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Further studies onvertical transmission ofreticuloendotheliosis virus inturkeysJ.S. McDougall a , R.W. Shilleto a & P.M. Biggs aa Houghton Poultry Research Station , HoughtonHuntingdon, Cambs., PE17 2DA, United KingdomPublished online: 12 Nov 2007.

To cite this article: J.S. McDougall , R.W. Shilleto & P.M. Biggs (1981) Further studieson vertical transmission of reticuloendotheliosis virus in turkeys, Avian Pathology, 10:2,163-169, DOI: 10.1080/03079458108418470

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Avian Pathology, 10: 163-169, 1981

FURTHER STUDIES ON VERTICAL TRANSMISSIONOF RETICULOENDOTHELIOSIS VIRUS IN TURKEYS

J.S. McDOUGALL, R.W. SHILLETO and P.M. BIGGS

Houghton Poultry Research Station, Hough tonHuntingdon, Cambs. PE17 2DA, United Kingdom

SUMMARY

Turkey hens were inseminated with infected semen from REV tolerantturkey stags at weekly intervals for 6 weeks, and subsequently with semenfrom uninfected control stags for a further 6 weeks. Cell cultures preparedfrom eggs produced during the experimental period were assayed for REVby an indirect fluorescent antibody test. Poults hatched from such eggswere also assayed for REV at 1 day of age or maintained to 28 weeks of ageto study lateral spread of virus to uninfected hatch mates.

REV was detected in 27.5% of embryos from hens inseminated with REVinfected semen and 8.7% of 1-day-old poults hatched were found to beviraemic. Following the change to insemination with uninfected semen,REV was detected in 0.7% of embryos examined. REV was isolated fromthe lower oviduct of two of 18 hens, 12 weeks after first insemination withinfected semen.

REV spread readily to uninfected poults maintained in contact with pro-geny of hens inseminated with REV-infected semen, in that 50% hadneutralising antibody by 8 weeks of age. At 28 weeks of age the leukosismortality in such poults was 16%.

INTRODUCTION

Reticuloendotheliosis virus (REV) has been isolated from outbreaks of neoplasticdisease in turkeys (Paul etal, 1976; McDougall etal, 1978). Experimental inocula-tion of 1-day-old turkey poults with the REV isolated by the latter workers produceda state of tolerance which persisted beyond 40 weeks of age (McDougall etal, 1980).REV was isolated from 8% of cell cultures prepared from embryonated eggs producedby viraemic turkey hens, and also from the semen of viraemic turkey stags (McDougallet al, 1980). Further studies reported in this paper have shown that turkey hens arereadily infected via the oviduct at artificial insemination resulting in the productionof infected progeny, which readily transmit the infection to uninfected hatchmates.

Received 23 October 1980Accepted 12 December 1980

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164 J.S. McDougall ef a/.

MATERIALS AND METHODS

Experimental turkeysTurkey eggs of a commercial strain were incubated and hatched and the poults weresubsequently reared in an isolation unit maintained under positive pressure with fil-tered air, and fed on commercial grower and breeder rations.

InoculumFor infection of 1-day-old poults to produce tolerant stags the isolate REV-HPRS-1previously described by McDougall et al. (1980) was used. Each bird was inoculatedintra-abdominally with 3.1 x 103 plaque forming units (pfu) of REV-infected tissueculture fluid.

Assays for REV viraemia and REV antibodyCitrated plasma and serum samples were assayed for viraemia and antibody by use ofthe plaque assay and plaque neutralisation test previously described (McDougall etai,1980).

Cell culturesChicken embryo fibroblast (CEF) cultures were prepared from embryonated eggsderived from the Houghton Poultry Research Station Brown Leghorn flock by themethod of Temin and Rubin (1958). Duck embryo fibroblast (DEF) and turkey em-bryo fibroblast (TEF) cultures were similarly prepared. The former were from hatch-ing eggs supplied by Cherry Valley Farms Ltd., Rothwell, Lincoln and the latter fromfertile eggs produced by the turkey hens in the experiment.

Experimental designFor vertical transmission studies on REV, 100 poults were hatched as parent stock.Of that number 20 poults were inoculated with REV-HPRS-1 at 1 day of age to es-tablish REV-tolerant males to provide infected semen for artificial insemination. Theremaining hatch mates were reared in isolation to 26 weeks of age when 35 femalesand 15 males were retained for REV transmission studies. From 30 weeks of age the35 turkey hens were separately maintained as two groups. One group of 18 was sub-sequently artificially inseminated weekly with pooled semen from the same four REVtolerant stags while the other group of 17 hens was inseminated with pooled semenfrom four to six randomly selected control uninfected stags. After the 6th week bothgroups were inseminated with semen from control uninfected stags. Serum andplasma samples from all birds were assayed for REV antibody and REV viraemia res-pectively, at 26 weeks of age, i.e. before commencing the experiment, and at 42 weeksof age when the experiment was terminated. Swabs taken from the everted oviduct ofboth groups of hens were cultured on DEF cells after five and 13 inseminations at 35and 42 weeks of age respectively.

TEF cell cultures were prepared from embryonated eggs produced by both groups on2 selected days each week, apart from the 5th and 6th weeks when all eggs were incu-bated to hatching. TEF cultures were passaged three times and assayed for REV bythe indirect fluorescent antibody (FA) test (Witter et al., 1970).

For studies on progeny, poults hatched from eggs laid by both groups during the 5thweek of the experiment were reared together from hatching to 28 weeks of age tostudy lateral spread of REV in turkey poults. Blood samples were assayed for REVantibody and viraemia at 4-week intervals from 4 to 28 weeks of age. Poults hatchedfrom eggs laid during the 6th week were killed at 1 day of age and plasma samplestested for the presence of REV.

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Vertical transmission of REV by infected turkey semen 165

RESULTS

Mortality and viraemia in poults inoculated with REV for supply of donor malesEleven of the 20 poults inoculated with REV at 1 day of age survived to 26 weeks ofage. Five of the nine turkeys that died had leukotic lesions similar to those seen innatural disease outbreaks. When tested for REV viraemia at 26 weeks of age, 10 ofthe 11 surviving birds were found to be viraemic and REV was also isolated from apooled semen sample of the four surviving viraemic male birds at 30 weeks of age.Semen from these was subsequently used to inseminate the group of 18 turkey hensweekly for 6 weeks.

Assay for REV antibody in inseminated turkey hensREV antibody was not detected in any of the hens selected for the experiment whentested at 26 weeks of age. Antibody was, however, detected in 15 or 17 hens follow-ing insemination with REV-infected semen for 6 weeks and subsequently with non-infected semen, in tests carried out at 42 weeks of age.

Isolation of REV from inseminated turkey hensREV was not isolated from plasma samples taken at 26 and 42 weeks of age, but wasisolated in cell culture from oviduct swabs from two of 18 hens 5 weeks after weeklyinsemination with REV-infected semen had began. REV was again isolated from theoviducts of the same two hens at the end of the experiment 12 weeks after primaryinsemination. Antibody was not detected in either of these birds and REV was notisolated from plasma in tests carried out at 42 weeks of age.

Isolation of REV from embryos from REV-inseminated turkey hensTwenty-five of 91 embryos (27.5%) collected during the first 4 weeks after primaryinsemination from the groups of hens inseminated with infected semen were infectedwith REV (Table 1). Following the change to the use of semen from uninfected stagsonly one of 138 embryos (0.7%) was found to be infected. No infected embryos weredetected in the 207 embryos derived from hens inseminated with semen from unin-fected turkey stags.

Table 1. Results of indirect FA tests on TEF cultures of embryos derivedfrom hens inseminated with REV-infected and uninfected semen.

Group No. of turkey hens Proportion of infected embryos

A Infected semenB Uninfected semen

1817

25/9la

0/9727.5D

0Results following insemination with uninfected control semen

1817

1/1380/110

0.70

a No. positive/No, tested,b Percentage positive.

Mortality in hatched turkey poultsThe overall mortality and leukosis mortality in progeny of turkey hens inseminatedwith REV infected semen and in turkeys maintained in contact with them to 28 weeksof age is given in Table 2. Total mortality was similar in both groups (32% and 30%respectively). In the REV-infected group two of eight birds which died had leukoticlesions; the other deaths occurred in unthrifty birds, some of which had severe pos-tural abnormalities. At autopsy at 28 weeks of age an additional two birds in the group

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166 J.S. McDougall et al.

Table 2. Mortality and incidence ofleukosis in progeny of turkey hensinseminated with REV-infected semen and in poults maintainedin contact.

Group

Infected progenyContact progenyUninfected controls

No. of poults

253012

No.Died

891

Incidence of leukosis

450

16.016.60

had generalised leukotic lesions. Five of eight contact exposed birds which died duringthe experimental period had leukosis. No lesions were detected in the survivors at 28weeks of age and leg problems were not a feature of REV infection in this group.

Assay for REV in hatched turkey poultsREV was isolated from two fo 23 plasma samples taken from progeny of hens insemi-nated with REV-infected semen and killed at 1 day of age. Of the hatch reared to 28weeks of age and tested at 4-weekly intervals (Table 3), REV was isolated from sixof 25 plasma samples at 4 weeks of age from the progeny of turkey hens inseminatedwith REV-infected semen. In two of the six, the viraemia was not detected at subse-quent tests. At 8 weeks of age a similar transient REV viraemia was detected in anadditional two birds in this group. Two of the four viraemic birds at the first testsurvived the 28-week experimental period and were viraemic at all tests. The othertwo died at 17 and 26 weeks of age and were viraemic at the preceding tests, i.e. at16 weeks and 24 weeks of age respectively. A transient viraemia was detected in twoof 30 contact exposed birds, viraemia being detected at 8 weeks of age but not atsubsequent tests (Table 3).

Table 3. Incidence of viraemia in the progeny of turkey hens inseminatedwith REV-infected semen and in uninfected poults maintained incontact with them.

Group

Infected progenyContact progenyControl progeny

Age in weeks

4

6/25a

0/300/12

8

6/242/300/12

12

4/240/290/12

16

4/240/270/11

20

3/210/270/11

24

3/200/250/11

28

2/170/210/11

a No. viraemic/No. tested.

Assay for REV antibody in hatched turkey poultsNeutralisation tests for REV antibody were not carried out on plasma samples fromthe hatch of poults killed for virus isolation at 1-day-old. In the groups of poultsreared to 28 weeks of age antibody to REV was present in 18 of 25 progeny ofturkey hens inseminated with REV-infected semen at the first test at 4 weeks of age(Table 4). Antibody was not detected in serum of 8 of these 18 poults at 8 weeks of agesuggesting that this antibody was maternally-derived. By 12 weeks of age only sixbirds in the group did not have antibody. Of these, four were viraemic birds and failedto develop REV antibody during the course of the experiment and two were poultswhich developed antibody later. REV antibody was not detected in the 30 contactpoults at 4 weeks of age. By 8 weeks 50% had neutralising antibody and at 16 weeksof age all had antibody.

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Vertical transmission of REV by infected turkey semen 167

Table 4. Incidence of REV neutralising antibody in the progeny of turkeyhens inseminated with REV-infected semen and in control unin-fected progeny maintained in contact.

Group

Infected progenyContact progenyControl progeny

Age in weeks4

18/25a

0/300/12

8

11/2415/300/12

12

18/2423/290/12

16

19/2427/270/11

20

18/2127/27

0/11

24

17/2025/250/11

28

15/1721/210/11

a No. positive/No, tested.

DISCUSSION

As reported previously (McDougall et al (1980) inoculation of 1-day-old poults withREV-HPRS-1 resulted in high mortality and the establishment of tolerance to thevirus. In the mature male turkey this induced state of tolerance was associated withthe production of REV-infected semen. In the present experiment the use of infectedsemen for artificial insemination of turkey hens resulted in infection of the hens viathe oviduct. In two of 18 hens a persistent infection of the oviduct was detected 6weeks after replacing infected semen with uninfected control semen. Neither REVantibody nor REV viraemia was detected in these two birds, though it is possiblethat a transient viraemia had been present prior to testing at 42 weeks of age and in-sufficient time had elapsed for the development of neutralising antibody.

REV was isolated from 27% of the embryos derived from eggs laid by 18 turkey hensduring the 4-week period of insemination with REV infected semen. Following thechange to insemination with uninfected semen from control stags REV was isolatedfrom only one of 138 embryos derived from the same 18 hens. Although the percen-tage of infected embryos might be expected to diminish as immunity developed inhens infected via the oviduct, REV was isolated from the oviducts of two of the 18at the end of the experiment and at that time both were found at autopsy to be infull lay. Despite this, no infected embryos were detected during the preceding 5weeks, suggesting that the higher rate of embryo infection following the use of infec-ted semen resulted directly from infection of the ovum at fertilisation by virus on orin the spermatozoa, rather than indirectly by infection of the hen. This view is sup-ported by the previous finding that only two of 25 embryos from viraemic hens wereinfected (McDougall etal, 1980), whereas 25 of 91 progeny embryos from hens in-seminated with REV-infected semen were infected in this study.

The importance of the congenitally infected poult in lateral spread of infection wasdemonstrated. Four of 25 progeny of hens inseminated with REV-infected semenwere found to be viraemic at all tests. Two of the four survived the experimentalperiod, constituting a reservoir of infection for uninfected progeny maintained incontact from 1 day of age. All the latter had neutralising antibody at 16 weeks ofage and at 28 weeks the leukosis mortality was 16%.

The detection of neutralising antibody at 4 weeks of age in 72% of progeny of hensinseminated with REV-infected semen indicates a high incidence of maternally-derived antibody. This did not appear to affect the outcome of infection with REVin that the overall mortality and the incidence of leukotic lesions were similar ingroups of poults with and without such antibody.

In this work, infection at 1 day of age resulted in a state of tolerance and the presence

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168 J.S. McDougall et al.

of REV in the semen of mature turkey stags. No information is available on virusshedding in the semen of turkey stags infected later in life during a period of transientviraemia or in birds with neutralising antibody. Further studies on these aspects ofREV infection are necessary in order to formulate control measures for dealing withdisease outbreaks in breeding flocks.

AcknowledgementThe authors wish to thank British United Turkeys Ltd. for supplying the turkeyhatching eggs for use in this experiment.

REFERENCES

McDougall, J.S., Biggs, P.M. and Shilleto, R. W. (1978). A leukosis in tuikeys associated with in-fection with reticuloendotheliosis virus. Avian Pathology, 7: 557-568.

McDougall, J.S., Shilleto, R. W. and Biggs, P.M. (1980). Experimental infection and vertical trans-mission of reticuloendotheliosis virus in the turkey. Avian Pathology, 9: 445-454.

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

Temin, H.M. and Rubin, H. (1958). Characteristics of an assay for Rous sarcoma cells in tissueculture. Virology, 6: 669-688.

Witter, R.L., Purchase, H.G. and Burgoyne, G.H. (1970). Peripheral nerve lesions similar to thoseof Marek's disease in chickens inoculated with reticuloendotheliosis virus. Journal of theNational Cancer Institute, 45: 567-577.

RESUME

Etude complémentaire de la transmission verticaledu virus de la réticuloendothéliose chez la dinde

Des dindes ont été inséminées six fois à une semaine d'intervalle avec de la semenceinfectée de mâles tolérants au REV et ensuite pendant à nouveau six semaines avecde la semence de mâles témoins non infectés. Des cultures cellulaires préparées àpartir des oeufs produits durant la période expérimentale ont été contrôlées pour laprésence de REV en immunofluorescence indirecte. La présence de REV a été égale-ment recherchée chez les dindonneaux d'un jour éclos de ces oeufs, ainsi que pendant28 semaines afin d'étudier la transmission horizontale du virus à des sujets témoinsnon infectés.

Le REV a été retrouvé chez 27,5% des embryons depoules inséminées avec la semenceinfectée et 8,7% des dindonneaux d'un jour ont été trouvés virémiques. Après insémi-nation avec la semence non infectée, le REV a été détecté chez 0,7% des embryonsexaminés. Douze semaines après la première insémination avec la semence infectée, leREV a été isolé de la partie inférieure de l'oviducte de 18 dindes.

Le REV se dissémine rapidement aux dindonneaux non infectés maintenus en contactde la descendance des dindes inséminées avec la semence infectée par le REV, 50% deces derniers ayant présenté une séroconversion à l'âge de 8 semaines. A 28 semainesd'âge, la mortalité par leucose chez ces dindes a été de 16%.

ZUSAMMENFASSUNG

Weitere Untersuchungen über die vertikale Übertragungdes Retikuloendotheliosevirus bei Puten

Putenhennen wurden mit einem infizierten Samen von Reotoleranten Putern 6 Wochenlang in wöchentlichen Abständen inseminiert und anschließend für weitere 6 Wochenmit einem Samen von nicht infizierten Kontrollputern. Zellkulturen aus Eiern, die

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Vertical transmission of REV by infected turkey semen 169

während der Versuchsperiode gelegt wurden, wurden mit einem indirekten Fluoreszenz-antikörpertest auf REV geprüft. Putenküken, die aus solchen Eiern erbrütet wordenwaren, wurden als Eintagsküken auf das Vorhandensein von REV überprüft oder 28Wochen gehalten, um die horizontale Verbreitung des Virus auf nichtinfizierte gleich-zeitig geschlüpfte Küken zu untersuchen. REV wurde in 27.5% der Embryonen vonHennen, die mit REV infiziertem Samen besamt worden waren, nachgewiesen und8.7% der geschlüpften Eintagsputen waren viraemisch. Nach dem Wechsel zur Be-samung mit nichtinfiziertem Samen war REV in 0.7% der überprüften Embryonenvorhanden. REV wurde 12 Wochen nach der ersten Besamung mit infiziertem Samenaus dem unteren Eileiter von 2 von 18 Hennen isoliert. REV verbreitete sich von derNachkommenschaft der mit infiziertem Samen besamten Hennen schnell auf nichtinfizierte Kontaktputenküken, von denen im Alter von 8 Wochen 50% neutralisierendeAntikörper aufwiesen. Im Alter von 28 Wochen betrug die Leukosemortalität beidiesen Puten 16%.

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