marek's in development of viral follicles...

INFlCTION AND IMMUNrrY, Apr. 1976, p. 1037-1045Copyright © 1976 American Society for Microbiology

Vol. 13, No. 4Printed in U.SA.

Marek's Disease in Chickens: Development of Viral Antigenin Feather Follicles and of Circulating Antibodies

F. STECK* AND H. U. HABERSTICH

Institute of Microbiology, School of Veterinary Medicine, University ofBerne, Switzerland

Received for publication 11 November 1975

The infection of young chickens with Marek's disease herpesvirus (MDHV)leads to the early appearance of viral antigen in the bursa Fabricii, in the kidneytubular epithelium, and particularly in the epithelial cells of growing featherfollicles (Calnek and Hitchner, 1969; Purchase, 1970). Viral antigen may persistin feather follicles over several months, whereas in neural lesions or in lymphoidtumors induced by MDHV viral antigen is either lacking or present only in a fewcells (Calnek and Hitchner, 1969; Spencer and Calnek, 1970; Purchase, 1970).Haider et al. (1976) have pointed out the high diagnostic value of the double-diffusion agar-gel precipitation test (Chubb and Churchill, 1968) using growingfeathers of MDHV-infected chickens as antigen.

In the following experiments we studied theappearance and persistence of feather follicle(FF) antigen after infection, as detected by thegel precipitation test, in unvaccinated chickensand in chickens vaccinated with the herpesvi-rus of turkeys (HVT). The presence of antigenwas compared with the formation of precipitat-ing and neutralizing antibodies and with thedevelopment of clinical disease.

MATERIALS AND METHODSChickens. A local commercial breed of white

Leghorn hybrid (Schweiz. Geflugelzuchtschule, Zol-likofen) was used in all experiments.

Infection experiments. A longitudinal study ofthe development viral antigen and circulating anti-body was conducted in vaccinated and unvaccinatedchickens after infection with virulent Marek's dis-ease virus (MDHV). The experiment is describedunder Results (iii).

For comparison, eight experimental groups ofbirds, all of the same breed and infected with thesame strain of MDHV at the age of 1 to 20 days andkilled after various time intervals up to the age ofabout 16 weeks, were evaluated together (Fig. 4;Tables 3 and 4).

Isolators. Groups of up to five chickens were heldin isolators under slight positive pressure with fil-tered ingoing and outgoing air, thermoregulation,and automatic water supply. A large food containerallowed feeding during the first 3 weeks withoutrefilling. Refilling was done from the outside with-out interruption of the isolation. Fecal matter wascollected in a bin with a 3-month holding capacityplaced under a wire-mesh tray covering the wholefloor.

Virus strains. A virulent strain of MDHV wasisolated by direct kidney culture (23) from a whiteLeghorn chicken with the classical form of Marek'sdisease (MD): neuritis of plexus ischiadicus with

moderate lymphoreticular infiltration. This viru-lent strain was used in the first or second passage incultures of embryonic chicken kidney cells or, insome instances, after one additional passage inchickens and reisolation by direct kidney cultures.

Infections were done by the intra-abdominal (i.a.)inoculation of infected culture cells. The number ofcells inoculated per bird corresponded to 10 to 100plaques in the original cell culture.The same virus isolated was passaged by cell

transfer in embryonic chicken kidney cultures. Itwas avirulent for 1-day-old chicks after passage 32in culture (MDHV-TC) (unpublished data).

MDHV-infected cell suspensions at low or hightissue culture passage level were preserved frozen at-196 C in liquid nitrogen after addition of 10% di-methyl sulfoxide (20).

Vaccine virus. HVT (live vaccine of the TAD Co.,Cuxhaven) was passaged by cell transfer six timesin chicken embryo cell cultures, liberated by ultra-sonic treatment, and stored as a cell-free suspensionat -70 C. For immunization, chickens were inocu-lated i.a. with about 2,000 plaque-forming units ofHVT.

Cell cultures. Direct kidney cultures for virusisolation from infected birds were done according tothe method described by Witter et al. (23).

Chicken embryo cell cultures of 10-day-old em-bryos and embryonic chicken kidney cultures of 18-day-old embryos were grown in Eagle minimal es-sential medium with 2 to 5% fetal bovine serum at38 C in an incubator with 5% CO2 in air.Serum neutralization. Serum neutralization was

done by plaque reduction against cell-free HVT inchicken embryo cell cultures (10). Chicken serumwith unknown titer of neutralizing antibodies wasdiluted 1:4 in phosphate-buffered saline inactivatedfor 30 min at 56 C, mixed in equal parts of HVTdiluted in SPGA solution (2), to contain about 200plaque-forming units/0.1 ml, and incubated for 1 hat room temperature. The mixture (0.2 ml) was inoc-

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1038 STECK AND HABERSTICH

ulated on a semiconfluent monolayer and coveredafter a 60-min adsorption at 38 C with a 2% carboxy-methyl-cellulose overlay. The cultures were washedand stained with 0.1% crystal violet after a 7-dayincubation at 38 C in a CO2 incubator. The numberof plaques was determined microscopically, and theserum neutralization effect expressed as X-foldplaque reduction was compared to the control cul-ture.

Agar-gel precipitation (4, 12). A microtest of thetwo-dimensional agar-gel precipitation adoptedfrom the method described by Crowle (5) was used.The agar-gel had the following composition: 1% No-ble agar (Difco), 8% NaCl, 1% NaN3 in 0.01 M phos-phate buffer at pH 7.2. The preparations were

stained with azokarmin (1.5 g of azokarmin per literof acetate buffer, pH 3.7).

Antigens for gel precipitation. Avirulent MDHVadapted to chicken embryo kidney cell cultures(MDHV-TC) was used at passage 32 or higher. In-fected cells were disrupted by ultrasonic treatment(Sonifier B-12, Branson Sonic Power Co.). Optimalyield ofantigen was obtained by sonication (6 x 30 s)with intervals of 1 min under cooling in ice water.The antigen was clarified by centrifugation for 30min at 12,000 x g and 4 C.FF antigen. FF antigen of MDHV was obtained

from soft, vascularized feather follicles of infectedchickens cut with scissors, suspended in phosphate-buffered saline, and homogenized with a high-speedblender (Polytron). The suspension was then soni-cated for 2 x 30 min under ice cooling and clarifiedby centrifugation (30 min, 12,000 x g at 4 C).

RESULTS

(i) Relationship between MDHV antigens oftissue culture and FF origin and HVT in gelprecipitation. For this reaction, a potent pre-

cipitating serum of chicken vaccinated withHVT on the first day after hatching and chal-lenged with MDHV 2 weeks later was used.The precipitation pattern obtained with im-mune serum against antigen from MDHV-in-fected feather follicles, embryonic chicken kid-ney tissue culture infected with high-passageMDHV-TC, on chicken embryo cell culturesinfected with HVT is shown in Fig. 1.The main precipitation band A obtained

against HVT and FF antigen was absent fromthe MDHV-TC antigen. On the other hand, thestrong precipitation band B obtained againstMDHV-TC was only weakly present in FF or inHVT antigen. The degree of cross-reactivitybetween the two main and additional minorantigenic components is shown in Table 1. Noreaction to uninfected tissue culture or FF ex-

tracts from uninfected chickens was recorded.Chickens infected experimentally with

MDHV reacted independently against one or

the other of the two main antigens of FF or

MDHV-TC origin.(ii) Distribution of FF antigen in MDHV-

B C(i)

FIG. 1. Relationship between MDHV-specific an-tigens from infected feather follicles (FF), after tissueculture adaption (MDHV-TC) and antigens fromHVT. Two-dimensional gel precipitation. Centerwell, serum from chicken immunized with HVT andafter challenge with virulent MDHV.

TABLE 1. Antigenic components of virulent (FFantigen), of tissue culture-adapted avirulent(MDHV-TC) Marek's disease virus, and of

herpesvirus of turkey (HVT) in gel precipitationa

Antigenic componentAntigens

A B C D

FF antigen +++ + - -MDHV-TC - + + + + +HVT +++ + + -

a Against hyperimmunized chicken serum (seeFig. 1). Symbols: (-) Not detected; (±) (+) weak;(+ + +) strong precipitation line.

infected chickens. In 110 chickens infectedwith virulent MDHV, the presence of FF anti-gen was searched for in feather tracts of ninedifferent areas of the skin: back, separately onleft and right side of neck, wings, externalcrural tract, and tail. It was found that theantigen was symmetrically distributed overboth sides of the body. Strongest reactions wereobserved in the large, growing, well-vascular-ized feather follicles of wings and tail.The results agree with the assumption that

the FF localization of viral antigen is the resultof a systemic dissemination of MDHV throughthe blood vessels.

(iii) Appearance and persistance of FF anti-gen. To study the appearance and persistanceof FF antigen and the development of circulat-ing antibody (see below) in MDHV-infectedchickens, the following experiments was con-ducted.

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VOL. 13, 1976

Twenty-eight chickens were immunized im-mediately after hatching with HVT by i.a. inoc-ulation. Twenty-eight chickens served as un-vaccinated controls. At the age of 2 weeks, halfof each group were infected abdominally withvirulent MDHV. Starting at week 2, two grow-ing feather follicles of each wing were taken atweekly intervals for antigen detection by gelprecipitation, and in 2-week intervals serumsamples were collected for serological tests. Allchickens dying in the course of the experimentwere autopsied and examined histologically.Surviving chickens were killed after 13 weeksand examined as above. In addition, direct kid-ney cultures were done for virus isolation. Thefollowing results were obtained.Unvaccinated chickens. In the 14 unvaccin-

ated chickens infected with MDHV at the age of3 weeks, FF antigen first became detectable 2weeks later in 13 of 14 birds. In the nine birdsremaining healthy the antigen gradually disap-peared. Only two ofthese nine chickens reactedpositively at the age of 13 weeks (Fig. 2). In fivechickens that developed the typcial disease,however, FF antigen remained present up tothe time of death (Fig. 3).

Similar observations were made in a larger

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FIG. 2. Appearance and repression ofMDHV FFantigen and formation of neutralizing antibodies inunvaccinated chickens remaining healthy after infec-tion with virulent MDHV. Symbol: (#) Standarddeviation.

MAREK'S DISEASE IN CHICKENS 1039

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FIG. 3. Persistence ofMDHV FF antigen and lackof formation of neutralizing antibodies in unvaccin-ated chickens succumbing to the infection with viru-lent MDHV.

group of 186 unvaccinated chickens infectedwith virulent MDHV in eight experiments. Thechickens were killed at various intervals afterinfection (Fig. 4). Again a significantly differ-ent development was found between chickensremaining healthy (clinically and histopatho-logically) and those that succumbed to MD.Twenty-nine percent of all chickens that died orwere killed showed clinical and histopathologi-cal signs of MD. Initially 1 to 5 weeks afterinfection, 8 of 11 diseased and 15 of 17 healthychickens showed the presence of FF antigen. Inthe chickens dying from MD between 6 to 15weeks after infection, more than 80% had FFantigen up to the time of death. In contrast,among the samples of clinically healthy chick-ens the percentage with FF antigen declinedfrom 89% 1 to 5 weeks after infection to 25% at16 weeks or later. This decline is highly signifi-cant, even if it was taken into account that thesick chickens have been gradually eliminatedfrom the group (x2, P < 0.01).Chickens vaccinated with HVT. None of the

15 chickens immunized with HVT without chal-lenge developed FF antigen. In all of 13 birdsfrom which direct kidney cultures were done at14 weeks, a persistent infection with HVT wasdetected. In the 15 vaccinated chickens chal-lenged with virulent MDHV, FF antigen waspresent in one bird at 2 weeks, in four birds at 3weeks, and in one again at 5 weeks after infec-tion. No antigen was detectable thereafter, andthe chickens remained healthy (Fig. 5).

Control chickens. None of the 15 unvaccin-ated and unchallenged control birds showed thepresence of FF antigen.

(iv) Development of precipitating and neu-


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ckinically affected by Mare

a clinically healthy

mentioned above infected i.a. with MDHV, itwas shown that those chickens with persistentMDHV infection or those affected by clinicaldisease had significantly lower levels of anti-bodies at the time of death or killing. The re-sults are summarized in Table 3 and 4. Thir-teen of fifteen (86%) chickens with no evidenceof persistent infection had neutralizing anti-bodies as compared to only 14 (32%) of those 42with persistent infections. Similarly only 3 of 16(18%) chickens coming down with MD had neu-tralizing antibodies as compared to 25 of 43

I eeks p.l.(58%) of those without gross evidence of the

_weeks6 disease. Both differences are significant (Table

16 3). Nevertheless, persistent infection was notalways associated with clinical disease duringthe observation period of 14 to 17 weeks.

ks disease Precipitating antibodies against MDHV-TCor FF antigens. The results on precipitatingantibodies against MDHV-TC or FF antigen

... -1 m__M l_L _ n _ u3 n A__ _ ___ _FIG. 4. Persistence ofMDHVFF antigen in chick-

ens succumbing to and clinically affected by Marek'sdisease and repression of FF antigen in those re-maining healthy after infection with virulent MDHV.Abbreviations: n, Number of chickens examined atthe various time intervals; P, significance of differ-ence between healthy and sick chickens by X2 analy-sis.

tralizing antibody. In the group of vaccinatedand unvaccinated chickens mentioned above(Results [iii]), the development of neutralizingantibodies (against HVT) and of precipitatingantibodies against MDHV-TC and FF antigenswas studied (Table 2 and 3).Neutralizing antibodies. In uninfected, un-

vaccinated control chickens a plaque reductionup to 50% was observed with a serum dilution of1:4. Plaque reduction up to this level was con-sidered nonspecific (Fig. 6).

In chickens vaccinated with HVT with orwithout subsequent challenge, significant lev-els of neutralizing antibody developed (Fig. 5and 6). Antibodies were already detectable 3weeks after vaccination and gradually in-creased up to the end of the experiment at 14weeks of age. One week after challenge withvirulent MDHV, the titers in the challengedbirds were lower than in the vaccinated con-trols.

In unvaccinated chickens infected with viru-lent MDHV, differences in the development ofneutralizing antibodies were seen which be-came significant from week 10 of life until thetime of death. Chickens succumbing to MD de-veloped much lower antibody titers than thepenmates surviving up to the end of the experi-ment (Fig. 2 and 3).

In the larger group of unvaccinated chickens

are shown in lamles 2 and 3. Again no reactionswere found in unvaccinated, uninfected con-trols.

Vaccination with HVT and/or challenge withvirulent MDHV led to the formation of precipi-

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FIG. 5. Formation of neutralizing antibodies inchickens vaccinated with HVT. Appearance andrepression ofFF antigen after challenge at 2 weeks ofage.

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TABLE 2. Precipitating antibodies against FF and MDHV-TC antigens

Precipitating anti- Age (weeks)body 2 3 5 7 9 10 12 13

Controls Anti-MDHV-TC 0/15a 0/9 0/9 0/9 0/9 0/9 0/9 0/15

Vaccinated with Anti-FF 0/14 0/10 0/10 2/10 5/10 7/10 7/10 10/14HVT

Anti-MDHV-TC 0/14 0/10 2/10 1/10 5/10 7/10 5/10 6/14

Infected with viru-lent MDHV'Surviving Anti-FF 0/9 0/9 8/9 7/9 d 8/8 9/9 7/7' 7/7

Anti-MDHV-TC 0/9 3/9 3/9 5/9 5/9 7/9 5/7 7/7

Clinically Anti-FF 0/5 1/5 3/5 0/4d 4/5 2/3affected

Anti-MDHV-TC 0/5 1/5 0/5 0/5 0/4 0/3

Vaccinated with Anti-FF 0/15 0/15 12/15 8/15d 11/15 12/15 12/15 13/15HVTb and chal- Anti-MDHV-TC 1/15 1/15 5/15 2/15d 6/15 11/15 10/15 11/15lenged withMDHV'

a Numerator, number of chickens reacting positively; denominator, number of chickens examined. Sixcontrol and four vaccinated chickens were held in uninterrupted isolation during the experiment. The otherbirds were taken out of the isolators for a few minutes for blood sampling at 1- to 2-week intervals.

b Vaccinated immediately after hatching with HVT.Infected with virulent MDHV at the age of 2 weeks.

d The lower percentage of serologically positive birds in many groups at week 7 is unexplained." Two chickens were lost by causes other than MD.

TABLE 3. Formation of humoral antibodies in chickens with or without persistent MDHV infectiona

(1) Chickens with (2) Chickens with-Humoral antibodies persistent MDHV out persistent Comparison of significance (1) vs (2)

infectionb MDHV infectionb

Precipitating antibodies against:FF antigen (A)

Positive 37 15 No significant differenceNegative 8 1 (x2, P > 0.25)

MDHV-TC antigen (B)Positive 17 13 Difference is highly significantNegative 28 3 (X2, P < 0.01)

Neutralizing antibodyPositive" 14 13 Difference is highly significantNegative 28 2 (x2, P < 0.002)

a Unvaccinated chickens infected intra-abdominally with virulent MDHV at 1 to 3 weeks of age.b Persistent infection with FF antigen detectable at time of death or killing and/or MDHV present in

direct kidney culture at the end of the experiment at the age of 3 to 4 months.' Plaque reduction at serum dilution (1:4) greater than 50%.

tating antibodies against both FF (componentA) and MDHV-TC (component B) antigens in a

large proportion of chickens, with the exceptionof one group: chickens clinically affected withMD developed antibodies against the FF anti-gen, but developed little, if any, antibodyagainst the MDHV-TC antigen.

A similar relationship was found in thelarger collection of unvaccinated birds fromseveral different experiments after i.a. in-fection with virulent MDHV. Among 66 un-vaccinated chickens, 86% developed antibodyagainst FF antigen, but only 49% developedantibody against MDHV-TC antigen. Precipi-

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tating antibodies against FF antigen (A) were

found with equal frequency in chickens withpersistent MDHV infection (FF antigen or kid-ney culture positive at time of killing) and inthose in which virus or viral antigen becameundetectable. Against MDHV-TC antigen (B),however, the percentage of chickens with anti-bodies was significantly lower in those withpersistent infection of the feather follicles thanin those without persistent infection (Table 3).

In chickens with gross evidence of MD, only 2of 18 were forming precipitating antibodiesagainst MDHV-TC, in contrast to many as 30 ofthose 47 without the disease. This difference ishighly significant. The formation of precipitat-ing antibodies against FF antigen was also re-

duced, but much less so.

100X

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20x

ifl5X5H

2x-

Control

0 2 3 5 7 9 10 12 13 agein weeks

FIG. 6. Formation of neutralizing antibodies inHVT-vaccinated chickens without challenge. Lack ofantibody formation in unvaccinated chickens withoutchallenge.

DISCUSSION

Eighty to one hundred percent of the chick-ens infected i.a. with virulent MDHV at 0 to 2weeks of age showed as early as 2 weeks later ageneralized infection of the feather follicles, asdetected in our studies by gel precipitation asdescribed by Haider et al. (7). The distributionof infected feather follicles more or less sym-metrically over the whole skin suggests anearly spread of the virus by the blood circula-tion.The further development of FF infection ap-

peared to be correlated with the fate of individ-ual birds. Chickens succumbing to the diseaseshowed in about 80% a persistent infection ofthe feather follicles up to the time of killing ordeath. An association between persistent vire-mia and MD lesions was also found by Witter etal. (24) in a long-term study of a naturallyinfected chicken flock. In infected chickens re-

maining clinically healthy in our study, how-ever, the FF antigen, which was initially pres-ent in the same proportion as in those whicheventually died, slowly disappeared. This dis-appearance is real and is not only due to thefact that MD-sick chickens were graduallyeliminated from the group.The early generalization of MDHV may be

responsible for inflammatory neural lesionswithout clinical signs, which we found in about85% of the infected chickens between 1 to 5weeks after infection. In about 30%, however,these lesions gradually disappeared up to andafter week 16 after infection. (Steck, unpub-lished data). This may be the same manifesta-

TABLE 4. Formation of humoral antibodies in chickens with or without Marek's diseasea

(1) Chickens af- (2) Chickens withHumoral antibodies fected by Marek's no gross evidence Comparison of significance (1) vs (2)

disease of Marek's di- Coprsnosiifcce()v(2seaseb

Precipitating antibodies againstFF antigen (A)

Positive 12 44 Difference is significantNegative 6 3 (x2, P < 0.02)

MDHV-TC antigenPositive 2 30 Difference is highly significantNegative 16 17 (X2, P < 0.001)

Neutralizing antibodyPositiver 3 25 Difference is significantNegative 13 18 (x2, P < 0.02)

Unvaccinated chicken infected intra-abdominally with virulent MDHV at 1 to 3 weeks of age.b Clinical signs of Marek's disease were confirmed by autopsy and histopathology. Experiment was

terminated at the age of 3 to 4 months.' Plaque reduction at serum dilution (1:4) greater than 50%.

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VOL. 13, 1976

tion of MDHV infection as the temporary pa-ralysis observed by Kenzy et al. (9). Thesuppression of the generalized infection inmany birds may reflect the age-related resist-ance observed by others (19, 22).

Infection with MD leads to the formation ofantibodies against several different viral anti-gens. We follQwed the development of precipi-tating antibodies against FF antigen (antigeniccomponent A), MDHV-TC antigen (antigeniccomponent B), and neutralizing antibodiesagainst HVT. It appears important to differen-tiate between antibodies against various anti-gens, since the antibody response against thosethree antigens is not going in parallel and onlythe last two mentioned appeared to be corre-lated with resistance against disease or recov-ery.

Most, i.e., 86% of the birds infected withvirulent MDHV or HVT, reacted with the for-mation of precipitating antibody against themain antigenic component A or FF antigen,irrespective of whether the virus infection per-sisted. In chickens with clinical MD the anti-body response was significantly lower than inhealthy chickens. Since, however, as many astwo-thirds of the sick chickens had antibodiesagainst the FF antigen up to the time of death,this antibody does not seem to be effective inresistance against disease, in accordance withthe results obtained by Sharma and Stone (18).

Neutralizing antibodies, measured againstHVT, and precipitating antibodies against theantigenic component B of MDHV-TC were lessreadily detectable in all birds than the antibodyagainst FF antigen mentioned above. However,they were more frequently seen in birds whichremained clinically healthy or where the infec-tion did not persist. Sharma and Stone (18) andCalnek (1) suggested that the formation of vi-rus-neutralizing antibodies may be involved inthe genetic resistance of chickens against MD.The situation is, however, not quite the same inrespect to persistent infection with MDHV or toclinical manifestation of infection.Most birds with no evidence of persistent

MDHV infection showed neutralizing and pre-cipitating (MDHV-TC) antibodies; only a fewwere without. However, about a third of thosewith persistent infection had also neutralizingor precipitating (MDHV-TC) antibodies. Sincethe experiments were terminated at the age of14 to 17 weeks, it is not possible to knowwhether these birds would have overcome thegeneralized infection at a later stage.

In the birds with clinical and pathologicalevidence ofMD the formation of antibodies wasreduced. This has also been found to be trueagainst antigens unrelated to MDHV (11, 16).


In particular, only a small fraction of thesebirds had precipitating antibodies againstMDHV-TC (component B) or neutralizing anti-bodies. In contrast, about two-thirds of the sur-viving chickens had both antibodies. The differ-ence between healthy chickens and those af-fected clinically by MD is highly significant. Itwas, however, not possible to establish a directcausal relationship. The two mentioned anti-bodies did not go strictly parallel in each birdand are probably directed against two differentantigenic components, but they both seem toreflect an immune status of the bird whichtends to overcome the persistent generalizedinfection and possibly also the development ofirreversible lesions. It has been shown by oth-ers that bursectomy or thymectomy had no ef-fect on the development of MD (13) in contrastto lymphoid leukosis, where removal of thebursa Fabricii prevents the development of thedisease (14). We found in unpublished experi-ments that the combination of surgical removalof the thymus before and of the bursa Fabriciiimmediately after hatching almost completelysuppressed the formation of circulating anti-bodies and led to a persistent infection in allbirds. We found, on the other hand, no evidencethat the antibodies studied were directly in-volved in the pathogenesis of lesions.The immunization of chickens with live HVT

did lead, in agreement with the results ob-tained by others (6, 17), to a persistent infectionwith the vaccine virus, but without localizationin the feather follicles detectable by gel precipi-tation. The vaccinated birds developed, as afterinfection with MDHV, precipitating antibodiesagainst MDHV-TC antigen, FF antigen, andneutralizing antibodies.

In immunized birds the challenge infectionwith virulent MDHV led to a generalizationonly in a few birds. The localization in featherfollicles, if it did occur, appeared to be onlytransitory. They were protected against clincialdisease.

It has been shown by Kaaden and Dietz-schold (8) that MDHV-infected cells acquire vi-rus-specific proteins in their plasma membranedetectable by immunodiffusion and indirectlydetectable by virus neutralization. At least oneof the most likely virus-induced components ofMDHV-infected chicken embryo fibroblastsstimulated the formation of cross-neutralizingantibodies against HVT.

It is conceivable that the neutralizing or pre-cipitating (against MDHV-TC) antibodies inour experiments associated with the regressionof lesions or the suppression of a generalizedinfection of feather follicles are not, or only inpart, directly effective by neutralizing free vi-


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rus. They may also interact with specific anti-gens located on the surface of virus-infectedcells. In addition, the role of cell-mediated im-munity in MD is still not elucidated. Since theelimination of virus-infected cells is probablyless efficient than the elimination of free virus,this would explain in part why the correlationbetween recovery and antibody formation is notvery strict.

In summary, chickens were infected with vir-ulent MDHV with or without prior immuniza-tion with HVT.The sequence of infection was followed by

studying the humoral immune response andthe appearance of precipitating antigen infeather follicles (FF antigen). It was differen-tiated between precipitating antibodies againstMDHV-FF antigen or against tissue culture-adapted MDHV and neutralizing antibodiestested against HVT.Beginning about 2 weeks after infection with

virulent MDHV 80 to 90% of all unvaccinatedchickens showed a generalized infection offeather follicles, which regressed again inabout one third of all chickens. The prior im-munization with HVT suppressed this localiza-tion of infection to a large extent.

In most chickens succumbing to MD the in-fection persisted in the feather follicles up tothe time of death, and only a small fraction ofthese birds had precipitating antibodies againstMDHV-TC or neutralizing antibodies.On the other hand, most of the chickens with

no evidence of persistent infection developedprecipitating antibodies against MDHV-TCand neutralizing antibodies.

In between was a group of chickens withpersistent infection with or without antibodiesbut with no evidence of MD. Since the experi-ments were terminated after observation pe-riods of 3 to 4 months, it is unknown whetherthese birds would eventually have come downwith the disease or would have overcome theinfection depending on their immune status.

In contrast to precipitating antibodiesagainst MDHV-TC and neutralizing antibod-ies, which appeared to reflect resistance to in-fection and disease, the precipitating antibod-ies against FF antigen were seen in most birdsregardless of their fate.

ACKNOWLEDGMENTSWe wish to thank Marlies Koller and Bea Scharen for

their excellent technical assistance and R. Fankhauser andL. Ryser, Institute of Comparative Neurology, Universityof Berne, for the histological preparations.

This study was supported by the Swiss National Founda-tion for Scientific Research, grant no. 4261.

LITERATURE CITED1. Calnek, B. W. 1972. Antibody development in chickens

exposed to Marek's disease virus, p. 129-138. In P. M.Biggs, G. de The, and L. N. Payne (ed.), Onco-genesis and herpesviruses. International Agency forResearch in Cancer, Publ. no. 2. World Health Orga-nization, Lyon.

2. Calnek, B. W., and H. K. Adidinger. 1971. Some char-acteristics of cell-free preparations of Marek's diseasevirus. Avian Dis. 15:508-517.

3. Calnek, B. W., and S. Hitchner. 1969. Localization ofviral antigen in chickens infected with Marek's dis-ease herpesvirus. J. Natl. Cancer Inst. 43:935-949.

4. Chubb, R. C., and A. E. Churchill. 1968. Precipitatingantibodies associated with Marek's disease. Vet. Rec.83:4-7.

5. Crowle, A. J. 1961. Immunodiffusion, p. 222-223. Aca-demic Press Inc., New York.

6. Eidson, C. S., 0. J. Fletcher, S. H. Kieven, and D. P.Anderson. 1971. Detection of Marek's disease antigenin feather follicle epithelium of chickens vaccinatedagainst Marek's disease. J. Natl. Cancer Inst. 47:113-120.

7. Haider, S. A., R. F. Lapen, and S. G. Kenzy. 1970. Useof feather in a gel precipitation test for Marek's dis-ease. Poult. Sci. 49:1654-1657.

8. Kaaden, 0. R., and B. Dietzschold. 1974. Alterations ofthe immunological specificity of plasma membranesfrom cells infected with Marek's disease and turkeyherpes viruses. J. Gen. Virol. 25:1-10.

9. Kenzy, S. G., B. R. Cho, and Y. Kim. 1973. OncogenicMarek's disease herpesvirus in avian encephalitis(temporary paralysis). J. Natl. Cancer Inst. 51:977-982.

10. Melchion, F. W., Jr., T. N. Fredrickson, and R. E.Luginbuhl. 1973. Neutralization studies with Ma-rek's disease virus and turkey herpesvirus. Appl.Microbiol. 26:925-933.

11. Nazarian, K. 1972. Virology and immunology of Ma-rek's disease (review), p. 59-73. In P. M. Biggs, Gde The, and L. N. Payne (ed.), Oncogenesis andherpesviruses. International Agency for Research onCancer, Publ. no. 2. World Health Organization,Lyon.

12. Okazaki, W., H. G. Purchase, and L. Noll. 1970. Effectof different conditions on precipitation in agar be-tween Marek's disease antigen and antibody. AvianDis. 14:532-537.

13. Payne, L. N., and M. Rennie. 1970. Lack of effect ofboursectomy on Marek's disease. J. Natl. CancerInst. 45:387-397.

14. Peterson, R. D. A., B. R. Burmester, T. N. Fredrick-son, H. G. Purchase, and R. A. Good. 1963. Effect ofbursectomy and thymectomy on the development ofvisceral lymphomatosis in the chicken. J. Natl. Can-cer Inst. 32:1343-1354.

15. Purchase, H. G. 1970. Virus specific immunofluores-cent and precipitin antigens and cell-free virus intissues of birds infected with Marek's disease. CancerRes. 30:1898-1908.

16. Purchase, H. G., R. E. Chubb, and P. M. Biggs. 1968.Effect of lymphoid leukosis and Marek's disease onthe immunological responsiveness of the chicken J.Natl. Cancer Inst. 40:583-592.

17. Purchase, H. G., and W. Okazaki. 1971. Effect of vacci-nation with herpesvirus of turkeys (HVT) on horizon-tal spread of Marek's disease herpesvirus. Avian Dis.15:391-397.

18. Sharma, J. M., and H. A. Stone. 1972. Genetic resist-ance to Marek's disease. Delineation of the responseof genetically resistant chickens to Marek's diseasevirus infection. Avian Dis. 16:894-906.

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19. Sharma, J. M., R. L. Witter, and B. R. Burmester.1973. Pathogenesis of Marek's disease in old chickens:lesion regression as the basis for age-related resist-ance. Infect. Immun. 8:715-724.

20. Spencer, J. L., and B. W. Calnek. 1967. Storage of cellsinfected with Rous sarcoma virus or JM strain avianlymphomatosis agent. Avian Dis. 11:274-287.

21. Spencer, J. L., and B. W. Calnek. 1970. Marek's dis-ease: application of immunofluorescence for detectionof antigen and antibody. Am. J. Vet. Res. 31:345-358.

22. Witter, R. L., J. M. Sharma, J. J. Solomon, and L. R.

Champion. 1973. An age-related resistance of chick-ens to Marek's disease, some preliminary observa-tions. Avian Pathol. 2:43-54.

23. Witter, R. L., J. J. Solomon, and G. H. Burgoyne. 1969.Cell culture techniques for primary isolation of Ma-rek's disease-associated herpesvirus. Avian Dis.13:101-118.

24. Witter, R. L., J. J. Solomon, L. R. Champion, and K.Nazarian. 1971. Long-term studies of Marek's diseaseinfection in individual chickens. Avian Dis. 15:346-365.

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marek's in development of viral follicles...

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