus became infected. The identification of immunoglobulinsinthebovine fetus (5) and the evidence that maternal
anti-bodywas not transferred to thefetus (1)
dem-onstratedtheimmunologicalcompetenceof the
fetus. Thus, the presence in fetal bovine sera
(FBS) of precolostral antibody against bovine
diarrhea virus
(8), parainfluenza
virus type 3(16,
18), and blue tongue virus (9) suggested that
virus spread could occur from the dam to the fetus.
On several occasions we have isolated BSV
fromfetalcalflung cells(FCL) whichwerebeing routinely subcultured. The BSV could have arisen fromeither oftwo
possible
sources.First,
cells may have become infected in utero, thevirusthenbeingcarried as alatentinfection,or
second,the commercial fetal serum which was
usedinthegrowthmedium may havecontained BSVas acontaminant. Sincecells from thesame frozenstocksdid notalwaysyield BSV,we con-sidered theadditional
possibility
that thecom-mercial fetal serum
might
contain BSVanti-body. Inthispaperweshow
by
indirect immu-nofluorescence thatall of the commercial seraexamined and 24% of individual fetalserafrom anabattoir did contain BSV antibody. Wealso
tPresent address: DepartmentofMicrobiology and
Im-munology,TheQueen'sUniversityofBelfast,BelfastBT12
6BN,NorthernIreland.
show that althoughRSV antibody wasnot
de-tected in commercial sera, 14% of
individually
tested fetal sera contained low but significant levels ofRSV antibody.MATERIALS
AND METHODSMedia.FCLwerecultured inEaglegrowthmedium
(Glasgowmodification; 11)containing10%FBSor10% newbornlamb serum, whereindicated,andmaintained
in Eagle medium containing 1% FBS. Although no
direct examinations were made to see if the FBS containedBSV,wehaveneverisolated this virusfrom othercells with thesamebatchesofserum.
FCL.FCLwereobtained from thelungsofhealthy
bovine fetuses by dispersion in 0.25% trypsin and cultivation in growth medium. Primary monolayers
were subcultured and examined for BSV and RSV antigenbyindirectimmunofluorescence (seebelow).
These cellsweresubcultured and used forall subse-quentwork.
Virusesand immunofluorescence. BSVwas iso-lated andcharacterizedasdescribedinResults.RSV
was obtained from J. Stott (Agricultural Research
Council, Compton, England). For immunofluores-cence,BSV-infectedFCLweremixed in
equal
propor-tions with uninfected FCL and dispensed on coverslips in5-cm dishes (l5/ml).The disheswere
incu-batedingrowthmediumat
370C
for2daysin5%C02.ForRSV, semiconfluent FCLcultureswere infected
with RSV at an estimated input multiplicity of20
plaque-forming units percoverslip. Afteradsorption
at room temperature for 2 h, 5 ml of maintenance
mediumwasadded,andthedisheswereincubatedat
370C
for 3 days in 5% C02. Infected and control noninfected cells were then washed three times inphosphate-buffered saline and fixed for 5 min with
acetone.Indirectimmunofluorescencewas
performed
by standardprocedures with fluorescein
isothiocya-233
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234 GOULD ET AL.
nate-labeledbovineantiglobulin (Nordic). For direct immunofluorescence, the fluorescein-labeled BSV
an-tiserumwasthatusedby Clarkeetal.(2).Thisserum
stainedBSV-infected FCL butnotuninfectedor RSV-infected FCL.Fluorescein-isothiocyanate labeled
an-tiserum prepared against simianfoamy virus (strain
MK5)was thatusedbyFlemingandClarke (7).
Specificityofstaining.Thespecificityof the
an-tiviral staining by fetalsera was established by the fact that the pattern ofpositivestainingwasidentical
tothat obtained with theappropriate hyperimmune
rabbitanti-BSV oranti-RSVserum andalso by the loss ofspecific staining after theabsorption of positive sera withacetone-fixedcells infected with eitherBSV
orRSV,respectively.
Radial immunodiffusion. Immunoglobulin (im-munoglobulin G [IgG],IgM, IgA) in fetalserumwas estimated by theprocedure of Mancinietal.(13) as
modified byFahey andMcKelvey (5); specific antisera werepreparedin NewZealand rabbits with the appro-priatepurified bovineimmunoglobulin. Priortouse, eachantiserum wasselectivelyadsorbedwith immu-noglobulin-free FBS and the appropriate classes of
immunoglobulin. Specificity was checked by immu-noelectrophoresis and radial immunodiffusion.
Electronmicroscopy.Forthepreparationofthin sections, infected cellswerefixedwithglutaraldehyde
followed by osmium and embedded in Spurr resin. Sectionswerestained withuranylacetatefollowedby
lead citrate.
Source ofsera.Pooled FBSwereobtained
com-mercially (Biocult, Flow Laboratories, and Grand Is-landBiologicalCo.) andweretested with andwithout
J. CLIN. MICROBIOL.
heat inactivation at560Cfor 30min. These sera were described asimmunoglobulin-free when tested by
im-munoelectrophoresisand freeof cytopathic and
non-cytopathicviruses. Individual fetal sera were obtained from an abattoir.
RESULTS
Identification of BSV in FCL. FCL, which
weresubcultured routinely,occasionallyshowed
spontaneous progressive degeneration,
produc-inga characteristic syncytial appearance, iden-tical to thatpreviously described by Clarke et
al. (2), who identified the causative agent as BSV. Passage of these infected cells was only
possible when uninfected FCLwereadded at the time of subculture. Within 2 to 3 days these
mixed cultures also showed characteristic
syn-cytia.
Electron microscopic examination of thin
sec-tions ofinfected cells showed particles (Fig. 1) which weretypical of the viral agent BSV (4). By directimmunofluorescence,infected cultures stained brightly with fluorescein-labeled BSV antiserum (Fig. 2), and the distribution of viral antigen was asdescribedfor BSV (12). Controls, whichwerestainedwithfluorescein-labeled
an-tiserum againstsimianfoamy virus strain MK5, showednofluorescence.
Detection of BSV and RSV antibody in FBS. Individual FBS andbatches of commercial
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`, :..FIG. 1. Thin-section electronmicrograph ofFCL infected with BSV; x85,000. Insetshows typical BSV
particle; X112,500.
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VIRUS ANTIBODY IN FBS 235
FIG. 2. Directimmunofluorescence of FCL infected with BSV showing a typical syncytiumwith nuclear
fluorescence.
FBSwere screened for BSV or RSV antibody
by indirect immunofluorescence, using FCL monolayersinfected with theappropriatevirus.
Thesera were tested atdilutionsup to 1:20. In addition, serafrom calvesandadultcowswere
also includedatthe above dilutions. All of the
commercial sera contained BSV antibody, but no RSV antibody was detected (Table 1). In
individual FBSthere was ahigher proportionof
BSV-positivefetal sera(24%) thanRSV-positive
sera (14%).OftheseRSV-positiveseraonly 50%
werepositiveat a1:20dilution, theothersbeing positiveat a 1:5dilution. Thehigherproportion of
BSV-positive
sera was also reflected in the adultsera(BSV,
82%;RSV, 64%). Six ofthe12RSV-positive sera were also positive for BSV. Onlysevenofthefetalsera
analyzed
werefrom fetuses youngerthan150days,and none of thesecontaineddetectable
antibody.
BSV antibody titers in fetal sera with known
high-titer
parent. Twelve dam serawith BSV antibody titers of at least 1:10,000 were available with the corresponding positive
fetalserum.These fetal sera weretherefore ti-trated for BSVantibody. Despite the very high
levels of antibody in the dam sera, the fetal
fluorescent-antibody titers varied (Table2). In
addition, 18damswhichwerenegativeforBSV antibodyproduced corresponding negativefetal sera.
Radialimmunodiffusion ofBSV-positive
TABLE 1. BSV andRSV antibody incommercial FBSandadult andindividualFBS
No.positive/no. tested (% positive) Source
BSVa RSVa
Commercial pooled 15/15 (100) 0/15 (0) fetal sera
Individual fetalsera 39/159 (24) 12/83 (14) Adult sera 82/100(82) 76/119(64)
aIndicatorvirus.
fetalsera.The 12positivefetal sera referred to
(Table 2) werealso examined
quantitatively by
radialimmunodiffusion for thepresenceofIgG, IgM, andIgA. IgM waspresent in all ofthose sera which containedIgG.
This suggests thatantigenic
stimulation had occurredin thefetus.However, there was no
correlation
betweenthe level ofIgGorIgMand the correspondingBSVantibodytiter. It isalsoworth
noting
thatserum4, which was
negative
by
radial immunodiffu-sion, nevertheless containedbothBSVandRSVantibody when tested byimmunofluorescence.
Similarly, commercial fetal serum, which
con-tained BSV
antibody by
immunofluorescence,
containedno detectableimmunoglobulin by
ra-dial
immunodiffusion
tests.Neutralizing
ability
ofcommercial FBS.Clarkeetal. (2) showedthatBSVremained
cell
associatedinbovine fetalcellcultureand stated that itwas notpossible
toobtaininfectiouscell-VOL.
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236 GOULD ET AL.
TABLE 2. BSVantibody titers andimmunoglobulin
levelsfrom fetuses borneof dams with high anti-BSVtitersa
Fluorescent- Immunoglobulin (mg/ml) Serumno.
antibody
titer
IgG IgM IgA1 640 0.05 0.03 -C
2 10 1.5 0.2 NDd
3 20 0.07 0.04 0.3
4 640 - -
-5 640 0.4 0.1 0.3
6 640 0.01 0.05 ND
7 10 0.01 0.06 ND
8 10 - -
-9 40 - -
-10 320 0.07 0.08 ND
il 20 - -
-12 1,280 0.05 0.03 0.1
aParenttiter 2 1:10,000.
bReciprocal of dilutionbeyond which fluorescence
was notdetected.
c-,Immunoglobulinnotdetected. dND,notdone.
free virus. Atfirst thiswasalsoour
experience.
However,thisproblemwas overcomeby theuse
of growth medium containing
BSV-antibody-freenewborn lambseruminsteadofFBS. BSV-infected FCLwere
scraped
into thegrowth
me-dium and then centrifuged at 1,000 xg for 10 min. The cell pellet was
resuspended
inEagle
medium containing 10% newborn lamb serum
andsonicallytreated for20 s. After
centrifuga-tion, the clear supernatant fluidwaseither fro-zen at -70°C or added to uninfected FCL and
incubatedfor3daysat37°C. These
monolayers
werethensubcultured
again
inmediumcontain-ingnewborn lambserum. Usually, syncytia
ap-pearedwithin a further 3 days. BSV infection was confirmed by immunofluorescence. When
FCL, infectedwithBSVin thepresenceoflamb serum, were then transferred to medium
con-taining FBS,theextentof
cytopathic
effect be-came noticeablydiminished during subsequent subculture. The effect was reversed when the infected cells were again cultured in medium withlamb serum, and because of the cytocidaleffect of the BSV it was difficult to maintain
these cultures formorethan2days. DISCUSSION
There is only one report ofthe presence of BSV antibody in FBS, and this was demon-stratedonlyafterexperimentalinutero inocula-tion (19). There have been no reports of RSV antibody in FBS. Whereas it is generally ac-cepted that maternal antibody transfer across the placenta does not occur in cattle, there is evidence thatantibody tootherviruses is pres-entinfetalsera (10).
The direct isolation of BSV from cultured bovine fetalcellspreviously free of BSV antigen and the presence ofantibody to BSV and RSV inFBSprovidestrong evidence thatcongenital
virus infections do occur. Thespontaneous ap-pearance of BSV from apparently uninfected
cells is likely toresult from the integration of
BSV into the cellulargenome,utilizingreverse transcriptase(15).Theexperimentsdescribed in this report,however,donotentirelyruleoutthe
possibility of virus in the serum, although the presence of BSV antibody in the fetal serum would almostcertainlyneutralize free infectious virus. The failure to detect RSV antibody in
batchesof commercial serum wasprobably be-causethe very low RSVantibodylevels in
indi-vidual samples would be diluted out in pooled commercialpreparations.It seemsunlikelythat theBSV antibodyinfetal sera arises from
ma-ternal antibody transferacrosstheplacenta, be-causeit isgenerallyaccepted thatthisdoes not
happenin cattle (1) and BSVwasisolated from FCL onlyaftertheyhad beensubcultured
sev-eral times. Thus, these cells presumably had been infected with BSV in utero.Thevariability
inBSV fetalantibody levelsfromhigh-titer par-ents may also argue against maternal transfer; onthe otherand,variabiitymight be due to the time ofcollection of thefetalsera or evendueto
placentalleakage.Thedetection ofIgM-specific
antibody in fetal sera will provide better evi-dence ofantigenicstimulationby viruses.
Quan-titative analysis of immunoglobulin levels by radial immunodiffusion showed that the BSV
antibodytiters did notcorrelatewith IgG, IgM, or IgA levels; thus, antigenic stimulation by
other microorganisms may have occurred, and this isfurtherborne outby theobservation that somefetal sera had antibodies toboth BSVand
RSV. In addition, it is known that some FBS possess antibodies tootherviruses such as bo-vine diarrhea virus (3), andunpublished obser-vations in thislaboratoryshowed thatfetalsera
have antibodies to parainfluenza virus type 3,
infectious bovinerhinotracheitis, and rotavirus. That some fetal sera were negative by radial immunodiffusion but positive by indirect
im-munofluorescence probably reflects the higher
sensitivity of the immunofluorescence
tech-nique.
It therefore seems that congenital infection withvariousunrelated viruses may occur, which couldexplain the presence of immunoglobulin in FBS (14). These observations have important implications for vaccine manufacturers, since the presence of virus-specific neutralizing
anti-body in fetal serum could participate in the
production ofchronic infections and may also mask the presence of latent infections already
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presentin the bovine cells.Finally, thepresence
of virus antibody in FBS could accountfor the
difficulty sometimesencounteredintheisolation
of virus frominfected animalspecimens.
ACKNOWLEDGMENTS
WearegratefultoS. E. Dermott for the electron
micros-copy,T. McLaughlinfor the photography, and J.J.O'Brien and staff of the Bacteriology Departmentfor theprovision of
theindividual fetalsera.
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