Characterization of InfectiousLaryngotracheitis Viruses, Antigenic Comparison

by Kinetics of Neutralization and Immunization Studies

R.G. Russell and A.J. Turner*

ABSTRACT

Five isolates of infectiouslaryngotracheitis virus werecompared by pock formation on

the chorioallantoic membraneof embryonated eggs, plaquesize in chicken embryo kidneytissue culture, and antigenicrelationship using reciprocalkinetics of neutralization.The A4557-5 strain of infec-

tious laryngotracheitis virus,which causes mild respiratorydisease, produced pocks with azone of edema on the chorioal-lantoic membrane. A virulentvirus (Virus 1), isolated from anoutbreak of severe disease char-acterized by a diphtheriticlaryngotracheitis, produced thelargest plaques in chickenembryo kidney cell culture.Other virulent viruses (Viruses2, 3 and V154) did not have uni-que growth characteristicswhen grown on the chorioallan-toic membrane or in chickenembryo kidney cell culture. Allviruses were closely relatedantigenically as shown by kinet-ics of neutralization but viruses2 and 3 were not homogeneouswith the other three viruseswhen neutralized by anti-V154chicken serum.Following aerosal infection,

chickens infected with theA4557-5 virus were immune tochallenge with virulent V154virus. However, in comparison

to SA-2 virus, this virus was a

less effective immunizing agentwhen administered by the ventor drinking water methods.

RPSUMt

Cette experience consistait acomparer cinq souches du virusde la laryngo-tracheite aviaireen se basant sur la formation delesions sur la membrane chorio-allantoldienne d'oeufs embryon-nes, les dimensions des plagesdans des cultures de cellulesrenales d'embryons de poulets etla relation antigenique entre cessouches, telle que demontree parla cinetique reciproque de laneutralisation.La souche A4557-5, qui cause

une maladie respiratoire be-nigne, produisit des lesions quiaffichaient une zone d'oedeme,sur la membrane chorio-al-lantoidienne. Une souche par-ticulierement virulente, appel6evirus #1 et isolee d'une eruptiongrave de la maladie qui se carac-terisait par une laryngo-trache-ite diphterique, produisit lesplages les plus volumineusesdans les cultures de cellulesrenales d'embryons de poulets.Les autres souches virulentes, asavoir les virus #2, #3 et V154,n'afficherent pas de caract6ris-tiques de croissance uniques,lorsqu'on les inocula sur lamembrane chorio-allantoidien-

ne d'oeufs embryonn6s ou dansdes cultures de cellules renalesd'embryons de poulets. Toutesces souches possedaient uneparente antigenique, comme ledemontra la cinetique de la neu-tralisation; les virus #2 et #3 ne sereve'lrent toutefois pas homo-genes avec les trois autres, lors-qu'on les soumit A l'epreuve deneutralisation avec l'antiserumde poulet specifique a la soucheV154.Apres une infection au moyen

d'aerosols, les poulets infectesavec la souche A4557-5 serevel'rent protge's contre uneinfection de d6fi avec la soucheV154. Par comparaison avec lasouche SA-2, le virus V154s'av6ra toutefois un antigtlnemoins efficace, lorsqu'on l'ad-ministra par le cloaque ou dansl'eau de boisson.

INTRODUCTION

Common antigens have beendemonstrated among isolates ofinfectious laryngotracheitis virus(ILTV) by neutralization tests andcross-protection studies in chick-ens (8, 19, 20, 22, 25, 28). Variationin the neutralization of ILTV vir-uses with heterologous antiserahas been reported (19, 25, 28) rais-ing questions about possible anti-genic differences among strains ofILTV. In some studies sufficientlysensitive quantitative methods

Can J Comp Med 1983; 47: 163-171.

*Victorian Department of Agriculture, Veterinary Research Institute, Park Drive, Parkville, Victoria, Australia 3052 and VictorianDepartment of Agriculture, Attwood Veterinary Research Station, Mickleham Road, West Meadows, Victoria, Australia 3047. Presentaddress of senior author: Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, P.O. Box 1071, Knoxville,Tennessee 37910.Submitted April 5, 1982.

were not used and further studiesof antigenic relatedness among iso-lates of ILTV are therefore war-ranted. For the experimentsreported in this paper, kinetics ofneutralization was used for anti-genic analysis of five isolates ofILTV.

Reciprocal neutralization kinet-ics is a sensitive serological methodfor studying the antigenic rela-tionship among closely relatedviruses (16). The method demon-strated antigenic subgroupsamong herpes simplex viruses thatshowed cross-neutralization, pro-viding a basis for classification ofherpes simplex virus types 1 and 2(2, 9). Using kinetics of neutraliza-tion, a spectrum of antigenic rela-tionships was proposed for cyto-megalovirus (1, 29). Neutrali-zation kinetics has been used toanalyze antigenic relatedness ofother herpesviruses includinginfectious bovine rhinotracheitis(3, 18), infectious equine rhino-pneumonitis (14), Marek's diseaseof chickens (12) and cytomegalovi-rus (1, 29).This study documents the biolog-

ical properties of five isolates ofILTV by their pock formation onthe chorioallantoic membrane(CAM) of embryonated eggs andcytopathology in chicken embryokidney (CEK) tissue culture. Theantigenic relationship of these vir-uses was compared by neutraliza-tion kinetics. The V154 virus is areference strain of virulent ILTV.Viruses 1, 2 and 3 were field iso-lates from severe clinical disease.The A4557-5 virus caused mildrespiratory infection in experi-mental chickens following aerosolinfection (24). Immunization stu-dies were undertaken to evaluatethe potential of this virus isolatefor application as a vaccinatingagent.

MATERIALSAND METHODS

VIRUSES

Virus V154 is a virulent isolateof ILTV causing necrotizing tra-cheitis. This virus was obtainedfrom the Veterinary Research Sta-tion, Glenfield, New South Wales

and was plaque purified threetimes in CEK. Aerosol infection ofexperimental chickens with V154virus caused a severe necrotizingtracheitis, acute suppurativebronchopneumonia with necrotiz-ing bronchiolitis and suppurativeairsacculitis (24).Viruses 1, 2 and 3 were isolated

from chickens in natural out-breaks of respiratory disease inVictoria. Viruses 1 and 3 causeddiphtheritic laryngitis in eight and14 week old chickens respectively.Significant mortality and highmorbidity occurred in both out-breaks. Virus 2 was isolated frombirds in a flock of six week oldchickens with high morbidity ofcoughing, conjunctivitis and mu-coid tracheitis; the mortality waslow but growth rate of affectedchickens was impaired.The A4557-5 was isolated from

chickens with mild upper respira-tory disease and was obtained fromthe Department of Virology, Uni-versity of Queensland. This viruscaused mild catarrhal tracheitiswith focal lymphocytic infiltrationin the lungs and mild focal lym-phocytic airsacculitis in experi-mental chickens infected by aero-sol (24).Virus SA-2 is used in commer-

cial vaccines throughout Austra-lia. It is a small plaque clone of aSouth Australian virus. The virushad been passaged approximatelysix times in embryonated eggsbefore plaque purification. It wasreceived as first passage stock ofthree times plaque purified virusfrom the National BiologicalSciences Laboratory, Parkville,Victoria. Electron microscopicexamination had shown it to befree of contaminating agents.

PROPAGATION OF VIRUSES

Field isolates were obtained onthe chorioallantoic membrane(CAM) of nine to 11 day oldembryonated eggs from trachealscrapings of infected chickens.Membranes with raised focalwhite pocks and confluent whitishthickening were harvested sixdays postinoculation.

Cell cultures of chicken embryokidney (CEK) were prepared from

18 to 20 day old embryos andgrown in medium 199 with 5% fetalcalf serum (7, 13). Cell monolayerswere prepared in 60 mm disposa-ble Petri dishes seeded with 4 x 106viable cells in 8 mL of medium andgrown at 37°C in a humidified CO2incubator. Stocks of each virus forcomparative studies were pre-pared from infected CAM becauseof the difficulty in obtaining hightiters of A4557-5 virus in cellcultures.

PLAQUE PURIFICATIONOF VIRUSES

Viruses were cloned three timesby selection of a single plaque in aCEK monolayer overlaid with5 mL of 1% nutrient agar inmedium 199 with 5% fetal calfserum (agar maintenancemedium).VIRUS NEUTRALIZATION

Viruses 1,2,3 and A4557-5 wereidentified by virus neutralization.Serial twofold dilutions of antise-rum prepared in rabbits againstV154 virus were mixed with anequal volume of virus containing800 PFU/mL and incubated at37°C. After two hours, virus-antiserum mixtures were titratedin CEK for residual virus.

ELECTRON MICROSCOPY

Chicken embryo kidney mono-layers grown in 10 mL plasticflasks were seeded with virus at0.01 multiplicity of infection andharvested at 70% cytopathology.After centrifugation at 3000 X gfor ten minutes the cells in thepellet were lysed with 0.5 mL ste-rile distilled water. The virus sus-pension was stained negativelywith 4% phosphotungstic acid (PH7.2) on 300 mesh formar coatedgrids and examined under theelectron microscope.CYTOPATHOLOGY IN CEK

Histological examination ofCEK monolayers was conductedusing coverslip preparations har-vested 24 to 48 hours after inocula-tion with virus and fixed in 10%buffered neutral formalin. Theywere stained with hemotoxylinand eosin and examined histologi-

164

cally for syncytia containing her-pesvirus inclusion bodies.

ETHER SENSITIVITY

The viruses were tested for ethersensitivity by adding one part ofdiethyl ether to four parts of virus.The mixture was shaken vigor-ously and held at 4°C overnighttogether with an untreated sus-pension of virus. Ether wasremoved by centrifugation andthen the virus suspension was heldfor evaporation to ensure all etherwas removed. Treated and un-treated samples were titrated forvirus using CED monolayers.

TITRATION OF INFECTIVITY

The titer of viruses was deter-mined in CEK by assaying0.25 mL of serial tenfold dilutionsof virus. The dishes were overlaidwith agar maintenance mediumand on the third day a stainingoverlay of 0.01% neutral red in 1%agar was applied, the plaques werecounted on the fourth day andexpressed as plaque forming unitsper mL (PFU/mL).COMPARISON OF POCKSON THE CAM

The pock characteristic of thefive viruses was compared fivedays after inoculation of the CAMwith 0.1 mL virus suspensionresulting in approximately 20pocks per membrane. The diame-ter of pocks was measured for eachvirus using photographs of theinfected CAM that were enlargedthree times. Statistical analysis ofpock diameters for the five viruseswas accomplished with one wayanalysis of variance. Differencesamong means were partitionedwith Duncan's multiple range testas modified by Kramer.

PLAQUE SIZE IN CEK CULTURESThe CEK monolayers in 60 mm

Petri dishes were inoculated withvirus suspension containing about50 PFU and overlaid with agarmaintenance medium. Photo-graphs of the plaques were takenfour days postinfection and en-larged three times. The diameterof plaques was measured on thephotographs. Statistical analysis

was by the procedure used to testpock diameter.

THERMOSTABILITY OF VIRUSES

For thermal inactivation of thefive viruses, a suspension of eachvirus was prepared containing 5 x103 PFU per 100 mL gelatin saline(28). The flasks containing virussuspension were tightly stopperedand maintained at 37°C in a waterbath. Initially and at three hourlyintervals over a period of 12 hours,1 mL aliquots of virus wereremoved for assay of residualinfectivity.PREPARATION OF SPECIFICANTISERA

Monospecific antisera againsteach of the five viruses were pre-pared in 23 week old specific-pathogen-free White Leghornchickens for kinetics of neutraliza-tion analyses. Chickens were inoc-ulated by intratracheal route with1 mL inoculum of the second pas-sage of a plaque purified virususing 1 x104 PFU of viruses 1, 2,3,V154 or 3 X 104 PFU of A4557-5.Chickens were bled by cardiacpuncture three weeks post-inoculation.Antiserum to the V154 strain

was prepared also in rabbits byintramuscular inoculation of0.5 mL of a virus suspension con-taining 1 x103 PFU emulsified inFreund's complete adjuvant.Serum was obtained three weekspostinoculation for virus neutrali-zation tests.

NEUTRALIZATION KINETICS

Antigenic comparison of the fiveviruses was made using neutrali-zation kinetics to measure the rateof virus neutralization by specificantisera prepared against each ofthe five viruses in chickens. Theviruses and antisera were pre-warmed to 37°C in a water bathand at 15 seconds prior to timezero, 1.0 mL of a virus suspensioncontaining 105-4 to 106 plaque form-ing units was added to 1.0 mL ofantisera (used at a dilution deter-mined by preliminary experi-ments to neutralize 90% of homolo-gous virus in 30 minutes at 37°C).The mixture was agitated and at

time zero, five, 10, 20 and 30 min-utes, 0.1 mL was removed and dil-uted in 9.9 mL of gelatin saline at4°C. This sample and an additional1:10 dilution was assayed for infec-tive virus in CEK monolayers. Ineach neutralization experimentthe antiserum against a givenvirus was tested against the fiveviruses on the same day. The neu-tralization kinetics of each anti-serum was repeated once. Exper-imental results were analyzed asfollows: K = D x 2.3 x log vo whereK is the neutralization rate con-stant, D is the dilution of serum, Vois virus titer at time zero and Vt istiter virus at time t (30 minutes)(16). The K value was obtained foreach serum virus interaction fromthe slope of the regression line andthe K values of different sera werecompared by normalizing the neu-tralization constant to obtain NKvalues. In this procedure the Kvalues of neutralization kineticsfor the homologous virus andserum is assigned a value of 100and heterologous K values areexpressed as proportions of homo-logous neutralization kinetics (16).Regression lines were analyzedstatistically by least squares anal-ysis of variance which containedthe classification variables (virusand day of test) and continuousvariables (log time and interactionof log time and virus).IMMUNIZATION STUDIES

Experimental chickens - Chick-ens were Rhode Island Red orAustralorpx White Leghorn ob-tained at day-old from a commer-cial hatchery. They were main-tained in wire cages throughoutthe experiments, except for thedrinking water vaccination trial,in which they were placed on litterone week before the experimentstarted.

Aerosol administration - Themethods used have been describedpreviously (24). Briefly, a virussuspension of allantoic fluid con-taining 1 X 105 PFU per mL wasaerosolized as 2.0,m particlesfrom a nebulizer. Fifteen chickenswere infected with A4557-5 andSA-2 viruses and ten chickens

165

received V154 virus. Ten chickensfrom each group and five controlchickens were sacrificed five dayspostinfection for pathologicalexamination.On the 15th day postinfection

convalescent sera were obtainedfrom the remaining five chickensinfected with A4557-5 and SA-2viruses. The following day theywere challenged with 10,000 PFUof V154 virus in 1 mL inoculum.The chickens were inspected dailyfor clinical signs. They were sacri-ficed and examined for grosspathological evidence of laryngo-tracheitis five days after thechallenge.

Vaccination withA4557-5 andSA -2 viruses by the vent method - Thechickens to be vaccinated were dis-tributed into 14 groups, each con-taining ten chickens. The 15groups were held isolated fromeach other.The various groups of chickens

were vaccinated with increasingdoses of 50, 500, 5 x 103,5 x104,5X105 and 5 X 106 PFU per mL. Viruswas applied to the vent mucosawith an abrasive match stick-likeapplicator which delivered ap-proximately 0.01 mL.On the 16th and 17th days post-

vaccination the vaccinated chick-ens and six unvaccinated controlswere challenged intratracheallywith 2500 PFU of V154 virus in0.5 mL inoculum. Five days post-challenge they were sacrificed andthe tracheas were examined.

Vaccination by the drinking waterwith A4557-5 and SA-2 viruses -One week prior to the experimentthe chickens to be vaccinated weremoved from the wire cages andplaced on deep litter in two groupsof eight chickens in separate pens.The five controls remained in wirecages and isolated.The viruses were administered

in PBS with 1.0% skim milkpowder to provide an estimatedconcentration of 5 X 105 PFU permL. Prior to vaccination thechickens were deprived of waterfor ten hours. They were allowed todrink the virus suspensions fromthe plastic base of a commercial

one gallon drinker over a period ofone and one-half hours. Samples ofthe virus suspension were passedthrough a 0.45,u millipore filterand assayed for virus after one anda half hours.Convalescent sera were obtained

18 days postvaccination. These 16chickens and the five susceptiblecontrols were challenged with2500 PFU of V154 in 0.5 mL inocu-lum. After five days they were sac-rificed and the tracheas examinedby gross pathology.

Humoral immunity following vac-cination - Serum antibody titerswere determined by the plaquereduction method using homolo-gous serum virus for neutraliza-tion. Serial twofold dilutions ofserum were made from a startingdilution of 1:25. An equal volume ofthe serum dilution and virus sus-pension containing 100 PFU permL was incubated at 37°C for half-hour neutralization. Two controltubes of virus in equal volume ofdiluent were maintained at 37°Cand 4°C. Chicken embryo kidneymonolayers were inoculated with0.25 mL of serum-virus mixture.Plaques were counted on thefourth day. The titre of neutraliz-ing antibody was calculated as thedilution of serum that neutralized50% of the virus.

RESULTSIDENTIFICATION OF VIRUSES

Viruses 1, 2,3 and A4557-5 wereshown to be strains of ILTV by thefollowing criteria: pock formationon the CAM, syncytia formation inCEK with eosinophilic intranu-clear inclusion bodies; neutraliza-tion of cytopathic effects in CEKby rabbit anti-V154 serum andether sensitivity. Enveloped her-

Fig. 1. Pocks produced by virus 2 on thechorioallantoic membrane.

pesvirus particles 200 nm diame-ter were demonstrated by electronmicroscopy in preparations of eachof these four strains of ILTV.

POCK MORPHOLOGY ON THE CAM

Viruses 1, 2, 3 and V154 pro-duced typical raised whitish pockson the CAM (Fig. 1). The A4557-5pocks were morphologically dis-tinct from the other viruses sincethey consisted of a raised centersurrounded by a clear zone (Fig. 2).The mean pock diameter of thisvirus was 3.4 mm with a range of1.5 to 6.0 mm, depending on thewidth of the edema halo. The pocksproduced by the other four viruseswere smaller (P<0.05), having amean diameter of 1.2-1.6 mm(Table I, Fig. 3).

PLAQUE SIZE IN CEK

The mean plaque diameter ofvirus 1 was 1.34 mm compared tothat of the other four viruses whosemean plaque diameters were 1.18to 1.22 mm, (Table II, Fig. 4).The plaque diameter of virus 1

was significantly larger thanA4557-5 and V154 viruses by sta-tistical analysis using Duncan'smultiple range test as modified by

TABLE I. Pock Size on the Chorioallantoic Membrane

Number of Number of Mean pock Standardpocks CAM per diameter deviation

Virus measured virus mm mm

Virus 1Virus 2Virus 3V154A4557-5

5633352426

10777

11

1.561.401.221.303.41

0.590.360.160.291.27

166

TABLE II. Plaque Size in Chicken Embryo Kidney Cell Culture

Number of Total number of Mean Diameter StandardVirus Cultures plaques measured mm Deviation mmVirus 1Virus 2Virus 3V154A4557-5

1514111413

146109123126112

1.341.221.231.191.18

0.220.140.240.110.09

1,~ ~ ~ .,

Fig. 2. Pocks (arrows) produced on thechorioallantoic membrane by A4557-5strain of ILTV.

Kramer. It was recognized how-ever that the plaque size of each ofthe viruses was heterogeneous. Inan attempt to stabilize subgroupvariances, logarithmic transfor-mation of the data was deemedappropriate. Subsequent analysisindicated no significant differen-

100.Virus

50

Virus 250

rD ~~~~~~~~~Virus350

0 _

_-

A4557-5S50.

0.6- I- 1.6- 21- 2.6- 3.1- 3.6- 4.1- 5.1-.0 1.5 2.0 2.5 30 3.5 4.0 5.0 6.0

Pock Diametermm

Fig. 3. Comparison of pock sizes on thechorioallantoic membrane by fivestrains of ILTV.

ces among the plaque sizes of thefive viruses.

THERMAL STABILITY

Thermal inactivation at 37°Cwas similar for the five strains ofILTV. The amount of infectivitywas inactivated by 88-95% of initialactivities during the test period of12 hours.

NEUTRALIZATION KINETICS

The results of neutralization100

50

50

G)Ca

c0)

01)a.

50

50

50

Virus

Virus 2

Virus 3

V154

A 4557- 5-.-0- 0.6- 1.1- i.6-0. 5 1.0 1.6 2.0

Ploque Diametermm

Fig. 4. Comparison of plaque sizes inchicken embryo kidney cell culture pro-duced by five strains of ILTV.

reactions are shown in Figs. 5-9.All neutralization reactions fol-lowed first-order kinetics as shownin semilogarithmic graphs. Neu-tralization rate constants (K) andnormalized K values (NK) arepresented in Table III. A closeantigenic relationship was demon-strated among the five viruses inneutralization kinetics test usingchicken antisera against four ofthe viruses (viruses 1, 2, 3 andA4557-5).However, with anti-V154 chick-

en serum, neutralization of virusesTime - Minutes

5 10 20 30

0 5-

-J

15

2.0

Anti-V154 serum.

T--------- Virus

...-o Virus 2

*-.-*-.4U Virus 3* _ V 154

_---6 A4557-5

Fig. 5. Neutralization kinetics slopesusing anti-V154 serum against fivestrains of ILTV.

TABLE III. Comparison of the Reciprocal Neutralization Rate of Five ILT Viruses

Anti-virus 1 Anti-virus 2 Anti-virus 3 Anti V154 Anti-A4557-5

Ka NKb K NK K NK K NK K NK

Virus 1 1.33 100.00 1.32 123.4 1.21 100.0 1.07 78.7 1.06 90.6Virus 2 0.97 72.9 1.07 100.0 1.04 86.0 0.79 58.0 1.00 85.5A4557-5 0.96 72.2 1.07 100.0 0.90 74.4 1.36 100.0 1.17 100.0Virus 3 1.07 80.5 1.07 100.0 1.21 100.0 0.69 50.7 1.11 94.9V154 1.15 86.5 1.36 127.1 1.21 100.0 1.36 100.0 0.91 78.7

aK Neutralization constant determined by equation K equals D/t 2.3 log 10 Vo/VtbNK Normalized constant (see reference 15)

167

2 and 3 gave NK values of 58 and 50which was lower than the otherthree viruses. The F statistics indi-cated a highly significant interac-tion between time and virus andtherefore the slopes of lines in Fig.5 cannot be considered homogene-ous. On further inspection the sta-tistical analysis showed that vir-uses 1, V154 and A4557-5 hadhomologous slopes, viruses 2 and 3also had homologous slopes to eachother, but these two viruses hadslopes which were measurably dif-ferent from the other three.

IMMUNIZATION STUDIES

Aerosol administration - Respi-ratory signs were not observed inthe group inoculated with A4557-5virus. Gross and histopathologicalexaminations confirmed a mildrespiratory disease (24).Chickens infected with SA-2

virus developed clinical signs bythe third day. They showed pro-nounced respiratory distress withheavy breathing, coughing andsneezing. Postmortem examina-tion showed a copious amount ofyellow mucous in the lumen of thetrachea, the wall was thickenedand there was necrosis of themucous membrane. The air sacswere moist and cloudy in severalchickens. Histological examina-tion showed marked regenerationhyperplasia of the epithelium inthe trachea and bronchi, suppura-tive brochopneumonia and airsac-culitis. The clinical signs andpathological findings were similarto those described previously (21).

Infection with V154 was used tomonitor the infectivity of aerosolexposure. Chickens infected withV154 showed a severe, necrotizinglaryngotracheitis, acute suppura-tive necrotizing bronchopneumo-nia and necrotizing airsacculitis.Therefore, adequate administra-tion of viruses was obtained withthe aerosol procedure (24).Antibody titres of sera taken 19

days postinoculation showed amean titre of 1:436 (range 1:390-1:500) in chickens infected withSA-2 virus. This was approxi-mately 12 times higher than theantibody titre of chickens infectedwith A4557-5 virus (mean 1:35;

TABLE IV. Immunity after Vent Vaccination with SA-2 and A4557-5

SA-2 A4557-5Vaccine Dose Number of Number Number of NumberPFU/mL Chickens Immune Chickens Immune

50500

5,00050,000

500,0005X106

101010101010

range 1:24-1:56). Chickens in bothgroups were challenged with10,000 PFU dose of V154 virus andwere all immune as determined byclinical observation and grosspathology five days after infection.

Vent vaccination - Significantimmunity was obtained with adose of vaccine containing 5 x 102PFU/mL of SA-2 virus. By com-parison the A4557-5 virus admin-istered at doses of 5 X 106 PFU permL protected three of ten chickensagainst intratracheal challengewith 2500 PFU of V154 virus(Table IV). Three chickens hadnormal tracheas at postmortemexamination five days after chal-lenge. Of the remaining sevenchickens two showed necrotizinghemorrhagic tracheitis. Two hadulceration and plaques of diphthe-ritic tracheitis, and the other threeshowed mucoid tracheitis. Threenonvaccinated chickens alsoshowed mucoid or diphtheritictracheitis, and two of the fivechickens died with necrotizinghemorrhagic tracheitis.

Vaccination by administration inthe drinking water - There wasvirtually no loss of virus in the dil-uent over the one and one-half hourperiod of vaccination. The esti-mated dose of virus administeredto both groups was similar, withchickens receiving 4.4x 105 ofA4557-5 and 5.1 X 105 of SA-2virus.

All of the eight chickens vacci-nated with SA-2 virus wereimmune to challenge with 2500PFU of V154 virus. Chickens vac-cinated with SA-2 virus showedhumoral immune response with amean titer of 1:111 (range 1:14-1:170) after vaccination. Approx-imately 50% of chickens adminis-

010101099

101010101010

003223

tered the A4557-5 virus in thedrinking water were fully pro-tected. All eight chickens survivedand three of the chickens had nor-mal tracheas at postmortem exam-ination. The remaining fivechickens had thickened tracheaswith moderate mucoid exudateand three of these chickens hadfocal areas of ulceration in the tra-chea or pharynx. This was a lesssevere tracheitis than occurred inthe nonvaccinated chickens afterchallenge with V154 virus. Threeof them showed diphtheritic tra-cheitis and two chickens died withhemorrhagic tracheitis. Only onechicken vaccinated with A4557-5virus had a significant rise inantibody titer (1:39). Anotherchicken had a marginal rise intiter (1:69). Notwithstanding thefailure to develop significant hum-oral antibody response, the resultsindicate that vaccination withA4557-5 via the drinking waterfully protected some chickens andconferred a partial immunity onthe remaining chickens.

DISCUSSION

The pocks produced by A4557-5on chorioallantoic membraneswere characteristic with a clearhalo of edema around the centralraised white foci. Differences inthe pock morphology and pockdiameter of ILTV have been doc-umented previously (4, 5, 10, 11,19, 20, 30). During serial passagesof an ILTV virus in CEK cultures,two populations of pocks with dif-ferent morphology were observed(10); the smaller foci were sur-rounded by a clear halo similar tothose reported here with A4557-5virus. This change in pock mor-phology was retained following

168

further passage in experimentalchickens (11). Another strain ofILTV with pocks similar to A4557-5 has been described (30). Thisstrain caused pocks which were 3-5 mm diameter with raised centralfoci and an outer edematous zone.However, unlike A4557-5 it causedsevere disease suggesting thatpock morphology among ILTV isnot correlated with pathogenicity.

Initial statistical analysis sug-gesting a significant difference inthe plaque size of virus 1 was notsubstantiated on further analysisby logarithmic transformation ofthe data. This implies that the con-siderable heterogeneity in plaquesize of each of the viruses, was ofsuch magnitude, that virus 1 couldnot be said with certainty to pro-duce plaques of larger diameterthan the other four viruses in CEK.Plaque variants have been re-ported with many viruses and insome instances this property isassociated with other characteris-tics of the virus. A strain of infec-tious bovine rhinotracheitis from acalf with meningoencephalitisproduced smaller plaques com-pared to isolates from the respira-tory and genital tracts (3). Anavirulent strain of Marek's diseasevirus produced large plaques whe-reas virulent strains did not (6). Allof the viruses in this study, with theexception of A4557-5, were viru-lent. In other studies of ILTV, bothsmall and large plaque variantshad similar virulence in experi-mental chickens (28).Viruses 2 and 3 were distinct

from viruses 1, V154 and A4557-5in the kinetics of neutralizationtest using anti-V154 serum. Otherworkers (1, 2, 9, 16, 17) have dis-cussed the reciprocal NK valuesconsidered to indicate homologousantigens and concluded that vir-uses with NK values of less than 60are antigentically distinct. Neu-tralization of viruses 2 and 3 withanti-V154 serum gave NK valuesof 58 and 50. They were signifi-cantly different from the otherthree viruses when regressionlines were analyzed by leastsquares analysis of variance. Anti-genic distinction was not shown inreciprocal neutralization tests

0.5

(00 0.o

i.5.

Time - Minutes5 10 20 30

kAnti - virus serum

a N.

.N.

N"

IY NNI

I

N

(J0-J

1.0

v-- Virus

0-.....0 Virus 2

1*--.-.- Virus 3

* V 154

~---6 A4557-5

zo2

Fig. 6. Neutralization kinetics slopesusing anti-virus 1 serum against fivestrains of ILTV.

using anti-virus 2 serum and anti-virus 3 serum for neutralization ofV154 virus (Figs. 6 and 7). The lackof differences in reciprocal neu-tralization tests suggests minorantigenic differences between theviruses. Whereas some virusesshow distinct antigenic differencesby reciprocal kinetics (2,3,9), withother viruses differences in neu-

0.5

(J0-J

1.0

1.5

Time - Minutes5 l0 20 30

Anti -virus 2 serum

a

Y

Time - Minutes

5 10 20 30

Anti-virus 3 serum

V------ Virus

o. 0 Virus 2

_---.4 Virus 3

*-* V154

&-----4 A 4557-5

a

201

Fig. 8. Neutralization kinetics slopesusing anti-virus 3 serum against fivestrains of ILTV.

tralization kinetics were not recip-rocal (17) and may indicate a spec-trum or mosaic of antigenicdiversity (1, 29). Neutralizationkinetics demonstrated a close rela-tionship with minor antigenic dif-ferences among the five strains ofILTV regardless of any differen-ces in their pathogenicity or pockmorphology on the CAM.

Time - Minutes

5 10 20 30

0.5

LI)

-

V -- Virus

..........0 Virus 2

U---. Virus 3

* -* V 154

6-----A A4557 -5

20J

Fig. 7. Neutralization kinetics slopesusing anti-virus 2 serum against fivestrains of ILTV.

1.0

i5

2.OJ

Anti A 4557 - 5 serum

v------v Virus

0. 0 Virus 2

_---. Virus3

._4 vitb----6 A 4557-5

Fig. 9. Neutralization kinetics slopesusing anti-A4557-5 serum against fivestrains of ILTV.

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The identification of strains ofILTV which are either of low viru-lence, or which induce immunitywithout clinical disease, may pro-vide valuable agents for vaccina-tion. At the present time, ILTVvaccines used in Australia containthe SA-2 virus which, because ofits pathogenicity is restricted tovent application. The gross andhistological findings followingaerosol administration of SA-2virus in this study were similar tothose described previously (21).The potential of A4557-5 virus forvaccination was investigatedbecause chickens infected by aero-sol showed only mild infection asdetermined by clinical observa-tions and pathological examina-tion (24). They were resistent tointratracheal challenge with highdoses of V154 virus.Vent vaccination with SA-2

virus gave immunity at a dose of500 PFU per mL, which is consid-erably lower than the minimumtiter of 106 PFU per mL recom-mended for commercial vaccinescontaining this virus (13). TheA4557-5 was a less effectiveimmunizing agent when adminis-tered by vent application.A safe and effective immuno-

genic agent for ILTV by routessuch as drinking water adminis-tration would be economical con-sidering the high labor costs inintensive poultry production. Itmay also provide a more directroute for stimulating local immun-ity of the respiratory tract. In thechicken, the predominant immuneglobulin in the respiratory tract isIgA (15). The drinking water routeof administration has been shownto be effective using SA-2 (26, 27)and other isolates (23). But it hasalso been reported to give only 37%protection with some isolates (11).The SA-2 virus caused adequateimmunity in experimental trials(26, 27), but when water vaccina-tion was tried under commercialconditions it was associated withsevere respiratory disease (Rus-sell, unpublished observations).This is consistent with the viru-lence demonstrated by aerosolinfection of experimental chickens(21) and confirmed in the aerosol

studies documented in this report.Vaccination with A4557-5 virus

via the drinking water did not pro-tect all chickens against intratra-cheal challenge with V154. Threeof the eight chickens were fullyprotected. The other five chickensappeared to have partial immunitybecause infection with virulentvirus was less severe than in non-vaccinated control chickens. Thedegree of protection was attaineddespite absence of a detectable risein humoral antibody in most of thechickens. This suggests that othercomponents of immune mecha-nisms such as local IgA immunityor cell mediated immunity mayhave contributed to host defense. Ithas been observed previously thatsome chickens which did notdevelop humoral antibodies wereimmune to challenge (13).The serum titer following aero-

sol and drinking water adminis-tration of A4557-5 virus was lowerthan with SA-2 virus. This may bean indication of differences in theimmunogenicity between theseviruses. The pathology studies ofA4557-5 virus reported previously(24) together with the observationsdocumented in this paper, demon-strate that the A4557-5 strain ofILTV causes mild respiratory dis-ease and is closely antigenicallyrelated to certain virulent virusesisolated from natural outbreaks ofsevere clinical disease. The immu-nization studies revealed that thisvirus was a less effective vaccinat-ing agent than SA-2 when admin-istered via the vent and drinkingwater.

ACKNOWLEDGMENTS

The authors wish to thank MsCarol Lieske for valuable technicalassistance and Dr. D.E. Hore foradvice.

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ERRATUM

Can J Comp Med 1983; 47: 57-63

Effect of Viral Dose on Experimental Pneumonia Causedby Aerosol Exposure of Calves to Bovine Herpesvirus 1

and Pasteurella haemolytica

On page 60 under the heading "Microbiology and Serology" line three ofthe second paragraph should read:

"> 1:400 in all except one calf,"On page 61 the magnification for Figure 3 should read X4.On page 62 the magnification for Figure 4 should read X40.

The editors apologize to the authors and the readers for any inconven-ience caused.

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