INFECTION AND IMMUNITY, Sept. 1974, p. 520-527Copyright ( 1974 American Society for Microbiology

Vol. 10, No. 3Printed in U.S.A.

Differentiation of Strains of Infectious Bovine RhinotracheitisVirus by Neutralization Kinetics with Late 19S Rabbit

Antibodies

LEON N. D. POTGIETER AND C. JOHN MARE

Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa StateUniversity, Ames, Iowa 50010

Received for publication 9 May 1974

Two vaccine, two respiratory (infectious bovine rhinotracheitis [IBR]), andtwo genital (infectious pustular vulvovaginitis [IPV ]) strains of infectious bovinerhinotracheitis virus were compared by neutralization kinetics using late 19Santibody (AB). The two vaccine strains were indistinguishable from one another,but were neutralized far more rapidly than the other four strains when eitheranti-IBR or anti-IPV 19S AB was used. The two IPV strains were indistinguisha-ble from one another, but were neutralized significantly more rapidly than thetwo IBR strains when anti-IBR 19S AB was used. The 2 IBR strains wereneutralized at a similar rate with the latter globulin preparation. Almostidentical results were obtained with anti-IPV 19S AB, except that one IPV strairwas neutralized at a rate similar to the IBR strains. However, when early and laterabbit 7S AB were used, IBR strains could not be distinguished from IPV strainsby neutralization kinetics. Preliminary experiments indicated that both earlyand late 19S rabbit antibodies neutralized the homologous strain more rapidlythan the heterologous strain, but the difference was more noticeable with late 19SAB. It was also determined that neutralization of IBR-IPV virus by specific earlyand late 19S rabbit AB and early 7S rabbit AB was markedly enhanced by guineapig complement. Neutralization of this virus by late 7S AB, however, was onlyslightly enhanced by complement. These results suggest that vaccine strains ofIBR-IPV virus may be distinguished by neutralization kinetics with late 19Srabbit AB, and that genital and respiratory strains may possibly also bedistinguishable with some 19S AB preparations.

Infection in cattle with the infectious bovinerhinotracheitis-infectious pustular vulvovagini-tis (IBR-IPV) virus has been associated withrespiratory disease (17), genital disease (13),conjunctivitis (1), encephalitis (5), and abortion(15). The IBR-IPV virus strains originatingfrom cattle with these varied disease syndromeshave been shown to be antigenically homogene-ous by reciprocal neutralization (1, 5, 6, 15,18-20, 23, 27), by immunodiffusion (19), and byneutralization kinetics (3) with specific anti-sera. In one study, both a genital and a respira-tory strain produced large and small plaques,and cloned populations of these variants wereneutralized equally well by antiserum to mixedand to large plaque variants (20).

Antigenic differences between some IBR-IPVvirus strains have been reported, but thesedifferences did not correlate with the origin ofthe isolates (4, 12). An intestinal strain wasrelated reciprocally to a brain isolate but non-reciprocally to a respiratory isolate when testedby reciprocal serum-virus neutralization (7).

This intestinal strain was found by neutraliza-tion kinetics to differ significantly from a respi-ratory strain, a vaccine strain, and a brainisolate (4). The latter three strains, however,had similar neutralization constants (4). Inanother neutralization kinetics study, two respi-ratory strains and one genital strain were closelyrelated, but differed significantly from a secondgenital strain (12). However, it has been re-ported that genital and respiratory strains ofIBR-IPV virus may be differentiated by carrier-free zone electrophoresis in a glucose densitygradient, even though the strains were serologi-cally identical (22, 26).The purpose of this study was to compare by

neutralization kinetics the capacity of variousglobulin fractions derived from specific rabbitantisera to detect antigenic differences betweenvaccine, genital, and respiratory strains of IBR-IPV virus.

MATERIALS AND METHODSCell cultures. The Madin Darby bovine kidney cell

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line (MDBK; 21) and a strain of embryonic bovinetracheal cells (EBT; supplied by P. C. Smith, Na-tional Animal Disease Laboratory, Ames, Iowa) wereused for the propagation and assay of viruses.

Cell medium. Cells were propagated in Eagleminimum essential medium with Earle salts, L-gluta-mine, and nonessential amino acids (MEM) (GrandIsland Biological Co., Grand Island, N.Y.). Where asemisolid medium was required for the plaquing ofviruses in cell monolayers, 0.6% Special Agar-Noble(Difco Laboratories, Detroit, Mich.) in Eagle basalmedium with Hanks salts and L-glutamine (GrandIsland Biological Co., Grand Island, N.Y.) was used.Ten percent fetal calf serum (FCS; Grand Island

Biological Co., Grand Island, N.Y.) was included inMEM after the initial seeding of cells, but 4% FCSwas used for the maintenance of cells and in agaroverlays.

Viruses. The virus strains used in this study arelisted in Table 1. All the viruses except the vaccinestrains were plaque purified three times before com-mencement of the experiments.

Virus assay. Viruses were assayed by the plaquemethod with EBT cell monolayers propagated in35-mm petri dishes. Serial 10-fold virus dilutions weremade in saline, which consisted of 0.11% glucose,0.8% NaCl, 0.04% KCI, 0.0153% Na2HPO4, 0.015%KH2PO4, 0.015% MgSO4-7H20, 0.1% lactalbuminhydrolysate, 0.0016% CaCl2 2H2O, and 0.00012%phenol red. The medium was removed from the petridishes containing the EBT cell monolayers, and 1 mlof every serial virus dilution was inoculated into eachof two dishes. Virus adsorption was allowed to takeplace for 1 h at 37 C, after which the virus preparationwas replaced with 2 ml of MEM supplemented with4% FCS and 0.5% specific IBR-IPV virus rabbitantiserum. These cells were then incubated for 30 to48 h for the plaques to develop and then fixed for 10min by the addition of 2 ml of 20% formalin into eachpetri dish. The fixed monolayers were rinsed withdistilled water and stained by pipetting 1 ml of 5%crystal violet in 20% ethanol into each petri dish. Thestain was rinsed out of the petri dishes after 5 min,and the plauqes were counted.

Neutralization kinetics. Kinetic neutralization ofthe viruses was carried out as described by McBride(16). Saline, supplemented with 1% heat-inac-tivated FCS, was used as a diluent in this procedure.Pooled fresh guinea pig serum diluted 1:3 was used as

TABLE. 1. Strains ofIBR-IPV virus used in this study

No. of cellStrain culture Origin Source

passages

650 8 Respiratory tract C. J. Mare1309 9 Respiratory tract C. J. MareK22 29 Genital tract D. G. McKercheraSteiner 11 Genital tract D. G. McKercherJensal 111 Vaccine JensalbDiamond 82 Vaccine Diamondc

a D. G. McKercher, School of Veterinary Medicine, Davis,Calif.

b Jensen-Salsbery Laboratories, Kansas City, Mo.c Diamond Laboratories Inc., Des Moines, Iowa.

a source of complement. Each batch of guinea pigserum was checked for nonspecific antiviral activityprior to its use in the test. The viruses were diluted tocontain 2 x 104 plaque-forming units per ml, whileantibody (AB) preparations were diluted so that 0.5ml neutralized approximately 9 x 103 plaque-formingunits (90% of a 0.5-ml preparation) of the homologousvirus in approximately 15 min. All the reagents werechilled to 4 C prior to the test. The test was carried outby rapidly mixing 0.5 ml of a virus preparation, 0.5 mlof rabbit antiserum or globulins derived from suchserum, and 0.1 ml of complement. A 0.1-ml portion ofthe mixture was immediately diluted 100-fold byblowing it into 9.9 ml of chilled diluent. The rest ofthe virus-antiserum mixture was incubated in a waterbath at 37 C for 15 min. Portions (0.1 ml) were takenevery 5 min from the incubating mixture and dilutedas above. The amount of virus present in each dilutedportion was then determined by the plaque method. Astatistical estimate of the rate of virus neutralizationwas calculated by linear regression and applying theStudent's t test. The neutralization rate constant (K)for a virus and a particular antiserum was calculatedby the method of McBride (16). "Normalized" neu-tralization rate constants (NK) were calculated foreach virus strain, where neutralization rate with thehomologous antiserum was arbitrarily set at 100 andneutralization of the other strains by the same anti-serum was rated proportionately according to their Kvalues.

Antiserum production. Viral preparations used forthe immunization of New Zealand albino rabbits werepropagated in 16 x 107 MDBK cells, partially purifiedby two cycles of differential centrifugation, and con-centrated to 4 ml by vacuum filtration throughdialysis tubing. The MDBK cells were infected at amultiplicity of 5 to 10 PFU per cell.One ml of the above viral antigen was emulsified in

1 ml of complete Freund adjuvant (Difco Laborato-ries, Detroit, Mich.), which was then inoculatedintradermally and into the rear footpads of a rabbit.Each footpad received 0.2 ml, and 0.1 ml was inocu-lated intradermally at 16 sites. Three weeks later asimilar inoculation was made, except that incompleteFreund adjuvant was used and the footpad inocula-tions were not repeated. After a further 3 weeks, 1 mlof the viral antigen was administered intravenously tothe rabbits. The rabbits were bled by cardiac punc-ture 10 days after the first inoculation, and again 10days after the last inoculation. The serum collectedfrom these blood samples was stored at -20 C andlabeled "early serum" and "late serum," respectively.

Rabbit globulin purification. Rabbit globulinswere prepared from the rabbit antisera by two cyclesof gel filtration through a Sephadex G200 (PharmaciaFine Chemicals Inc., Piscataway, N.J.) column. Each19S globulin preparation was tested for contamina-tion with 7S globulins by immunoelectrophoresis,using sheep anti-rabbit globulin (Pentex Biochemical,Kankakee, Ill.). The 7S globulin preparation wastested for 19S contamination in the same way. Eachglobulin fraction was concentrated by vacuum filtra-tion through dialysis tubing to the original volume ofthe serum from which it was prepared. All theglobulin preparations were dialyzed against the saline

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diluent prior to use in the neutralization kineticsprocedure. The globulins were filtered through450-nm membrane filters (Millipore Corp., Bedford,Mass.) and stored at 4 C for a maximum of 4 weeks.To prevent aggregation of the 19 S globulins, an equalvolume of FCS was added to the preparations prior tofiltration, and then the preparations were subjected toultrasonic treatment for 30 s with a Bronwill Biosoniksonicator (Will Scientific, Inc., Rochester, N.Y.) setat the 10% position.Antiserum absorption. In some instances, antisera

and globulins derived from such sera were absorbedone to three times with either 12 x 10' EBT cells or 20x 106 MDBK cells per ml of serum. Prior to theabsorption procedure, the cells were subjected tothree cycles of rapid freezing and thawing. Absorptionwas allowed to take place for 2 h at room temperaturebefore the cell debris was removed by centrifugationfor 20 min at 800 x g, followed by filtration through a220-nm filter.

RESULTSVirus assay procedure. The method of

achieving plaque formation with a liquid me-dium containing specific antiserum (plaquingmedium) was found to be very convenient andresulted in clearer plaques than with an agaroverlay. The concentration of late rabbit anti-IBR-IPV serum in the plaquing medium wasfound to be important. The minimal antiserumconcentration which still allowed plaque forma-tion was 0.125 to 0.25%. Concentrations ofantiserum in liquid overlays below this mini-mum resulted in indistinct plaques which weredifficult to count. Antiserum concentrationsgreater than 2% were found to be toxic to thecells. However, absorption of antiserum withEBT cells prior to its use in the liquid overlayconsiderably reduced the toxic effects of theantiserum.A 0.5% concentration of unabsorbed anti-

serum was subsequently routinely used in theplaquing medium for the assay of the IBR-IPVvirus strains. Plaques first became visible underthe liquid plaquing medium after approxi-mately 30 to 36 h of incubation at 37 C, andwere usually counted at 48 h postinfection.Such plaquing medium has been stored for upto 11 months at 4 C and was still usable. Anexample of typical virus plaques which devel-oped under plaquing medium may be seen inFig. 1.Neutralization kinetics. Preliminary inves-

tigations were conducted with neutralizationkinetics to examine the role of complement inthe neutralization of IBR-IPV virus by specificearly and late rabbit antisera and by purifiedglobulins derived from such sera. The guineapig sera used in these trials did not affect the

titer of IBR-IPV virus significantly in the pres-ence of normal rabbit serum or saline.

Early 19S, late 19S, and early 7S AB requiredcomplement for the neutralization of IBR-IPVvirus (Fig. 2, 3, and 4). In contrast, late 7S ABefficiently neutralized this virus in the absenceof complement, but the rate of neutralizationwas nevertheless still slightly enhanced by com-plement (Fig. 5). The complement require-ments of whole antiserum resembled that of 7SAB. Early antiserum was complement depend-ent, whereas late antiserum efficiently neutral-

*~

, . _ N. h _

Z , 4

FIG. 1. Plaques produced by 650 virus (IBR) in EBTcells using plaquing medium with 0.5% anti-650 virusserum. xl.9.

1.5.

LOG

PFU

0.5[

2 MINUTES 10FIG. 2. Neutralization of 650 virus (IBR) by early

homologous 19S AB (immunoglobulin M [IgM]).Complement (c) significantly increased the neutral-ization rate. Little neutralization occurred in theabsence of complement.

early IgM

no c

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DIFFERENTIATION OF STRAINS OF IBR VIRUS

LOG

PFU

2 MINUTES 10FIG. 3. Neutralization of 650 virus (IBR) by late

homologous 19S (immunoglobulin M [IgM]). Littleneutralization occurred in the absence of complement(c), but rapid neutralization was observed in thepresence of complement.

early IgG

1.5 no

LOG\

PFU\

0.5

2 MINUTES 10FIG. 4. Neutralization of 650 virus (IBR) by early

homologous 7S AB (immunoglobulin G [IgG]). Thevirus was rapidly neutralized but only in the presenceof complement (c).

ized the virus in the absence of complement,and the addition of complement only slightlyenhanced the rate at which the virus wasneutralized by the latter serum.As a result of these findings, complement was

routinely included whenever early 7S, early 19S,and late 19S AB were used. Experiments werethen conducted to determine which globulintype possessed the greatest potential for distin-guishing strains of IBR-IPV virus by neutraliza-tion kinetics. The neutralization rate of a respi-ratory strain (650) was similar to that of a

genital strain (K22) in the presence of earlyanti-K22 7S antibody. These two viruses werealso neutralized at almost an equal rate byanti-650 early 7S AB. These viruses were alsoindistinguishable by their rates of neutraliza-tion when tested with late anti-K22 7S AB orlate anti-650 7S AB. In contrast, K22 virus wasneutralized at a greater rate than 650 virus byearly anti-K22 19S globulin. This difference inneutralization rate was even more pronouncedwhen late anti-K22 19S AB was used. Conse-quently, late 19S AB derived from IBR-IPVantisera was used in the subsequent neutraliza-tion kinetics trials in which the IBR-IPV strainswere compared.The rates at which late anti-650 19S AB

neutralized IBR-IPV virus strains is presentedin Fig. 6. The K and NK values representingneutralization by this globulin preparation arelisted in Table 2. A 1:3 dilution of late anti-65019S AB was used in these trials. The two genitalstrains (K22 and Steiner) could not be distin-guished from one another, nor could the tworespiratory strains (650 and 1309) be separatedon the basis of their neutralization rates. Signif-icantly, the genital strains were neutralizedmore rapidly (P < 0.05) than the respiratorystrains. The two vaccine strains were also indis-tinguishable from one another, and were neu-tralized far more rapidly than either the respira-tory or the genital strains.The rates of neutralization of IBR-IPV virus

strains by late anti-K22 19S AB are given in Fig.7. The K and NK values of the virus strains

2 MINUTES 10FIG. 5. Neutralization of 650 virus (IBR) by late

homologous 7S AB (immunoglobulin G [IgG]). Thevirus was rapidly neutralized in the absence of com-plement (c), but complement did seem to slightlyenhance the neutralization rate.

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FIG. 6. Neutralization of IBR-IPV virus strains bylate anti-650 (anti-IBR) 19S globulins (immunoglobulinM[IgM]). Note that the genital strains (K22 andSteiner) were neutralized more rapidly than therespiratory strains (650 and 1309). The vaccine strains(Diamond and Jensal), however, were neutralizedmore rapidly than the genital strains.

TABLE 2. Neutralization rate constants (K) and nor-malized neutralization rate constants (NK) of IBR-

IPV virus strains using late anti-650 19S AB

Virus K NK

K22 0.078 169.6Steiner 0.069 150.0650 0.046 100.01309 0.047 102.2Jensal 0.149 323.9Diamond 0.156 339.1

with this AB preparation are listed in Table 3. A1:2 dilution of late anti-K22 19S AB was found,in preliminary tests, to be the most suitabledilution for use in these kinetic neutralizationtrials. The two respiratory strains (650 and1309) and the one genital strain (Steiner) wereneutralized at approximately the same rate,whereas the homologous genital strain (K22)was neutralized significantly more rapidly (P <0.05) than the former three strains. The two

vaccine strains were neutralized at a similarrate, and both were neutralized much morerapidly than either the genital or the respiratorystrains.

DISCUSSIONPreliminary results suggested that IBR-IPV

virus strains could not be differentiated by their

FIG. 7. Neutralization of IBR-IPV virus strains bylate anti-K22 (anti-IPV) 19S globulins (immunoglobu-lin M[IgM]). The two vaccine strains (Jensal and Dia-mond) were neutralized at a similar rate, whereas thetwo respiratory strains (1309 and 650) and one genitalstrain (Steiner) were neutralized at a similar rate. Theother genital strain (K22) was neutralized more rap-idly than the respiratory strains but slower than thevaccine strains.

TABLE. 3. Neutralization rate constants (K) andnormalized neutralization rate constants (NK) ofIBR-IPV virus strains using late anti-K22 19S AB

Virus K NK

K22 0.080 100.0Steiner 0.041 51.3650 0.049 61.31309 0.045 56.3Jensal 0.222 277.5Diamond 0.265 331.5

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neutralization rate using either early or late 7SAB. In contrast, both early and late 19S ABneutralized the homologous strain more rapidlythan the heterologous strain. Late 19S AB wasthus used for comparing the various virusstrains by neutralization kinetics, since largeramounts of late antiserum was available andthe difference in neutralization rate between thehomologous and heterologous strain seemedgreater with the late 19S AB. In addition,Hampar et al. (10) reported that late 19S ABwas four to eight times more efficient than early7S, late 7S, or early 19S AB in distinguishingherpes simplex virus strains by neutralizationkinetics. Similar results were also observedwhen late 19S AB were compared with the otherimmunoglobulins in distinguishing strains ofsimian cytomegaloviruses (9) and in differenti-ating between SA8 herpesvirus and herpes sim-plex virus (24). The results reported here withIBR-IPV virus were similar except that early19S AB seemed to approach the specificity oflate 19S AB.

Neutralization kinetics with IBR-IPV virusstrains using late 19S AB revealed significantdifferences between some of the strains. Anunexpected and striking observation was theextremely rapid neutralization of the vaccinestrains by both the antigenital (anti-K22) andthe antirespiratory (anti-650) globulin prepara-tions. The two vaccine strains could not, how-ever, be distinguished from one another by theirrates of neutralization with either anti-650 oranti-K22 19S AB. The two respiratory strains,650 and 1309, had similar neutralization rateswhen anti-650 19S AB was used. The somewhatsurprizing result with the genital strains (K22and Steiner) was that they were neutralizedsignificantly more rapidly than the homologousrespiratory strain. These genital strains were,however, indistinguishable using the later glob-ulin preparation. Thus, with anti-650 19S glob-ulin, the respiratory, genital, and vaccinestrains each had their distinctive rates of neu-tralization. It seems that with this globulinpreparation the potential does exist for distin-guishing strains of IBR-IPV virus by neutraliza-tion kinetics according to their origin. However,before a definitive position can be adopted, astudy including a larger number of strainsshould be undertaken. Such a study is presentlybeing considered.The distinction between strains was not as

clear when anti-K22 19S AB was used. Onegenital strain (Steiner) behaved like the tworespiratory strains. The homologous virus (K22)was, however, neutralized more rapidly than

the two respiratory strains and the other genitalstrain.

In this study, several heterologous viruseswere neutralized more rapidly than homologousstrains, but the reason for this is not clear.Apart from the origin of these virus strains, theonly other known difference between them istheir passage level in cell cultures. The vaccinestrains had been passaged 82 to 111 times inbovine cell cultures and in cells derived fromother animal species. Steiner virus had beenpassaged approximately 11 times and K22 virus29 times in bovine cell cultures. The two respi-ratory strains had been passaged the smallestnumber of times (approximately eight to ninetimes in bovine cell cultures). It is tempting tospeculate that the rate by which these viruseswere neutralized by late 19S AB was, in someway, a function of the number of passages theyhad undergone in cell cultures. This conclusionseems to correlate with the results of neutraliza-tion kinetics with anti-K22 19S AB, sinceSteiner virus was neutralized at a rate similar tothe two respiratory strains. The passage level ofSteiner virus is approximately equal to that ofthe respiratory strains, but less than half thanthat of K22, the other genital strain. In ad-dition, the two vaccine strains were neutral-ized considerably more rapidly than the homol-ogous virus when either anti-650 or anti-K2219S AB was used. In an earlier study with wholeantiserum, Buening and Gratzek (4) also ob-served the phenomenon where a heterologousstrain of IBR-IPV virus was neutralized morerapidly than the homologous strain. However,the cell culture passage levels of the virusstrains were not considered in their study. Theabove theory would require that cell adaptationof a virus enhances the rate at which it isneutralized by AB to an unadapted strain. Howcell adaptation could enhance neutralization ofa virus is not known, but it is recognized thatherpesviruses may acquire new antigens afternumerous passages through cell cultures (8). Itdoes not seem possible, however, that newantigens can influence the neutralization of avirus to antibodies which do not have a specific-ity directed against these antigens. The answermay lie in the reported alteration of the virionsurface charge of viruses after passage in cellcultures (22). Since the binding of AB to anti-gen is known to be influenced by the relativecharge on each (2), it is possible that thealteration in the viral charge due to passage incell cultures could enhance the binding ofspecific neutralizing AB to the virus.Many workers have reported that IBR-IPV

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virus strains form an antigenically homogeneousgroup (1, 5, 6, 15, 18-20, 23, 27), but some didfind strain differences by neutralization kinetics(4, 12). In these latter studies, whole serum wasemployed and strains could not be classifiedaccording to their origin. Matheka and Straub(22) and Straub et al. (26) reported that genitaland respiratory strains were distinguishable bycarrier-free zone electrophoresis based on differ-ences in the surface charges of the virions.However, they found that these charge differ-ences disappeared after several cell culturepassages. The unique antigenic nature of thevaccine strains of IBR-IPV virus which weobserved has apparently not been reported inthe literature.The action of complement on the neutraliza-

tion of IBR-IPV virus by globulins purified fromspecific rabbit antisera was similar to what hasbeen reported for herpes simplex virus (11).Early 7S, early 19S, and late 19S globulinsderived from specific rabbit antisera were al-most completely dependent on complement forthe neutralization of IBR-IPV virus. In contrast,late 7S AB efficiently neutralized this virus inthe absence of complement. Complement didappear, however, to slightly increase the rate bywhich IBR-IPV virus was neutralized by late 7SAB.

Neutralization of IBR-IPV virus by earlywhole antiserum was also complement depend-ent. This was expected, since both 7S and 19SAB derived from early antisera were comple-ment dependent. It is not known, however, towhat extent either of the latter two globulintypes in whole serum contributes to the neutral-ization of a virus in the presence of complement.The dependence of early antisera on comple-ment for virus neutralization has also beenobserved with herpes simplex virus (14). In thepresent study, late immune sera were not de-pendent on complement for neutralization ofIBR-IPV virus, indicating that the 7S globulincomponent of these sera dominated over the 19Scomponent. The rate at which IBR-IPV viruswas neutralized by late antiserum was slightlyenhanced by complement, which is similar towhat was observed with 7S globulin derivedfrom such serum.The use of specific antiserum in liquid me-

dium to achieve plaquing with IBR-IPV viruswas preferred to an agar overlay, since theformer method was more convenient, clearerplaques were produced, and the results weremore reproducible. Stevens and Groman (25)observed that secondary IBR-IPV virus plaquesdeveloped when this method was used, which

they attributed to detachment, translocation,and reattachment of infected cells. They were,however, able to distinguish between the pri-mary and the considerably smaller secondaryplaques. In this study very few of the secondaryplaques were observed, since the concentrationof antiserum used in the plaquing medium(0.5%) allowed the counting of the plaques aftera relatively short postinfection period (30 to 48h). Antiserum concentrations above 2% resultedin a degeneration of the cells, which was proba-bly due to antibodies against cellular antigens,since absorption of antisera with cells prior touse in the liquid overlay markedly reduced thistoxicity. It was not necessary to absorb theantiserum when it was used at the workingdilution of 0.5%, and such plaquing mediumresulted in rapid plaque formation even after 11months of storage at 4 C.

In conclusion, it does seem that the distinc-tive neutralization of the vaccine strains ofIBR-IPV virus by specific rabbit 19S AB may beused to distinguish attenuated strains from wildstrains of IBR-IPV virus. Notwithstanding thepossibility that neutralization kinetics with 19SAB may be detecting antigens somehow relatedto cell culture adaptation, some preparationsapparently have the potential of distinguishingbetween genital and respiratory strains of IBR-IPV virus.

ACKNOWLEDGMENTWe are grateful for the able technical assistance of Anna

Yeung.

LITERATURE CITED

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4. Buening, G. M., and J. B. Gratzek. 1967. Comparison ofselected characteristics of four strains of infectiousbovine rhinotracheitis virus. Amer. J. Vet. Res.28:1257-1267.

5. French, E. L. 1962. Relationship between infectiousbovine rhinotracheitis (IBR) virus and a virus isolatedfrom calves with encephalitis. Aust. Vet. J. 38:555-556.

7. Gillespie, J. H., J. A. Baker, and W. C. Wagner. 1958.The relationship of infectious pustular vulvovaginitisvirus to infectious bovine rhinotracheitis virus. Proc.U.S. Livestock Sanit. Ass. 62:119-126.

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14. Linscott, W. C., and W. E. Levinson. 1969. Complementcomponents required for virus neutralization by earlyimmunoglobulin antibody. Proc. Nat. Acad. Sci.U.S.A. 64:520-527.

15. Lukas, G. N., S. J. Weidenbach, K. G. Palmer, C. W.Dickie, R. F. Duncan, and J. Barrera. 1963. A bovinefetal viral isolate neutralized by IBR immune serum asa cause of abortion in cattle. Proc. U.S. LivestockSanti. Ass. 67:108-128.

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