This article was downloaded by: [California Poly Pomona University]On: 28 October 2014, At: 17:54Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UK

Avian PathologyPublication details, including instructions for authorsand subscription information:http://www.tandfonline.com/loi/cavp20

Reticuloendotheliosis antigen forthe agar gel precipitation testM. Ianconescu aa Department of Avian Pathology , Kimron VeterinaryInstitute , Bet‐Dagan, P.O.B. 12, IsraelPublished online: 12 Nov 2007.

To cite this article: M. Ianconescu (1977) Reticuloendotheliosis antigen for the agar gelprecipitation test, Avian Pathology, 6:3, 259-267, DOI: 10.1080/03079457708418234

To link to this article: http://dx.doi.org/10.1080/03079457708418234

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all theinformation (the “Content”) contained in the publications on our platform.However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, orsuitability for any purpose of the Content. Any opinions and views expressedin this publication are the opinions and views of the authors, and are not theviews of or endorsed by Taylor & Francis. The accuracy of the Content shouldnot be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions,claims, proceedings, demands, costs, expenses, damages, and other liabilitieswhatsoever or howsoever caused arising directly or indirectly in connectionwith, in relation to or arising out of the use of the Content.

This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly

forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4

Avian Pathology, 6: 259-267, 1977

RETICULOENDOTHELIOSIS ANTIGEN FOR THE AGAR GEL

PRECIPITATION TEST

M. IANCONESCU

Department of Avian Pathology, Kimron Veterinary Institute1

Bet-Dagan, P.O.B. 12, Israel

SUMMARY

An antigen for gel precipitation was prepared from chick embryo fibroblastcultures inoculated with the Reticuloendotheliosis virus. The specificity ofthe reaction was confirmed with reference Reticuloendotheliosis and Spleennecrosis sera. No precipitation reactions occurred between this antigen andMarek's disease, Newcastle disease, Infectious bronchitis, Pasteurella Multo-cida and Haemorrhagic enteritis antisera.

INTRODUCTION

Reticuloendotheliosis virus (REV) was originally isolated in 1958 from a turkey withgross enlargement of the liver and spleen (Robinson and Twiehaus, 1974). This virus,together with Chick Syncytial, Duck Infectious Anaemia and Spleen Necrosis viruses,belong to a new group of oncogenic viruses termed the reticuloendotheliosis virusgroup. In fluorescent antibody and virus neutralization experiments it has been shownthat they are all serologically related (Purchase et al., 1973; Purchase and Witter,1975).

Under experimental conditions REV was found to be pathogenic for chickens, poults(Robinson and Twiehaus, 1974; Sevoian et al., 1964), Japanese quail (Theilen et al.,1966; Ziegel et ai, 1966), goslings and ducklings (Taylor and Olson, 1972; Purchaseet al., 1973), and other birds, but not for mammals or mammalian cells in cultures(Calnekera/., 1969).

The original REV isolate, designated as strain T (REV-T) was studied in several lab-oratories, and due to a various number of passages in vivo and in vitro in these labora-tories some of its properties have apparently changed. The viruses derived from the Tstrain are known as strain C (REV-C), strain F (REV-F) and strain S (REV-S).

Besides infection of turkeys (Paul et al., 1976) REV antibodies have been recordedin chickens (Aulisio and Shelokov, 1969). The differential diagnosis between reticu-loendotheliosis (RE) and lymphoid leukosis, Marek's disease and lymphoproliferativedisease of turkeys (Biggs etal., 191 A) is difficult based on macroscopic lesions alone. For a

Received 28 October 1976Accepted 17 January 19771Affiliated with the Tel-Aviv University

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4

260 M. Ianconescu

correct diagnosis a careful histopathological examination is essential. RE diagnosismay be verified by the isolation and identification of the virus and by serologicalstudies (Purchase, 1975). The serological methods used for the identification of REVare the virus-neutralization test in vivo, in ovo, or in cell cultures. The purpose of thispaper is to describe an agar gel precipitation test and the preparation of antigen forthis reaction. The possibility of using the agar gel precipitation test instead, or with,the fluorescent antibody and neutralization tests can simplify the serological studiesand allow more comprehensive serological surveys.

MATERIALS AND METHODS

RE virusREV-F was received from Dr. H.G. Purchase, Poultry Research Laboratory, EastLansing, Mich, and was grown in chick embryo fibroblast cell cultures (CEF) preparedfrom Specific Pathogen Free (SPF) (SPF eggs from Lohmann, W. Germany) eggs.After eight serial passages in CEF, infected cells were harvested, resuspended in Eagle'sMEM medium with 10% DMSO and 15% calf serum and stored in liquid nitrogen. Thismaterial, designated RE-cells, was used as the source of virus in this study.

RE antigen for gel precipitationPrimary CEF cultures were inoculated with RE-cells 1 to 2 hours after seeding the cellsinto culture flasks. After 24 or 48 hours, when a confluent cell monolayer was formed,the cells were harvested with trypsin-versene and reseeded at the rate of one flask totwo new flasks without addition of fresh cells. A new passage followed after 48 hoursand when the monolayer was complete the growth medium was replaced with main-tenance medium. This medium was harvested 3-4 days later and was used for antigenpreparation. In some experiments the cells were passed again and the same procedurewas repeated.

The growth medium used was Eagle's MEM plus 10% calf serum and 1% L-glutamin(0.2 M) and the maintenance medium was M 199 plus 0.05% bovine serum albumin.

The antigen was prepared by concentrating the medium from the REV inoculatedcell cultures six to 10 times. This concentration was obtained by three methods:

1. Precipitation with ammonium sulphate by adding slowly 26 g ammoniumsulphate to 100 ml medium with continuous stirring. The pH was maintainedat 7.2 to 7.4 by adding sodium bicarbonate solution. After adding all the am-monium sulphate the stirring was continued for 1 hour after which it wasstored overnight at 4°C. The precipitate was separated next day by centrifu-gation at 5000 x g for 30 min and redissolved in 0.2 M borate buffer at pH8.2 in a volume 10 times less than the original volume of medium. The re-maining ammonium sulphate was eliminated by dialysis against the samebuffer.

2. Evaporation by placing the medium in visking tubes in front of a fan untilits volume was reduced to 1/6 to 1/10 of the original. This concentrationcould be obtained at room temperature (22°C) in 6 to 8 hours.

3. Lyophilisation by freeze-drying the medium and redissolving it in a volumeof distilled water necessary to reach the desired concentration. This operationwas done in an Edwards Mini Fast Freeze Dryer at -40°C in 12 hours. Usually,5 ml vials were filled with 3 ml medium and redissolved before use in 0.5 mldistilled water.

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4

REV agar gel precipitation 261

In order to avoid the deep red colour of the antigen due to the concomitant concen-tration of the phenol red, the cell culture media was dialysed against saline prior toits concentration by lyophilisation. When the medium was concentrated by evapora-tion dialysis was done on the concentrated antigen.

The three types of antigen were stored at 4°C and remained effective for more than5 months. For the production of control "antigen" uninfected cell cultures from thesame batches of CEF used for antigen production were passaged in a similar mannerand the media were concentrated by the same methods.

AntiseraAntisera were prepared by intramuscular inoculation of RE-cells of 1-day and 7-days-old SPF chickens, 3-weeks-old turkeys and rock partridges of uncertain age. Afterthawing and before inoculation the RE-cells were washed twice in PBS in order toeliminate the DMSO and calf serum. In some experiments the inoculation was repeat-ed 1 week later.

Reference sera against REV-C, REV-F, REV-S and spleen necrosis virus were kindlyprovided by Dr. R.L. Witter and Dr. J.J. Solomon, Poultry Research Laboratory, EastLansing.

Antisera against Newcastle disease, Infectious bronchitis, Marek's disease, and Haem-orrhagic enteritis viruses and to Pasteurella multocida type 1 and 3 from our collec-tion were used together with homologous antigens to control the specificity of theRE reactions in the agar gel precipitation tests.

Agar gel precipitation test (A GPTjThe AGPT was performed in 1% Noble agar containing 8% NaCl at pH 7.4 on slidesin Gelman frames (Gelman, Ann Arbour, Michigan U.S.A.). Instead of the standardGelman punch set, a self made punch was used which had the same pattern of 6 + 1wells but each well had a diameter of 4 mm and the distance between the centres ofthe wells was 8 mm. The results were read after 24 hours incubation at 37°C and re-checked after a further 24 hours.

Fluorescent antibody testsCover-slip CEF cultures inoculated with RE-cells were fixed for 2 min in cold acetone,dried and stored at -20°C in ajar containing blue silica gel.

The indirect staining method was used (Purchase, 1969) and the cover-slips weretreated with 1 in 10 dilutions of chicken or turkey RE antisera or with reference REantisera, washed in PBS and finally treated with fluorescein-conjugated anti-chick(Roboz Surgical Instruments, Washington D.C. 20006, U.S.A.) or anti-turkey (CappelLab. Dowingtown Pa., 19335, U.S.A.) gammaglobulin. For control, cover-slips withuninoculated CEF cultures were treated with RE antisera and the respective anti-gammaglobulin, and cover-slips with RE inoculated CEF cultures were treated with"normal" chicken or turkey sera collected from birds reared in isolation.

The preparations were examined in a dark field with a 100 W halogen bulb as thelight source with the appropriate interference filter.

RESULTS

Confirmation of presence of REVThe presence and the quantity of REV in the various passages of CEF inoculated withthe RE-cell material, was monitored with the help of the fluorescent antibody test.The proportion of cells showing a granular cytoplasmic fluorescence was 30 to 40%

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4

262 M. Ianconescu

in the primary culture, but reached 60% and nearly 100% by the second and thirdpassage. In cultures which were not passaged, even if they were maintained for thesame length of time, the number of fluorescent cells was lower than in the culturespassaged every 24 or 48 hours. No difference was observed in the number offluorescent cells or in the intensity of their fluorescence when cover-slips from thesame passage level were treated with the reference antisera (REV strain C, F, S andspleen necrosis) or antisera from chickens and turkeys immunised with RE cells.

Control uninfected cultures treated with the same sera and inoculated culturestreated with "normal" sera did not fluoresce.

Agar gel precipitationSera from birds inoculated with RE-cells were tested for precipitins against RE anti-gens prepared by all three methods. The results are presented in Table 1.

Table 1. Agar gel precipitation between RE antigens and sera fromimmunised and control birds.

Bird

ChickenChickenf

Rock partridgeRock partridgef

TurkeyTurkeyf

Immunised with

RE cellsnoneRE cellsnoneRE cellsnone

RE antigensAsa

4/4e

0/46/60/33/40/6

Evb

4/40/46/60/33/40/6

Lyc

4/40/46/60/33/40/6

Contd

0/40/40/60/30/40/6

a Antigen prepared with ammonium sulphateb Antigen prepared by evaporationc Antigen prepared by lyophilisationd Control antigens prepared from uninoculated CEF cultures by ammonium sulphate,

evaporation or lyophilisatione No. positive/No, testedf Control unimmunised birds

No line of precipitation was present between any of these sera and the control antigensprepared from uninfected cultures, or between RE antigens and sera from control un-immunised birds.

RE antigens prepared by the three methods produced a common precipitation linewhen reacted with each of a number of RE antisera (Fig.l).

Control of the specificity of the reactionTable 2 presents the results of agar gel precipitation tests between RE antigens andantisera, and the antigens and antisera for Newcastle disease, Infectious bronchitis,Marek's disease, Pasteurella multocida and Haemorrhagic enteritis. With the exceptionof some of the anti Newcastle disease sera which reacted not only against Newcastledisease antigen but also against Marek's disease antigen, all the antisera reacted onlywith the homologous antigen. Neither of the RE antisera reacted with antigens otherthan the RE antigens and none of the other antisera reacted with the RE antigen.

The bovine serum albumin (BSA) was included in the tests because the maintenancemedium of the CEF cultures from which the antigen was prepared contained thissubstance. A small number of sera from immunised birds reacted also with BSA, due

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4

REV agar gel precipitation 263

Fig. 1. Precipitation in agar gel using RE antigens and antisera.Ch.RE - Chicken RE antiserum;T.RE - Turkey RE antiserum;Ref.RE-S - Reference RE strain S antiserum;Ag.RE/L, P, E — RE antigens concentrated by lyophilisation,precipitation or evaporation respectively.

Fig. 2. Precipitation in agar gel using RE antigen preparedby evaporation (Ag RE) and reference REV antisera(REV-F and REVS) and chicken (Ch.S.RE), turkey(T.S.RE) and rock partridge (Rp.S.RE) sera preparedby immunisation with RE-cells. Ch.C. is serum froman unimmunised control chicken.

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4

Rev agar gel precipitation

Table 2. Agar gel precipitation tests between RE and other antigens andtheir antisera.

265

Immune sera

Chick-RERock Partridge-REChick-NDVChick-MDChick-IBTurkey-HETurkey-Pas teurella

multocida

Antigena

RE

4/4b

4/40/40/50/20/50/2

ND

0/40/44/40/50/20/50/2

IB

0/40/40/40/52/20/50/2

Past.M.

0/40/40/40/50/20/52/2

MD

0/40/43/45/50/20/50/2

HE

0/40/40/40/50/25/50/2

BSA

0/40/40/40/50/20/50/2

a RE — Reticuloendotheliosis antigen;ND — Newcastle disease; IB - Infectious bronchitis;Past.M. -Pasteurella multocida types 1 and 3;MD - Marek's disease; HE - Haemorrhagic enteritis;BSA — Bovine serum albumin,

b Number of sera positive/number of sera tested.

probably to traces of calf serum on the RE-cells used for immunisation. By adsorptionof such sera with calf serum the precipitation reaction with BSA could be abolishedwithout changing the reaction for the RE antigens.

The antigens were also tested with RE reference antisera obtained from another lab-oratory. A precipitation line was obtained between the RE antigens and antisera tostrains F and S REV and the spleen necrosis virus antiserum, but no precipitation linewas observed with strain C REV antiserum.

The reactions were identical whether the antigens were prepared by precipitation,evaporation or lyophilisation, and the reference sera gave a common line with the anti-sera prepared in this laboratory (Fig.2).

DISCUSSION

The growth of REV in cell culture has been reported to be dependent on active cellmultiplication (Temin and Kessner, 1974). With the aim of getting a high rate of in-fection, the cells in this study were inoculated with REV soon after seeding the cul-ture flasks so that the virus was present during the early growth phase when the cellswere multiplying at a high rate. For the same reason confluent monolayers werepassaged early.

The fluorescent antibody experiments confirmed that the number of infected cellswas much higher in cultures which were passaged every 24 to 48 hours than in cul-tures maintained for the same total length of time but unpassaged.

The specificity of the reaction was checked in various ways in order to exclude thepossibility that the strain of REV used for antigen and antisera preparation was con-taminated by an adventitious virus.

The presence of REV in the CEF cultures inoculated with RE-cells was confirmed bythe results of the fluorescent antibody tests using reference REV antisera.

The specificity of the RE antigen in agar gel precipitation reactions was demonstrated

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4

266 M. Ianconescu

by the line of identity between the reference REV antisera and the antisera preparedin this laboratory. No precipitation line appeared when the RE antigen was reactedwith precipitating antisera against various avian viral and bacterial antigens, or be-tween the RE antisera and heterologous antigens or control antigens prepared fromuninoculated CEF cultures.

The strain C REV antiserum gave positive results in the fluorescent antibody test butfailed to give a precipitation line against RE antigens in the agar gel precipitation test.No definitive explanation can be given for this result but it could have been due tothe serum having a low titre.

The differentiation of the viruses belonging to the RE group by precipitation in agargel and the use of this test for serological field surveys is under study.

A cknowledgementsThe skilful technical assistance in this work of Miss Raya Blumenkrantz is gratefullyacknowledged.This work was helped by an ICRETT grant for cancer research, awarded by theInternational Union Against Cancer.

REFERENCES

Aulisio, C.G. and Shelokov, A. (1969). Prevalence of RE in chickens: Immunofluorescencestudies. Proceedings of the Society of Experimental Biology and Medicine, 130: 178-181.

Biggs, P.M., Milne, B.S., Frazier, J.A., McDougall, J.S. and Stuart, J.C. (1974). Lymphoprolifera-tive disease of turkeys. Proceedings XV World's Poultry Congress, pp.55-57. New Orleans,U.S.A.

Calnek, B. W., Madin, S.H. and Kniozeff, A.J. (1969). Susceptibility of cultured mammalian cellsto infection with a herpesvirus from Marek's disease and T-virus from reticuloendothelio-sis of chickens. American Journal of Veterinary Research, 30: 1403-1412.

Paul, P.S., Pomeroy, K.A., Sarma, P.S., Johnson, K.H., Barnes, D.M., Kumar, M.C. and Pomeroy,B.S. (1976). Naturally occurring reticuloendotheliosis in turkeys. Transmission. Journalof the National Cancer Institute, 56: 419-422.

Purchase, H.G. (1969). Immunofluorescence in the study of Marek's disease. Journal ofVirology, 3: 557-565.

Purchase, H.G. (1975). Reticuloendotheliosis. In: Isolation and identification of avian pathogens,pp.155-159. American Association of Avian Pathologists.

Purchase, H.G. and Witter, R.L. (1975). The reticuloendotheliosis viruses. Current Topics inMicrobiology and Immunology, 71: 104-124. Springer Verlag.

Purchase, H. G., Lud ford, C., Nazerian, K. and Cox, H. W. (1973). A new group of Oncogenicviruses: Reticuloendotheliosis, Chick Syncytial, Duck Infectious Anemia and SpleenNecrosis Viruses. Journal of the National Cancer Institute, 51: 489-499.

Robinson, F.R. and Twiehaus, M.J. (1974). Isolation of the Avian Reticuloendotheliosis virus(strain T). Avian Diseases, 18: 278-288.

Sevoion, M., Larose, R.N. and Chamberlain, D.M. (1964). Avian Lymphomatosis. VI A virusof unusual potency and pathogenicity. Avian Diseases, 8: 336-347.

Taylor, H. W. and Olson, L.D. (1972). Spectrum of infectivity and transmission of the T-virus.Avian Diseases, 16: 330-335.

Temin, H.M. and Kessner, V.K. (1974). Replication of REV in cell culture: Acute infection.Journal of Virology, 13: 291-297.

Theilen, G.H., Ziegel, R.F. and Twiehaus, M.J. (1966). Biological studies with RE virus(strain T) that induces reticuloendotheliosis in turkeys, chickens and Japanese quail.Journal of the National Cancer Institute, 37: 731-743.

Ziegel, R.F., Theilen, G.H. and Twiehaus, M.J. (1966). Electron microscope observations onRE virus (strain T) that induces reticuloendotheliosis in turkeys, chickens and Japanesequail. Journal of the National Cancer Institute, 37: 709-729.

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4

REV agar gel precipitation 267

RESUME

Essai de précipitation en gel avec l'antigène Reticuloendotheliosis

Un antigène pour la précipitation en gel a été préparé en partant des cultures d'em-bryons de poulets inoculés avec le virus de la Réticuloendotheliose.

La spécificité de la réaction a été confirmée avec des sérums de référence de Réti-culoendotheliose et de nécrose de la rate.Cet antigène n'a donné aucune précipitation avec des sérums de la maladie de Marek,Newcastle, bronchite infectieuse, Pasteurella et intérite hémorragique.

ZUSAMMENFASSUNG

Retikulose-Antigen für den Agargelpraezipitationstest

Aus Kükenembryofibroblastenkulturen, die mit dem Reticuloendotheliose-Virusinokuliert worden waren, wurde ein Antigen für die Agargelpraezipitation hergestellt.Die Spezifität der Reaktion wurde mittels Reticuloendotheliose- und Milznekrose-referenzsera nachgewiesen. Zwischen diesem Antigen und Antisera gegen Marek'scheKrankheit, Newcastle-Disease, Infektiöse Bronchitis, Pasteurella multocida undHaemorrhagische Enteritis erfolgt keine Praezipitation.

Dow

nloa

ded

by [

Cal

ifor

nia

Poly

Pom

ona

Uni

vers

ity]

at 1

7:54

28

Oct

ober

201

4