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On epizootiology and control of lymphoid leukosis in chickens
Comp. Imrnun. Microbiol. infect. Dis., Vol. 1, pp. 93-106. © PergamonPressLtd., 1978.Printedin GreatBritain
0147-9571/78/0701-0093502.00/0
O N EPIZOOTIOLOGY A N D C O N T R O L OF LYMPHOID LEUKOSIS IN C H I C K E N S G. F. DE BOER, J. VAN VLOTEN, J. E. GROENENDAL a n d H . J. L. MAAS Central Veterinary Institute, Virology Department, 39 Houtribweg, Lelystad, The Netherlands and A . HOOGERBRUGGE University of Utrecht, Institute of Zootechnic, Yalelaan, De Uithof, Utrecht, The Netherlands Abstract--Sera and organ extracts from ten different commercial stocks of layer chickens were examined for the presence of lymphoid leukosis (LL) viruses. Virus was recovered from 40.8% of the cockerels between three and six weeks of age. Their female hatch mates were examined at the age of 20 months. A mean of 11.3% of these laying hens was positive in the NP activation test. Lymphoid leukosis was successfully controlled in three inbred strains of White Leghorn chickens and in a commercial White Plymouth Rock line. All flocks were kept in a filtered air positive pressure (FAPP) house during the first two months of life and thereafter transferred to a conventional environment. The control method is based on three elements: - - f r o m an infected flock, hens are selected in whose eggs no avian lymphoid leukosis viruses can be detected by examination of pooled extracts of groups of embryos; - - o n l y eggs from hens that are shown not to shed congenitally virus in their eggs are used for the production of progeny. The offspring are reared in isolation until two months of age at which time the age-related resistance against tumour formation appears to be sufficiently developed; - - t h e chickens are subsequently intramuscularly inoculated with lymphoid leukosis viruses of subgroups A and B and transferred to a conventional chicken house. The inoculated birds become persistently viremic and resist horizontal virus exposure and intramuscular challenge infections. Horizontal virus transmission was observed to take place when virus-free non-vaccinated chickens were reared in isolation for two months and then exposed under field conditions. Efficiency of virus recovery was considerably improved when washed buffy coat cells were cocultivated with chick embryo fibroblasts or explant cultures were prepared from various tissues before testing with the NP activation test. Key words: Lymphoid leukosis, epizootiology, congenital infection, horizontal transmission
EPIZOOTIOLOGIE
ET ERADICATION DE LA LEUCOSE LYMPHOIDE POULETS
CHEZ
Rtsume--L'incidence des infections du virus de la leucose lympho'ide (LL) dans 10 lots du commerce de poulets destints /l la ponte /~ 6t6 recherchte par des techniques virologiques. Les poussins m~.les ont 6t6 examints entre 3 et 6 semaines apr~s rtclosion et les femelles ont 6t6 testtes apr/~s 20 mois. La frtquence moyenne des animaux porteurs de virus 6tait de 40.8% chez les poussins males et de 11.3% chez les pondeuses de m~me origine fi l'5.ge de 20 mois. La frtquence des sympttmes eliniques pendant 20 mois &ait de 0.43%. Les virus isolts appartiennent essentiellement au sous-groupe A et seulement 3% appartiennent au sous-groupe B. L'isolement du virus est amtlior6 quand les leucocytes apr~s lavage sont mis en coculture avec des fibroblastes d'embryons de poulet ou d'explants primaires /t partir de divers tissus avant le test dit "NP activation". La leucose lymphode h 6t6 6radiqute dans trois ligntes de poulet Leghorn et dans une lignte commerciale "White Plymouth Rock". La m&hode de contrSle appliqute est baste sur trois 616ments: - - d e s pondeuses produisant des oeufs indemnes de LL ont 6t6 ehoisies parmi un troupeau infectt. A 93
94
G. F. DE BOER, J. VAN VLOTEN, J. E. GROENENDAL, H. J. L. MAAS and A. HOOGERBRUGGE l'aide des tests dits "NP activation" des extraits mtlangts de diff&ents groupes d'embryons ont et~ examints de faqon ~t dtterminer une 6ventuelle transmission congtnitale de virus; - - s e u l s les oeufs provenant des animaux virus-ntgatifs sont utilis& pour la reproduction. La descendance est 61evte dans un poulailler en surpression d'air et muni de filtres (FAPP) jusqu' ~i deux mois, ~.ge ~. partir duquel la rtsistance h la leucose lympho'ide est suffisamment dtveloppte; - - l e s poulets sont ensuite inocults par vole intramusculaire avec le virus de la leucose lymphoide de sous-groupes A e t Bet mis ensuite dans un poulailler conventionnel. Les animaux inocults ne developpent pas de sympt6mes cliniques de LL, restent porteurs de virus et resistent h la transmission horizontale du virus. Et surtout, chez des pondeuses traittes par les mesures prtc&tentes la transmission par l'oeuf de virus de LL ne pouvait pas &re d~celte. Quand la transmission congtnitale a. 6t6 61iminte par les mesures prtctdentes la diffusion horizontale du virus joue un r61e important dans l'epizootiologie de la leucose lymphoi'de dans les quelques groupes non-vaccints.
Mots-clefs: Leucose lymphoide, epizootiologie, infection congtnitale, transmission horizontale
INTRODUCTION It has long been recognised that infections with avian lymphoid leukosis (LL) viruses are wide-spread all over the world. Specific antibodies are present in most chicken flocks. A limited number of publications, however, is available on the prevalence of viremic birds in commercial poultry [5, 9, 19]. The eradication of lymphoid leukosis from chicken flocks may be pursued by eliminating all viremic and serologically positive birds from a given population [4, 20] or by selecting dams that do not excrete virus in their eggs and using only LL virus-free eggs to hatch the next generation. The latter procedure has better prospects for successful control since the natural flow of infection is interrupted at the stage which results in the highest incidence of clinical disease and the most severe spreading of LL viruses into the environment [6, 8]. Congenitally infected birds are considered a primary source of contagion. Our investigations to develop a control programme were primarily directed at the elimination of hens excreting LL viruses in their eggs. In order to make the control method applicable under field conditions, the chickens hatched from virus-free eggs were reared in isolation until the agerelated resistance against tumour formation was present and were inoculated with LL viruses of subgroups A and B before being transferred to a conventional environment. The procedure resulted in birds persistently infected with LL viruses, but in which no clinical disease was observed and none produced LL virus-infected eggs. Preliminary data are reported on virus recovery from buffy coat cells or explant cultures of various tissues of inoculated chickens.
MATERIALS AND METHODS
Epizootiologiestudy In 1974 and 1975 ten different commercial stocks of layer chickens were examined for the presence of LL viruses. Eggs of each layer stock were hatched in a formalin disinfected incubator. The cockerels were transferred to a filtered air positive pressure (FAPP) house directly after hatching. Positive pressure was maintained at 5 mm water column and the incoming air was passed through high efficiency particulate air (HEPA) filters (Camfil AB, type 1J-1000, class 100, Trosa, Sweden). The stocks were subsequently tested during a period of three weeks starting at the age of three weeks. Serum samples and homogenates of
On epizootiologyand control of lymphoid leukosis in chickens
95
spleen, liver, pancreas and bursa were examined. All female chicks were transferred to a conventional chicken house for a random sample egg production test. These birds were reared under the same conditions and placed in individual cages at the age of five months. At 20 months of age the incidence of LL virus infections was recorded by testing of serum samples.*
Experimental chickens Investigations of control methods for LL were initiated in 1969 with three strains of inbred White Leghorn (WL) chickens: A, B and 98A. In 1972 a commercial White Plymouth Rock line (WPR), in use for producing hybrid females for broilers, was included in the study.t
Holding conditions Each generation of WL chickens consisted of 3 0 - 5 0 adult birds. The chickens were kept in a pen type chicken house. Different age groups were kept in the same house, usually chickens of other breeds were present. The various groups were held in pens on a dirt floor. The pens were separated from each other by a single wire netting. Each pen of 12 m 2 held 3 0 - 5 0 adult chickens. Before introduction of a new group of chickens the litter was removed and the pen cleaned and disinfected. The chickens were fed with commercial all mash meal feeds. Drinking water was supplied with non-flowing water fountains. Eggs were collected in trap nests. All chickens were vaccinated at day-old against Marek's disease with cell-associated CVI 988 vaccine [15]. Vaccination against Newcastle Disease (ND) and infectious bronchitis (IB) was performed at three weeks of age with a combined vaccine based on the ND-Hitchner B 1 and I.B.-H 120 virus strains. At seven weeks of age the chickens were revaccinated with NDLaSota vaccine. Vaccinations against avian encephalomyelitis and fowlpox were performed in the second part of the rearing period. Prior to 1972 each new generation was hatched from eggs from LL virus-negative hens. After hatching the chickens were raised under conventional conditions. From 1972 on progeny from virus-negative hens were kept in a FAPP house for the first two months of life. While in isolation the chickens were fed pelleted feeds and each flock had its own attendant. During the entire period of the investigation a WL strain A control flock was maintained but progeny were hatched from eggs from unselected hens. This flock was constantly kept in the conventional chicken house. Except for the two-month periods of isolation, the WPR chickens were kept on litter during rearing and in individual cages during the laying period. An all-in all-out type of management was practised at the basic breeding farm. The normal selection procedures used for commercial pedigree chickens were applied in addition to those aimed at eliminating LL. Vaccination procedures against other diseases were those in use for commercial laying flocks in The Netherlands.
Procedures for the control of LL In order to detect viremic hens and hens excreting virus with their eggs, sera and pooled extracts of 11-day embryos from individual hens were tested for the presence of LL viruses. * The co-operation of the Koninklijke Vereniging ter Bevordering van de Pluimveehouderij in Zeeland (V.P.Z.), Wissenkerke and of Dr. O. J. H. Devos, Provincial Animal Health Servicein Goes, is gratefullyacknowledged. t The authors thank Drs. F. Borm, A. van Hedel and Mr. A. W. Manders for their co-operationin this part of our study.
96
G. F. DE BOER, J. VAN VLOTEN, J. E. GROENENDAL, H. J. L. MAAS and A. HOOGERBRUGGE
Hens of the WL flocks were examined repeatedly. Eggs collected during two-week periods were pooled and examined as one sample. Between the sampling periods were intervals of two to three weeks. Eggs from the WPR grand parent flock were collected during two 14-day periods, at least three weeks apart. WPR hens producing less than seven living embryos per 14-day observation period were not included in the test and thus not used for the production of progeny. From 1972 on chickens hatched from eggs of LL virus-negative dams were reared in the FAPP house for a period of two months. Thereafter the chicks were transferred to the conventional chicken houses described above after having been inoculated with LL viruses of subgroups A and B.
Collection and processing of specimens Blood samples were collected by venipuncture. The sera were harvested after 10 min. centrifugation at 1000g and were stored a t - 7 0 ° C . Eggs were collected individually and stored at 15 °C until the entire production of each flock obtained during a 14-day period was available for incubation as one batch at 37.5°C. Embryos were harvested after 11 days and pooled extracts were prepared of all embryos available from each hen. The embryos were collected aseptically, washed with Hanks' buffered saline solution (BSS) and antibiotics and squeezed through a 20 ml syringe. An equal amount of Hanks' BSS plus 10% calf serum and antibiotics was added and the samples were stored at - 7 0 ° C . They were ready for virus assay after two cycles of freezing and thawing and centrifugation for 10 min. at I000 g. For the epidemiologic study organ extracts were prepared of spleen, liver, pancreas and bursa. The organs were aseptically removed and ground in a Griffith tube. A third volume of Hanks' BSS plus calf serum and antibiotics was added and the suspensions were centrifuged for 10 min. at 1000g.
Cell cultures and media Chick embryo fibroblasts (CEF) derived from embryos of specific pathogen free chickens of the WL strain A, which have been shown to provide cell cultures of the C/E phenotype, duck embryo fibroblast (DEF) cultures and Rous sarcoma virus-induced non producer (NP) cells were used throughout. The preparation and maintenance of the cell cultures and the various media have been described previously [14, 15].
Virus assays The NP activation test [12, 13] was used throughout. The test is based on the specific activation of non-producing Rous sarcoma cells (NP cells) and detects, without differentiation, all subgroups of lymphoid leukosis viruses (subgroups A, B, C and D, since CEF cells of the C/E phenotype were used). Subgroup determination of LL isolates was performed by the RSV resistance inducing factor (RIF) test [16]. Incidentally the RIF test and the indirect immunofluorescence technique for group-specific antigens of avian tumour viruses were used for virus assay as well. Since the use of the NP activation test was preferred because of easier performance and greater capacity, the immunofluorescence test was omitted in the later stages of the investigation.
On epizootiologyand control of lymphoid leukosis in chickens
97
Serum neutralisation test Neutralising antibodies were assayed with a focus reduction test with 100 F F U of BHRSV (RAV-1) and BH-RSV (RAV-2) viruses. The neutralisation titre is the reciprocal of the dilution which reduces the number of foci by 90% or more. Viruses used for immunisation Originally, each chicken received an inoculum of 0.5 ml containing a mixture of 105 T C I D Rous associated virus CVI-1 (a subgroup A leukosis virus), 103 TCIDso Rous associated virus (RAV-2) of subgroup B and about 3 × 105 TCIDs0 of three LL virus isolates (presumably of subgroup A) of the WL chicken strains A, B and 98A respectively. The CVI1 virus strain was isolated according to the method of Rubin and Vogt [18] from a stock of Bryan standard strain Rous sarcoma virus that was kindly supplied by Dr. P. M. Biggs, Houghton Poultry Research Station, Houghton, England. This avian leukosis subgroup A virus is probably similar to the HPRS-2 isolate of Chubb and Biggs [7]. The RAV-2 virus was separated with the same procedures from a stock of BH-RSV (RAV-2) virus obtained from Dr. B. R. Burmester, Regional Poultry Research Laboratory, USDA, East Lansing, Michigan. From 1974 on the inoculum contained 105 TCIDs0 o f R A V CVI-1 and the dose of RAV-2 was increased to 105 TCIDs0. All virus stocks were grown in CEF cultures.
Virus isolation procedures Material to be tested for virus received one passage on CEF monolayers prior to further testing. The processing of serum samples, embryo and organ extracts has been described above. In the later stage of the study virus isolation from blood of a group of WL line A chickens was done by cocultivation of washed buffy coat cells with CEF cultures and virus cultivation from tissue suspensions was substituted by the application of explant cultures of infected tissues. The white blood cells were taken from heparinised blood samples and washed three times by centrifugation for I0 min. at 1000 g in PBS. This resulted in a plasma dilution factor of x 10.000. CEF monolayers, 24 hr old, were inoculated with the washed cells. Maintenance medium was added after 1 hr incubation at 38°C. The cultures were harvested after six days. Usually one cell passage was made in between. The cells and supernatant were collected by scraping off with a silicone policeman and stored at - 7 0 ° C until further assayed. Explants of the various tissues were prepared with two scalpels in a few drops of growth medium. After a gentle wash, fragments of 1 to 2 m m 3 were placed in a dry plastic Petri dish. The explants became attached to the bottom by leaving the Petri dish on the bench for about 45 min. Subsequently, growth medium was added and the dishes placed in a humidified CO2 incubator at 38°C. A change of medium was performed twice a week. The cultures were harvested when a significant outgrowth of cells around the explants was observed. This required six to ten days. After storage at - 7 0 ° C , the samples were tested using the NP activation test. The comparative trials for virus isolation from plasma and buffy coat were conducted with experimental WL strain A chickens which were inoculated with subgroup A and B viruses at ages varying between 0 and 10 weeks. The various age groups were inoculated at the same time and kept in the FAPP house for a period of one and a half years. At the age of about 16 months 4
98
G . F . DE BOER, J. VAN VLOTEN, J. E. GROENENDAL, H. J. L. MAAS and A. HOOGERBRUGGE
these chicks were challenge-infected with 105 TCIDs0 R A V - C V I - 1 or R A V - 2 and samples were taken at about 20 months o f age. RESULTS A mean frequency o f infection with L L viruses of 40.8% was demonstrated in the commercial stocks of chickens examined between three and six weeks o f age (Table 1). A total o f 196 virus isolates was obtained. Virus was recovered from 152 serum samples taken from cockerels of the various commercial stocks. Organ extracts of the same birds were also positive but an additional 44 isolates were recovered from organs only. Interference assays were done with 135 isolates. L L virus of subgroup A was most prevalent (97%) and 3% of our isolates belonged to subgroup B. O n l y one bird yielded both subgroups A and B. A b o u t 20 months later, sera from female chickens o f the same hatch were tested (Table 2). N o w 80 of 706 samples were found positive (11.3%). In contrast with the frequency o f viremic birds an unusually low incidence o f clinical disease was observed in the commercial layer chicks under study. During an observation period o f 420 days a total mortality o f 13.7% was Table 1. Virus isolation from 3- to 6-week-old cockerels of 10 commercial layer stocks Sera 1 2 3 4 5 6 7 8 9 10
35/47* 8/40 17/48 12/50 5/48 0/44 14/51 5/51 35/51 21/51
1-10
152/481
Organ extracts 74.5% 20.0% 35.4% 24.0% 10.4% 27.4% 9.8% 41.2%
38/47* 12/40 24/47 20/50 10/47 2/44 22/51 9/50 36/51 23/51
80.8% 30.0% 51.1% 40.0% 21.3% 4.5% 43.1% 18.0% 70.6% 45.1%
31.6%
196/478
40.8%
68.6%
* Total positive chickens/No, of specimens examined. Table 2. Virus isolation from laying hens of 10 commercial stocks at 20 months of age Sera 1 2 3 4 5 6 7 8 9 10
17/72* 13/71 7/69 4/67 7/72 2/72 2/69 3/71 17/72 8/71
23.6% 18.3% 10.1% 6.096 9.7% 2.8% 2.9% 4.2% 23.6% 11.3%
1-10
80/706
11.3%
* Total positive chickens/No, of sera examined.
99
On epizootiologyand controlof lymphoidleukosis in chickens
observed, but in only 0.43% of the laying hens overt lymphoid leukosis was diagnosed by macroseopical examination. In the period 1969-1972 the three WL strains were examined for LL viruses with the indirect immunofluorescent staining technique and the NP activation test [11-13]. Both the sera of adult chickens and embryo extracts prepared from eggs of individual hens were tested. The two techniques yielded identical results. Each new generation was hatched from eggs obtained from hens that had been found negative in all tests. With this procedure no significant reduction of the number of virus isolations from serum samples and embryo extracts was obtained for three generations. Table 3. Elimination of lymphoidleukosis fromtwo WL flocks, strains B and 98 A White Leghorn strain Generation number No. of hens
WL B
Age in months 9 Pooled embryo
extracts of 14-dayperiods
WL 98 A
I 23
/ ~'10 J 11
II* 42
II* 39 Virus Age in Virus Age in Virus Age in Virus recovery months recovery months recovery months recovery 2/20t 2/19 2/21
~ 2/22~t ~.
8 9
I 33
0/35 0/37
8 9
6/30 5/27
9 10
0/33 0/35
10
0/36
10
3/22
11
0/35
12 13 14 15
0/31 0/27 0/25 0/21
13 14 15 16
0/35 0/35 0/37 0/38
7/32
0/39
0/38
* Reared in isolation and "vaccinated" at 2 months of age. 1"Number of pooled embryoextracts from whichvirus was recovered/numberof samples examined. :~Numberof positive hens/total number of hens examined. From 1972 on chickens destined for breeding were reared in isolation for two months. They were subsequently transferred to the conventional chicken house after being inoculated intramuscularly with LL viruses of subgroups A and B. From that time on a significant reduction of the number of virus-excreting hens was observed. No eggs infected with LL viruses were found in WL strains B and 98A second generations (Table 3), but in strain A, virus was still demonstrated in embryo extracts of 5 of 94 (5/94) hens (Table 4). No virus was however recovered from the succeeding generation of this flock when the same procedures were again applied. The eggs of hens of the second generation of the WL strains B and 98A and of the second generation of strain A were collected when the birds were between 8 and 16 months of age comprising a period of 70-98 days of the laying period. Intermittent virus excretion was occasionally encountered, e.g. virus was recovered four times from embryo extracts prepared from eggs of one of the five positive hens of the W L strain A generation (Table 4). The embryo extracts collected in the 14-day productions at the age of 9, 10, 15 and 16 months were positive but not those obtained in month 8, 12, 13 and 14. In the W P R flock egg transmission of LL viruses was absent in the first generation that was hatched from eggs from virus-negative dams and reared in the F A P P house (Table 5). At the age of one month the sera of the cockerels, that were culled from the flock, were examined. Virus was recovered from three of them. Their sisters were also removed from the
100
G. F. DE BOER, J. VANVLOTEN, J. E. GROENENDAL, H. J. L. MAASand A. HOOGERBRUGGE Table 4. Eliminationof lymphoidleukosis from WL chickens, strain A
Generation number
I
No. of hens
II*
41 Age in months
Serum samples
Pooled embryo extracts of 14-day periods
Virus recovery
. I 9 10
.
l
III*
47
. ll/41t 8/36
Age in months .
37
Virus recovery
Age in months
Virus recovery
7 9 10 11 14 16
0/37t 0/34 0/33 0/33 0/29 0/30
. 8 9 10 12 13 14 15 16
11/41~
49
0/41 1/44 1/43 0/40 0/38 0/36 1/33 1/35
0/38 2/45 1/46 2/33 3/30 0/31
1/46
0/35
4/48 5/94
* Reared in isolation and "'vaccinated" at 2 months of age. t Number of positive samples/numberof samples examined. $ Number of positivehens/total number of hens examined.
Table 5. Control of egg-transmissionof LL virus in a commercial White Plymouth Rock line Generation number
I
No. of chickens
II*
500 Age in months
Serum samples
-
Pooled embryo extracts of 14-day periods
f 7 J 8
III*
410 ~ + 560 ~
Virus Age in recovery months 9/'500 9/437 13/437§
1084 ~ + 60 0~c~
Virus Age in recovery months
It
3/560~:
7 8
0/368 0/359
Virus recovery
2
0/257
8 12
0/552 0/175
0/359
0/175
* Reared in isolation and "vaccinated" at 2 months of age. t Of cockerels that were culledfrom the flock at 1 month of age. $ Number of samples from which virus was recovered~numberof samples examined. § Number of positive hens/number of hens which was examinedtwice.
flock but serum samples of these chickens were negative for virus. The examination of the third generation yielded negative results as well [2]. The data of Table 6 give an indication of the extent in which horizontal transmission of LL viruses may occur. Since these chickens were full sibs of the third generation of W L strain A and were hatched from eggs from virus-negative hens (Table 4), the virus shedding in these groups must be ascribed to an infection contracted after hatching. The 47 chickens in the left side of Table 6 were kept in isolation for a period of two months and were not "vaccinated". Therefore the infection is likely to have been acquired after the transfer to the conventional environment. Virus containing embryo extracts were obtained from 13/36 hens. At the end of
On epizootiology and control of lymphoid leukosis in chickens
101
the laying period serum samples as well as organ extracts of the hens were examined. LL virus was demonstrated in the serum sample and the organ extract of only one bird. The 49 chickens of the same origin (right side, Table 6) were exposed to the infected environment at day-old. At the age of six months, LL virus was detected in two serum samples of this flock and eggexcretion was demonstrated in six of these hens (6/41). Table 6. Horizontal transmission of LL virus in WL strain A chickens hatched from virus-free eggs No. of chickens*
37 ~ Age in months
Serum samples
7
Pooled embryo extracts of 14-day periods
I 9 10 11 ~ 14 ~ 16 17
+ 10 ~ d ' t Virus recovery
41 9Q + 8 ~ : [ : Age in months
Virus recovery
0/46§
6
2/49
5/33 4/32 4/30 6/31 1/23 4/21
8 10 11 14 15 16
4/37 2/36 2/36 3/31 3/31 1/27
13/36** Sera and organ
18
6/41
1/36
extracts * Derived from the same negative hens as the third generation in Table 2.
t Reared in isolation for 2 months. Reared in a conventional chicken house. § Number of positive samples/numberof samplesexamined. ** Number of positivehens/total number of hens examined. During the entire period o f the study, the untreated WL strain A control flocks were examined with the same techniques. Results for the flocks that were housed together with the treated groups of the same chicken strain (second and third generations of Table 4) are presented in Table 7. By examination of embryo extracts 4 0 - 5 0 % of the birds were found to be shedding virus. The second generation had been hatched from eggs of 11 virus-positive and 17 virus-negative hens. Viremia was demonstrated in respectively 13/20 and 1/36 chickens of this generation at 7 months of age. The total scores for the embryo extracts were 18/22 and 13/38, respectively. In contrast with the high number of viremic and congenitally transmitting chickens in this flock, the incidence of clinically and by autopsy detected cases of LL was rather low (2/60 and 3/78 in the first and second generation, respectively). A group of 50 unselected hens of the WL strain A control flock was "vaccinated" at two months of age, after being reared in the conventional chicken house. This treatment did not seem to influence the number of hens excreting virus during the laying period. Since the NP activation test does not differentiate between the subgroups of LL virus, the positive samples were again examined with the RIF test. Only viruses of subgroup A were recovered from the flocks under study. Starting with the first generation in which vertical virus transmission was absent, no symptoms of LL were observed in any of the chickens of the flocks reported in this study. All virus isolations from the birds described above were done with sera or organ extracts.
~02
G.F. DE BOER, J. VAN VLOTEN,J. E. GROENENDAL,H. J. L. MAASand A. HOOGERBRUGGE
The vaccine viruses were only recovered from serum samples of the inoculated birds in the first two to three weeks. In the later stage o f our study a definite improvement o f the technique for virus detection was obtained by cocultivation o f washed buffy coat cells with C E F cultures or by explanting various tissues. These cultures were then assayed with the N P activation test. With these techniques vaccine viruses were in a few cases recovered from birds which had been inoculated one and a half years before. Virus was recovered from only one o f the four birds inoculated at the age of 8 weeks (Table 8). This chicken was challengeinfected with subgroup B virus at the age o f 16 months. Both subgroups A and B of L L virus were present in the virus isolate at the age o f 20 months. Table 7. Virus recovery from untreated and unselected flocks of WL strain A chickens Generation number No. of hens
Serum samples Pooled embryo extracts of l4-day periods
I 50
II 60 Hatched~omeggs of: 17 neg. hens 11 pos. hens
Agein months
Virus recovery
-
-
" 8 9 10 11 14 15 16 17
I
Agein months
12/42" 12/43 9/39 10/37 8/34 6/31 7/31 7/31
Virus recovery
Virus recovery
7
1/36"
13/20
9 10 12 15 16
9/37 9/36 9/33 9/29 2/16
12/18 11/15 10/15 9/14 0/ 5
13/38
18/22 31/60
19/48t * Number of samples which yielded virus/number of samples examined. "~Number of positive hens/total number of hens examined.
The persistent virus infection was nearly always demonstrated after one and a half years if the chickens underwent the controlled exposure below the age of four weeks. Virus isolation from the chicks of Table 8 was invariably negative when tissue homogenates were tested.
DISCUSSION As in other countries, L L viruses o f subgroup A were the most prevalent in the chickens under study [5, 9, 19]. Only 3% o f the virus isolates which we recovered belonged to subgroup B. A mean percentage of infection o f 40.8 was observed in young chickens and one of 11.3 was present in the commercial laying hens at the end of their laying period. These viruses were recovered from sera and organ extracts. Presumably, the score would have been even higher if washed buffy coat fractions or explant cultures were used. Since subgroup C or D viruses have not been detected in The Netherlands, it seems appropriate to have restricted the
On epizootiology and control of lymphoid leukosis in chickens
103
procedures for control to the subgroups A and B. M a n y more chickens become infected with L L viruses than the number that actually develops a lymphoma. The reported method for the control o f lymphoid leukosis is primarily directed to the elimination from the breeder flock of those hens which are capable of congenitally transmitting L L virus to their embryos. The in o v o infected chickens are known to develop L L with high frequency and to disseminate large quantities of virus [6, 17]. A high proportion
Table 8. Virus isolation from chickens "vaccinated" and challenge-infectedwith LL-viruses of subgroups A and B Vaccination* Hen No. at the age of (weeks)
1
2 5 6 9 10 13 14 17 18 21 22 3 4 7 8 11 12 15 16 19 20 23 24 25 26
Challenget at the age of + 16 months with LL virus of subgroup
Plasma m
1°l
10 8 8 6 6 4 4 2 2 day 1 day 1 10 10 8 8 6 6 4 4 2 2 day 1 day 1 day 1 day 1
Virus isolation at the age of +_20 m o n t h s
S u b g r o u p Neutralisingantibodies:~
Buffycoat m
m
A
B
no challenge
--
--
--
+
--
+
A+B
--
+
A+B
+
+
A+B
--
+
A+B
-
+
A+B
--
--
--
+
--
+
--
+
A+B
--
+
A+B
--
+
A+B
--
+
A+B
--
+
A+B
--
+
A+B
B
A+B B
Anti A
Anti B
>8192 512 8192 2048 2048 8192 1024 1024 38192 38192 >8192 128 1024 4096 4096
3
4096 >8192 256 >8192 128
3
2048 512
32 32 16 3 32 1024 < 2 32 3 32 < 2 1024 1024 < 2 16 256 512 32 32 1024 4096 1024
8
32
* Vaccination with 105 TCIDs0 of LL viruses RAV-CVI-1 and RAV-2. ~"Challenge-infectionwith 105 TCIDs0 of LL virus RAV-C VI-1 or RAV-2. :~390% reduction of 100 FFU RSV RAV-1 or RSV RAV-2.
of these birds is immunologically tolerant for L L viruses and is persistently viremic. The epidemiologically most important flow o f infection is interrupted when virus-free eggs are used to hatch the succeeding generation. Initially sera as well as pooled embryo extracts prepared from eggs collected during twoweek periods from individual hens were examined. Very little correlation was observed between the results obtained with both types o f material from the same hens. A l t h o u g h the time lapse between the samplings has to be considered, in our hands the examination o f sera
104
G. t7. DE BOEK,J. VAN VLOTEN,J. E. GROENENDAL,H. J. L. MAASand A. HOOGERBRUGGE
did not seem a reliable method for the elimination of hens which excrete virus congenitally. However Zander et al. [21] recently reported on a successful LL control programme in spf breeding flocks that was based on the elimination of viremic birds. In order to make the control method applicable under field conditions the chicks were immunised by a controlled exposure of LL viruses before being transferred to conventional chicken houses. Virus strains used in the "vaccine" will cause lymphoid leukosis in chickens when exposed shortly after hatching but due to the age-related resistance the disease incidence is significantly reduced in older birds. The number of cells susceptible for tumour formation decreases with time [ 1]. Burmester et al. [3] reported on the age-related resistance against infection with the R P L I 2 strain administered by various routes. Although these authors observed a greater resistance against infection via oral and intranasal avenues of infection, in our study the intramuscular route was chosen for practical reasons. In contrast with Brumester's results obtained with two month-old chickens, no clinical cases of LL were observed in the "vaccinated" generations of the four flocks under study. However one should always take into account that tumour formation may occur in an occasional chicken. (In another experiment, designed to study the age-related resistance at various ages, overt disease symptoms were observed in one chick of a group inoculated at eight weeks of age.) Presumably our virus strains used for "vaccination" are less virulent than the RPL 12 strain. It is also possible that Burmester's residual cases of LL after infection at older age are due to the extreme susceptibility of the WL line 151. The age-related resistance is not effective against LL virus infections acquired by horizontal exposure. The flock of 47 WL strain A chickens hatched from virus-free eggs (left side, Table 6) were transferred to the conventional chicken house at two months of age without being "vaccinated". Since these chickens were hatched from virus-negative eggs, they can only have been infected by horizontal route. Their exposure differs from that of their "vaccinated" sisters (Table 4) in that they were infected by natural route after two months of age and probably received less virus than had they been vaccinated. Presumably immunity is only acquired with a large antigenic stimulus. Furthermore, the inoculation results in non virusexcreting hens only when the "vaccination" is the first contact with the LL viruses involved. The treatment at two months of age did not affect shedding of previously infected hens of the WL strain A control flock. Intermittent virus-excretion was occasionally encountered in this study, e.g. one of the WL strain A hens had an interval of 4~ months between positive samples (Table 4). Such intermittent virus-shedders are easily missed when the hens are only examined for two 14-day periods of egg production. However, with large numbers of chickens more frequent testing becomes too laborious. The hens that excrete LL viruses intermittently represent the Achilles' heel of this control method. The data of Tables 6 and 7 indicate that the conventional environment of the three WL strains is highly contaminated with LL virus. These chickens were separated from virusinfected birds by a wire netting, but the same non-flowing drinking fountains were used in adjacent pens. Therefore ample opportunity for virus spreading was present. The incidence of virus-excreting birds may be less on farms with an all-in all-out type of management, but one cannot wish better circumstances for the purpose of this study. The relatively high percentage of virus-excreting hens in Table 6 indicates the role that horizontal transmission may play in the epizootiology of LL. The reported data emphasise the value of the FAPP isolation facilities and make it unlikely that a method of LL control based on maternal immunity
On epizootiology and control of lymphoid leukosis in chickens
105
o b t a i n e d via active i m m u n i s a t i o n o f the hens as p r o p o s e d by L61iger and V o n d e m H a g e n [ 10] will be effective u n d e r c o n v e n t i o n a l conditions. E x p e r i m e n t s h a v e been designed to d e t e r m i n e m o r e precisely the age-related resistance at v a r i o u s ages and to better define the m e c h a n i s m o f i m m u n i t y . N e u t r a l i s i n g antibodies are present after a b o u t t w o w e e k s p.i. and persist for the entire life o f the bird. A n t i b o d i e s m a y be e x p e c t e d to h a v e an adverse effect on virus r e c o v e r y . T h e t e c h n i q u e for virus detection was c o n s i d e r a b l y i m p r o v e d w h e n the samples were treated to a v o i d virus inhibitory activity. W a s h e d buffy c o a t cells p r o v e d to be v e r y useful and viruses could only be r e c o v e r e d f r o m o r g a n s by the use o f explant cultures. T h e controlled e x p o s u r e p r e s u m a b l y results in a persistent infection during the entire life o f the chicken. H o w e v e r , o f p a r a m o u n t i m p o r t a n c e is the o b s e r v a t i o n that the i n o c u l a t e d viruses are not excreted in the eggs. W h e n at the end of the egg-laying period " v a c c i n a t e d " hens were i n o c u l a t e d i n t r a t r a c h e a l l y or i n t r a m u s c u l a r l y with 105 T C I D s 0 o f R A V - C V I - 1 or R A V - 2 viruses, no egg-transmission o f these viruses could be d e m o n s t r a t e d . Acknowledgements--The authors wish to thank Mrs. H. Kappert, A. W. Manders, A. P. C. H. van Beek, H. M.
Boerrigter, C. van Engelen, L. Hartog, G. Petersen and J. IJzerman for their technical assistance. We thank Drs. B. R. Burmester and J. G. van Bekkum for helpful criticism of the manuscript. REFERENCES 1. Baluda, M. A. and Jamieson, P. P., In vivo infectivity studies with avian myeloblastosis virus. Virology 14, 33-45 (1961). 2. De Boer, G. F., Van Vloten, J., Groenendal, J. E., M ~ s , H. J. L., Borm, F., Hoogerbrugge, A. and Krasselt, M., The control of lymphoid leukosis in a commercial White Plymouth Rock line. Neth. J. Vet. Sci. (in press). 3. Burmester, B. R., Fontes, A. K. and Walter, W. G., Pathogenicity of a viral strain (RPL 12) causing avian visceral lymphomatosis and related neoplasms. III. Influence of host age and route of inoculation. J. hath. Cancerlnst. 24, 1423-1442 (1960). 4. Calnek, B. W., Lesions in young chickens induced by lymphoid leukosis virus. Avian Dis. 12, 111-129 (1968). 5. Calnek, B. W., Lymphoid leukosis virus: A survey of commercial breeding flocks for genetic resistance and incidence of embryo infection. Avian Dis. 12, 105-111 (1968). 6. Calnek, B. W., Bankowski, R. A., Beasly, J. N., Biggs, P. M., Burmester, B. R., Cho, B. R., Cole, R. K., Eidson, C. S., Furrow, R. D., Helmboldt, C. F., Hughes, W. F., Kenzy, S. C., Luginbuhl, R. E., Nazerian, K., Purchase, H. G., Rispens, B. H., Schmitfle, S. C., Siccardi, F. and Witter, R. L., Report of the AAAPsponsored leukosis workshop. Avian Dis. 11,694-702 (1967). 7. Chubb, R. C. and Biggs, P. M., The neutralization of Rous sarcoma virus. J. gen. ViroL 3, 87-96 (1968). 8. Di Stefano, H. S. and Dougherty, R. M., Mechanisms for congenital transmission of avian leukosis virus. J. hath. Cancer Inst. 37, 869-883 (1966). 9. Katz, D., Luft, A. and Kohn, A., Incidence of Marek's disease and lymphoid leukosis in poultry flocks in Israel. Refuah Vet. 25, 20-25 (1971). 10. L61iger, H. Ch. und Von dem Hagen, D., Versuche zur Immunisierung yon Legehennen zum Schutz vor Leukoseerkrankungen unter den Nachkommen. Arch. Geflfigelk. 5, 190-194 (1974). 11. Rispens, B. H., De Boer, G. F., Hoogerbrugge, A. and Van Vloten, J., A method for the control of lymphoid leukosis in chickens. Z hath. Cancer Inst. 57, 1151-1156 (1976). 12. Rispens, B. H. and Long, Ph. A., The non-producer cell activation test in avian leukosis virus assay. Comp. Leukemia Res., B ibl. haemat, no. 36, Dutcher, R. M. (Ed.), pp. 192-197. K arger, Basel (1970). 13. Rispens, B. H., Long, Ph. A., Okazaki, W. and Burmester, B. R., The NP activation test for assay of avian leukosis/sarcoma viruses. Avian Dis. 14, 738-751 (1970). 14. Rispens, B. H., Van Vloten, J. and Maas, H. J. L., Some virological and serological observations in Marek's disease. Br. vet. J. 125,445-453 (1969). 15. Rispens, B. H., Van Vloten, J., Mastenbroek, N., Maas, H. J. L. and Schat, K. A., Control of Marek's disease in The Netherlands. II. Field trials on vaccination with an avirulent strain (CVI 988) of Marek's disease virus. Avian Dis. 16, 126-138 (1972). 16. Rubin, H., A virus in chick embryos which induces resistance in vitro to infection with Rous sarcoma virus. Proc. hath. Aead. Sci., U.S.A. 46, 1105-1119 (1960).
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17. Rubin, H., Cornelius, A. and Fanshier, L., The pattern of congenital transmission of an avian leukosis virus. Proc. natn. Acad. Sci., U.S.A. 47, 1058-1060 (1961). 18. Rubin, H. and Vogt, P. K., An avian leukosis virus associated with stocks of Rous sarcoma virus. Virology 17, 184-194 (1962). 19. Sandelin, K. and Estola, T., Occurrence of different subgroups of avian leukosis virus in Finnish poultry. Avian Path. 3, 159-168 (1974). 20. Selenski, W. P., L6sungswege zu Fragen der Diagnostik und Bek~mpfung der Geflfigelleukose. Mhft. Vet. Med. 27, 647-651 (1972). 21. Zander, D. V., Raymond, R. G., McClary, C. F. and Goodwin, K., Eradication of subgroup A and B lymphoid leukosis virus from commercial poultry breeding flocks. Avian Dis. 19, 408-423 (1975).
0147-9571/78/0701-0093502.00/0
O N EPIZOOTIOLOGY A N D C O N T R O L OF LYMPHOID LEUKOSIS IN C H I C K E N S G. F. DE BOER, J. VAN VLOTEN, J. E. GROENENDAL a n d H . J. L. MAAS Central Veterinary Institute, Virology Department, 39 Houtribweg, Lelystad, The Netherlands and A . HOOGERBRUGGE University of Utrecht, Institute of Zootechnic, Yalelaan, De Uithof, Utrecht, The Netherlands Abstract--Sera and organ extracts from ten different commercial stocks of layer chickens were examined for the presence of lymphoid leukosis (LL) viruses. Virus was recovered from 40.8% of the cockerels between three and six weeks of age. Their female hatch mates were examined at the age of 20 months. A mean of 11.3% of these laying hens was positive in the NP activation test. Lymphoid leukosis was successfully controlled in three inbred strains of White Leghorn chickens and in a commercial White Plymouth Rock line. All flocks were kept in a filtered air positive pressure (FAPP) house during the first two months of life and thereafter transferred to a conventional environment. The control method is based on three elements: - - f r o m an infected flock, hens are selected in whose eggs no avian lymphoid leukosis viruses can be detected by examination of pooled extracts of groups of embryos; - - o n l y eggs from hens that are shown not to shed congenitally virus in their eggs are used for the production of progeny. The offspring are reared in isolation until two months of age at which time the age-related resistance against tumour formation appears to be sufficiently developed; - - t h e chickens are subsequently intramuscularly inoculated with lymphoid leukosis viruses of subgroups A and B and transferred to a conventional chicken house. The inoculated birds become persistently viremic and resist horizontal virus exposure and intramuscular challenge infections. Horizontal virus transmission was observed to take place when virus-free non-vaccinated chickens were reared in isolation for two months and then exposed under field conditions. Efficiency of virus recovery was considerably improved when washed buffy coat cells were cocultivated with chick embryo fibroblasts or explant cultures were prepared from various tissues before testing with the NP activation test. Key words: Lymphoid leukosis, epizootiology, congenital infection, horizontal transmission
EPIZOOTIOLOGIE
ET ERADICATION DE LA LEUCOSE LYMPHOIDE POULETS
CHEZ
Rtsume--L'incidence des infections du virus de la leucose lympho'ide (LL) dans 10 lots du commerce de poulets destints /l la ponte /~ 6t6 recherchte par des techniques virologiques. Les poussins m~.les ont 6t6 examints entre 3 et 6 semaines apr~s rtclosion et les femelles ont 6t6 testtes apr/~s 20 mois. La frtquence moyenne des animaux porteurs de virus 6tait de 40.8% chez les poussins males et de 11.3% chez les pondeuses de m~me origine fi l'5.ge de 20 mois. La frtquence des sympttmes eliniques pendant 20 mois &ait de 0.43%. Les virus isolts appartiennent essentiellement au sous-groupe A et seulement 3% appartiennent au sous-groupe B. L'isolement du virus est amtlior6 quand les leucocytes apr~s lavage sont mis en coculture avec des fibroblastes d'embryons de poulet ou d'explants primaires /t partir de divers tissus avant le test dit "NP activation". La leucose lymphode h 6t6 6radiqute dans trois ligntes de poulet Leghorn et dans une lignte commerciale "White Plymouth Rock". La m&hode de contrSle appliqute est baste sur trois 616ments: - - d e s pondeuses produisant des oeufs indemnes de LL ont 6t6 ehoisies parmi un troupeau infectt. A 93
94
G. F. DE BOER, J. VAN VLOTEN, J. E. GROENENDAL, H. J. L. MAAS and A. HOOGERBRUGGE l'aide des tests dits "NP activation" des extraits mtlangts de diff&ents groupes d'embryons ont et~ examints de faqon ~t dtterminer une 6ventuelle transmission congtnitale de virus; - - s e u l s les oeufs provenant des animaux virus-ntgatifs sont utilis& pour la reproduction. La descendance est 61evte dans un poulailler en surpression d'air et muni de filtres (FAPP) jusqu' ~i deux mois, ~.ge ~. partir duquel la rtsistance h la leucose lympho'ide est suffisamment dtveloppte; - - l e s poulets sont ensuite inocults par vole intramusculaire avec le virus de la leucose lymphoide de sous-groupes A e t Bet mis ensuite dans un poulailler conventionnel. Les animaux inocults ne developpent pas de sympt6mes cliniques de LL, restent porteurs de virus et resistent h la transmission horizontale du virus. Et surtout, chez des pondeuses traittes par les mesures prtc&tentes la transmission par l'oeuf de virus de LL ne pouvait pas &re d~celte. Quand la transmission congtnitale a. 6t6 61iminte par les mesures prtctdentes la diffusion horizontale du virus joue un r61e important dans l'epizootiologie de la leucose lymphoi'de dans les quelques groupes non-vaccints.
Mots-clefs: Leucose lymphoide, epizootiologie, infection congtnitale, transmission horizontale
INTRODUCTION It has long been recognised that infections with avian lymphoid leukosis (LL) viruses are wide-spread all over the world. Specific antibodies are present in most chicken flocks. A limited number of publications, however, is available on the prevalence of viremic birds in commercial poultry [5, 9, 19]. The eradication of lymphoid leukosis from chicken flocks may be pursued by eliminating all viremic and serologically positive birds from a given population [4, 20] or by selecting dams that do not excrete virus in their eggs and using only LL virus-free eggs to hatch the next generation. The latter procedure has better prospects for successful control since the natural flow of infection is interrupted at the stage which results in the highest incidence of clinical disease and the most severe spreading of LL viruses into the environment [6, 8]. Congenitally infected birds are considered a primary source of contagion. Our investigations to develop a control programme were primarily directed at the elimination of hens excreting LL viruses in their eggs. In order to make the control method applicable under field conditions, the chickens hatched from virus-free eggs were reared in isolation until the agerelated resistance against tumour formation was present and were inoculated with LL viruses of subgroups A and B before being transferred to a conventional environment. The procedure resulted in birds persistently infected with LL viruses, but in which no clinical disease was observed and none produced LL virus-infected eggs. Preliminary data are reported on virus recovery from buffy coat cells or explant cultures of various tissues of inoculated chickens.
MATERIALS AND METHODS
Epizootiologiestudy In 1974 and 1975 ten different commercial stocks of layer chickens were examined for the presence of LL viruses. Eggs of each layer stock were hatched in a formalin disinfected incubator. The cockerels were transferred to a filtered air positive pressure (FAPP) house directly after hatching. Positive pressure was maintained at 5 mm water column and the incoming air was passed through high efficiency particulate air (HEPA) filters (Camfil AB, type 1J-1000, class 100, Trosa, Sweden). The stocks were subsequently tested during a period of three weeks starting at the age of three weeks. Serum samples and homogenates of
On epizootiologyand control of lymphoid leukosis in chickens
95
spleen, liver, pancreas and bursa were examined. All female chicks were transferred to a conventional chicken house for a random sample egg production test. These birds were reared under the same conditions and placed in individual cages at the age of five months. At 20 months of age the incidence of LL virus infections was recorded by testing of serum samples.*
Experimental chickens Investigations of control methods for LL were initiated in 1969 with three strains of inbred White Leghorn (WL) chickens: A, B and 98A. In 1972 a commercial White Plymouth Rock line (WPR), in use for producing hybrid females for broilers, was included in the study.t
Holding conditions Each generation of WL chickens consisted of 3 0 - 5 0 adult birds. The chickens were kept in a pen type chicken house. Different age groups were kept in the same house, usually chickens of other breeds were present. The various groups were held in pens on a dirt floor. The pens were separated from each other by a single wire netting. Each pen of 12 m 2 held 3 0 - 5 0 adult chickens. Before introduction of a new group of chickens the litter was removed and the pen cleaned and disinfected. The chickens were fed with commercial all mash meal feeds. Drinking water was supplied with non-flowing water fountains. Eggs were collected in trap nests. All chickens were vaccinated at day-old against Marek's disease with cell-associated CVI 988 vaccine [15]. Vaccination against Newcastle Disease (ND) and infectious bronchitis (IB) was performed at three weeks of age with a combined vaccine based on the ND-Hitchner B 1 and I.B.-H 120 virus strains. At seven weeks of age the chickens were revaccinated with NDLaSota vaccine. Vaccinations against avian encephalomyelitis and fowlpox were performed in the second part of the rearing period. Prior to 1972 each new generation was hatched from eggs from LL virus-negative hens. After hatching the chickens were raised under conventional conditions. From 1972 on progeny from virus-negative hens were kept in a FAPP house for the first two months of life. While in isolation the chickens were fed pelleted feeds and each flock had its own attendant. During the entire period of the investigation a WL strain A control flock was maintained but progeny were hatched from eggs from unselected hens. This flock was constantly kept in the conventional chicken house. Except for the two-month periods of isolation, the WPR chickens were kept on litter during rearing and in individual cages during the laying period. An all-in all-out type of management was practised at the basic breeding farm. The normal selection procedures used for commercial pedigree chickens were applied in addition to those aimed at eliminating LL. Vaccination procedures against other diseases were those in use for commercial laying flocks in The Netherlands.
Procedures for the control of LL In order to detect viremic hens and hens excreting virus with their eggs, sera and pooled extracts of 11-day embryos from individual hens were tested for the presence of LL viruses. * The co-operation of the Koninklijke Vereniging ter Bevordering van de Pluimveehouderij in Zeeland (V.P.Z.), Wissenkerke and of Dr. O. J. H. Devos, Provincial Animal Health Servicein Goes, is gratefullyacknowledged. t The authors thank Drs. F. Borm, A. van Hedel and Mr. A. W. Manders for their co-operationin this part of our study.
96
G. F. DE BOER, J. VAN VLOTEN, J. E. GROENENDAL, H. J. L. MAAS and A. HOOGERBRUGGE
Hens of the WL flocks were examined repeatedly. Eggs collected during two-week periods were pooled and examined as one sample. Between the sampling periods were intervals of two to three weeks. Eggs from the WPR grand parent flock were collected during two 14-day periods, at least three weeks apart. WPR hens producing less than seven living embryos per 14-day observation period were not included in the test and thus not used for the production of progeny. From 1972 on chickens hatched from eggs of LL virus-negative dams were reared in the FAPP house for a period of two months. Thereafter the chicks were transferred to the conventional chicken houses described above after having been inoculated with LL viruses of subgroups A and B.
Collection and processing of specimens Blood samples were collected by venipuncture. The sera were harvested after 10 min. centrifugation at 1000g and were stored a t - 7 0 ° C . Eggs were collected individually and stored at 15 °C until the entire production of each flock obtained during a 14-day period was available for incubation as one batch at 37.5°C. Embryos were harvested after 11 days and pooled extracts were prepared of all embryos available from each hen. The embryos were collected aseptically, washed with Hanks' buffered saline solution (BSS) and antibiotics and squeezed through a 20 ml syringe. An equal amount of Hanks' BSS plus 10% calf serum and antibiotics was added and the samples were stored at - 7 0 ° C . They were ready for virus assay after two cycles of freezing and thawing and centrifugation for 10 min. at I000 g. For the epidemiologic study organ extracts were prepared of spleen, liver, pancreas and bursa. The organs were aseptically removed and ground in a Griffith tube. A third volume of Hanks' BSS plus calf serum and antibiotics was added and the suspensions were centrifuged for 10 min. at 1000g.
Cell cultures and media Chick embryo fibroblasts (CEF) derived from embryos of specific pathogen free chickens of the WL strain A, which have been shown to provide cell cultures of the C/E phenotype, duck embryo fibroblast (DEF) cultures and Rous sarcoma virus-induced non producer (NP) cells were used throughout. The preparation and maintenance of the cell cultures and the various media have been described previously [14, 15].
Virus assays The NP activation test [12, 13] was used throughout. The test is based on the specific activation of non-producing Rous sarcoma cells (NP cells) and detects, without differentiation, all subgroups of lymphoid leukosis viruses (subgroups A, B, C and D, since CEF cells of the C/E phenotype were used). Subgroup determination of LL isolates was performed by the RSV resistance inducing factor (RIF) test [16]. Incidentally the RIF test and the indirect immunofluorescence technique for group-specific antigens of avian tumour viruses were used for virus assay as well. Since the use of the NP activation test was preferred because of easier performance and greater capacity, the immunofluorescence test was omitted in the later stages of the investigation.
On epizootiologyand control of lymphoid leukosis in chickens
97
Serum neutralisation test Neutralising antibodies were assayed with a focus reduction test with 100 F F U of BHRSV (RAV-1) and BH-RSV (RAV-2) viruses. The neutralisation titre is the reciprocal of the dilution which reduces the number of foci by 90% or more. Viruses used for immunisation Originally, each chicken received an inoculum of 0.5 ml containing a mixture of 105 T C I D Rous associated virus CVI-1 (a subgroup A leukosis virus), 103 TCIDso Rous associated virus (RAV-2) of subgroup B and about 3 × 105 TCIDs0 of three LL virus isolates (presumably of subgroup A) of the WL chicken strains A, B and 98A respectively. The CVI1 virus strain was isolated according to the method of Rubin and Vogt [18] from a stock of Bryan standard strain Rous sarcoma virus that was kindly supplied by Dr. P. M. Biggs, Houghton Poultry Research Station, Houghton, England. This avian leukosis subgroup A virus is probably similar to the HPRS-2 isolate of Chubb and Biggs [7]. The RAV-2 virus was separated with the same procedures from a stock of BH-RSV (RAV-2) virus obtained from Dr. B. R. Burmester, Regional Poultry Research Laboratory, USDA, East Lansing, Michigan. From 1974 on the inoculum contained 105 TCIDs0 o f R A V CVI-1 and the dose of RAV-2 was increased to 105 TCIDs0. All virus stocks were grown in CEF cultures.
Virus isolation procedures Material to be tested for virus received one passage on CEF monolayers prior to further testing. The processing of serum samples, embryo and organ extracts has been described above. In the later stage of the study virus isolation from blood of a group of WL line A chickens was done by cocultivation of washed buffy coat cells with CEF cultures and virus cultivation from tissue suspensions was substituted by the application of explant cultures of infected tissues. The white blood cells were taken from heparinised blood samples and washed three times by centrifugation for I0 min. at 1000 g in PBS. This resulted in a plasma dilution factor of x 10.000. CEF monolayers, 24 hr old, were inoculated with the washed cells. Maintenance medium was added after 1 hr incubation at 38°C. The cultures were harvested after six days. Usually one cell passage was made in between. The cells and supernatant were collected by scraping off with a silicone policeman and stored at - 7 0 ° C until further assayed. Explants of the various tissues were prepared with two scalpels in a few drops of growth medium. After a gentle wash, fragments of 1 to 2 m m 3 were placed in a dry plastic Petri dish. The explants became attached to the bottom by leaving the Petri dish on the bench for about 45 min. Subsequently, growth medium was added and the dishes placed in a humidified CO2 incubator at 38°C. A change of medium was performed twice a week. The cultures were harvested when a significant outgrowth of cells around the explants was observed. This required six to ten days. After storage at - 7 0 ° C , the samples were tested using the NP activation test. The comparative trials for virus isolation from plasma and buffy coat were conducted with experimental WL strain A chickens which were inoculated with subgroup A and B viruses at ages varying between 0 and 10 weeks. The various age groups were inoculated at the same time and kept in the FAPP house for a period of one and a half years. At the age of about 16 months 4
98
G . F . DE BOER, J. VAN VLOTEN, J. E. GROENENDAL, H. J. L. MAAS and A. HOOGERBRUGGE
these chicks were challenge-infected with 105 TCIDs0 R A V - C V I - 1 or R A V - 2 and samples were taken at about 20 months o f age. RESULTS A mean frequency o f infection with L L viruses of 40.8% was demonstrated in the commercial stocks of chickens examined between three and six weeks o f age (Table 1). A total o f 196 virus isolates was obtained. Virus was recovered from 152 serum samples taken from cockerels of the various commercial stocks. Organ extracts of the same birds were also positive but an additional 44 isolates were recovered from organs only. Interference assays were done with 135 isolates. L L virus of subgroup A was most prevalent (97%) and 3% of our isolates belonged to subgroup B. O n l y one bird yielded both subgroups A and B. A b o u t 20 months later, sera from female chickens o f the same hatch were tested (Table 2). N o w 80 of 706 samples were found positive (11.3%). In contrast with the frequency o f viremic birds an unusually low incidence o f clinical disease was observed in the commercial layer chicks under study. During an observation period o f 420 days a total mortality o f 13.7% was Table 1. Virus isolation from 3- to 6-week-old cockerels of 10 commercial layer stocks Sera 1 2 3 4 5 6 7 8 9 10
35/47* 8/40 17/48 12/50 5/48 0/44 14/51 5/51 35/51 21/51
1-10
152/481
Organ extracts 74.5% 20.0% 35.4% 24.0% 10.4% 27.4% 9.8% 41.2%
38/47* 12/40 24/47 20/50 10/47 2/44 22/51 9/50 36/51 23/51
80.8% 30.0% 51.1% 40.0% 21.3% 4.5% 43.1% 18.0% 70.6% 45.1%
31.6%
196/478
40.8%
68.6%
* Total positive chickens/No, of specimens examined. Table 2. Virus isolation from laying hens of 10 commercial stocks at 20 months of age Sera 1 2 3 4 5 6 7 8 9 10
17/72* 13/71 7/69 4/67 7/72 2/72 2/69 3/71 17/72 8/71
23.6% 18.3% 10.1% 6.096 9.7% 2.8% 2.9% 4.2% 23.6% 11.3%
1-10
80/706
11.3%
* Total positive chickens/No, of sera examined.
99
On epizootiologyand controlof lymphoidleukosis in chickens
observed, but in only 0.43% of the laying hens overt lymphoid leukosis was diagnosed by macroseopical examination. In the period 1969-1972 the three WL strains were examined for LL viruses with the indirect immunofluorescent staining technique and the NP activation test [11-13]. Both the sera of adult chickens and embryo extracts prepared from eggs of individual hens were tested. The two techniques yielded identical results. Each new generation was hatched from eggs obtained from hens that had been found negative in all tests. With this procedure no significant reduction of the number of virus isolations from serum samples and embryo extracts was obtained for three generations. Table 3. Elimination of lymphoidleukosis fromtwo WL flocks, strains B and 98 A White Leghorn strain Generation number No. of hens
WL B
Age in months 9 Pooled embryo
extracts of 14-dayperiods
WL 98 A
I 23
/ ~'10 J 11
II* 42
II* 39 Virus Age in Virus Age in Virus Age in Virus recovery months recovery months recovery months recovery 2/20t 2/19 2/21
~ 2/22~t ~.
8 9
I 33
0/35 0/37
8 9
6/30 5/27
9 10
0/33 0/35
10
0/36
10
3/22
11
0/35
12 13 14 15
0/31 0/27 0/25 0/21
13 14 15 16
0/35 0/35 0/37 0/38
7/32
0/39
0/38
* Reared in isolation and "vaccinated" at 2 months of age. 1"Number of pooled embryoextracts from whichvirus was recovered/numberof samples examined. :~Numberof positive hens/total number of hens examined. From 1972 on chickens destined for breeding were reared in isolation for two months. They were subsequently transferred to the conventional chicken house after being inoculated intramuscularly with LL viruses of subgroups A and B. From that time on a significant reduction of the number of virus-excreting hens was observed. No eggs infected with LL viruses were found in WL strains B and 98A second generations (Table 3), but in strain A, virus was still demonstrated in embryo extracts of 5 of 94 (5/94) hens (Table 4). No virus was however recovered from the succeeding generation of this flock when the same procedures were again applied. The eggs of hens of the second generation of the WL strains B and 98A and of the second generation of strain A were collected when the birds were between 8 and 16 months of age comprising a period of 70-98 days of the laying period. Intermittent virus excretion was occasionally encountered, e.g. virus was recovered four times from embryo extracts prepared from eggs of one of the five positive hens of the W L strain A generation (Table 4). The embryo extracts collected in the 14-day productions at the age of 9, 10, 15 and 16 months were positive but not those obtained in month 8, 12, 13 and 14. In the W P R flock egg transmission of LL viruses was absent in the first generation that was hatched from eggs from virus-negative dams and reared in the F A P P house (Table 5). At the age of one month the sera of the cockerels, that were culled from the flock, were examined. Virus was recovered from three of them. Their sisters were also removed from the
100
G. F. DE BOER, J. VANVLOTEN, J. E. GROENENDAL, H. J. L. MAASand A. HOOGERBRUGGE Table 4. Eliminationof lymphoidleukosis from WL chickens, strain A
Generation number
I
No. of hens
II*
41 Age in months
Serum samples
Pooled embryo extracts of 14-day periods
Virus recovery
. I 9 10
.
l
III*
47
. ll/41t 8/36
Age in months .
37
Virus recovery
Age in months
Virus recovery
7 9 10 11 14 16
0/37t 0/34 0/33 0/33 0/29 0/30
. 8 9 10 12 13 14 15 16
11/41~
49
0/41 1/44 1/43 0/40 0/38 0/36 1/33 1/35
0/38 2/45 1/46 2/33 3/30 0/31
1/46
0/35
4/48 5/94
* Reared in isolation and "'vaccinated" at 2 months of age. t Number of positive samples/numberof samples examined. $ Number of positivehens/total number of hens examined.
Table 5. Control of egg-transmissionof LL virus in a commercial White Plymouth Rock line Generation number
I
No. of chickens
II*
500 Age in months
Serum samples
-
Pooled embryo extracts of 14-day periods
f 7 J 8
III*
410 ~ + 560 ~
Virus Age in recovery months 9/'500 9/437 13/437§
1084 ~ + 60 0~c~
Virus Age in recovery months
It
3/560~:
7 8
0/368 0/359
Virus recovery
2
0/257
8 12
0/552 0/175
0/359
0/175
* Reared in isolation and "vaccinated" at 2 months of age. t Of cockerels that were culledfrom the flock at 1 month of age. $ Number of samples from which virus was recovered~numberof samples examined. § Number of positive hens/number of hens which was examinedtwice.
flock but serum samples of these chickens were negative for virus. The examination of the third generation yielded negative results as well [2]. The data of Table 6 give an indication of the extent in which horizontal transmission of LL viruses may occur. Since these chickens were full sibs of the third generation of W L strain A and were hatched from eggs from virus-negative hens (Table 4), the virus shedding in these groups must be ascribed to an infection contracted after hatching. The 47 chickens in the left side of Table 6 were kept in isolation for a period of two months and were not "vaccinated". Therefore the infection is likely to have been acquired after the transfer to the conventional environment. Virus containing embryo extracts were obtained from 13/36 hens. At the end of
On epizootiology and control of lymphoid leukosis in chickens
101
the laying period serum samples as well as organ extracts of the hens were examined. LL virus was demonstrated in the serum sample and the organ extract of only one bird. The 49 chickens of the same origin (right side, Table 6) were exposed to the infected environment at day-old. At the age of six months, LL virus was detected in two serum samples of this flock and eggexcretion was demonstrated in six of these hens (6/41). Table 6. Horizontal transmission of LL virus in WL strain A chickens hatched from virus-free eggs No. of chickens*
37 ~ Age in months
Serum samples
7
Pooled embryo extracts of 14-day periods
I 9 10 11 ~ 14 ~ 16 17
+ 10 ~ d ' t Virus recovery
41 9Q + 8 ~ : [ : Age in months
Virus recovery
0/46§
6
2/49
5/33 4/32 4/30 6/31 1/23 4/21
8 10 11 14 15 16
4/37 2/36 2/36 3/31 3/31 1/27
13/36** Sera and organ
18
6/41
1/36
extracts * Derived from the same negative hens as the third generation in Table 2.
t Reared in isolation for 2 months. Reared in a conventional chicken house. § Number of positive samples/numberof samplesexamined. ** Number of positivehens/total number of hens examined. During the entire period o f the study, the untreated WL strain A control flocks were examined with the same techniques. Results for the flocks that were housed together with the treated groups of the same chicken strain (second and third generations of Table 4) are presented in Table 7. By examination of embryo extracts 4 0 - 5 0 % of the birds were found to be shedding virus. The second generation had been hatched from eggs of 11 virus-positive and 17 virus-negative hens. Viremia was demonstrated in respectively 13/20 and 1/36 chickens of this generation at 7 months of age. The total scores for the embryo extracts were 18/22 and 13/38, respectively. In contrast with the high number of viremic and congenitally transmitting chickens in this flock, the incidence of clinically and by autopsy detected cases of LL was rather low (2/60 and 3/78 in the first and second generation, respectively). A group of 50 unselected hens of the WL strain A control flock was "vaccinated" at two months of age, after being reared in the conventional chicken house. This treatment did not seem to influence the number of hens excreting virus during the laying period. Since the NP activation test does not differentiate between the subgroups of LL virus, the positive samples were again examined with the RIF test. Only viruses of subgroup A were recovered from the flocks under study. Starting with the first generation in which vertical virus transmission was absent, no symptoms of LL were observed in any of the chickens of the flocks reported in this study. All virus isolations from the birds described above were done with sera or organ extracts.
~02
G.F. DE BOER, J. VAN VLOTEN,J. E. GROENENDAL,H. J. L. MAASand A. HOOGERBRUGGE
The vaccine viruses were only recovered from serum samples of the inoculated birds in the first two to three weeks. In the later stage o f our study a definite improvement o f the technique for virus detection was obtained by cocultivation o f washed buffy coat cells with C E F cultures or by explanting various tissues. These cultures were then assayed with the N P activation test. With these techniques vaccine viruses were in a few cases recovered from birds which had been inoculated one and a half years before. Virus was recovered from only one o f the four birds inoculated at the age of 8 weeks (Table 8). This chicken was challengeinfected with subgroup B virus at the age o f 16 months. Both subgroups A and B of L L virus were present in the virus isolate at the age o f 20 months. Table 7. Virus recovery from untreated and unselected flocks of WL strain A chickens Generation number No. of hens
Serum samples Pooled embryo extracts of l4-day periods
I 50
II 60 Hatched~omeggs of: 17 neg. hens 11 pos. hens
Agein months
Virus recovery
-
-
" 8 9 10 11 14 15 16 17
I
Agein months
12/42" 12/43 9/39 10/37 8/34 6/31 7/31 7/31
Virus recovery
Virus recovery
7
1/36"
13/20
9 10 12 15 16
9/37 9/36 9/33 9/29 2/16
12/18 11/15 10/15 9/14 0/ 5
13/38
18/22 31/60
19/48t * Number of samples which yielded virus/number of samples examined. "~Number of positive hens/total number of hens examined.
The persistent virus infection was nearly always demonstrated after one and a half years if the chickens underwent the controlled exposure below the age of four weeks. Virus isolation from the chicks of Table 8 was invariably negative when tissue homogenates were tested.
DISCUSSION As in other countries, L L viruses o f subgroup A were the most prevalent in the chickens under study [5, 9, 19]. Only 3% o f the virus isolates which we recovered belonged to subgroup B. A mean percentage of infection o f 40.8 was observed in young chickens and one of 11.3 was present in the commercial laying hens at the end of their laying period. These viruses were recovered from sera and organ extracts. Presumably, the score would have been even higher if washed buffy coat fractions or explant cultures were used. Since subgroup C or D viruses have not been detected in The Netherlands, it seems appropriate to have restricted the
On epizootiology and control of lymphoid leukosis in chickens
103
procedures for control to the subgroups A and B. M a n y more chickens become infected with L L viruses than the number that actually develops a lymphoma. The reported method for the control o f lymphoid leukosis is primarily directed to the elimination from the breeder flock of those hens which are capable of congenitally transmitting L L virus to their embryos. The in o v o infected chickens are known to develop L L with high frequency and to disseminate large quantities of virus [6, 17]. A high proportion
Table 8. Virus isolation from chickens "vaccinated" and challenge-infectedwith LL-viruses of subgroups A and B Vaccination* Hen No. at the age of (weeks)
1
2 5 6 9 10 13 14 17 18 21 22 3 4 7 8 11 12 15 16 19 20 23 24 25 26
Challenget at the age of + 16 months with LL virus of subgroup
Plasma m
1°l
10 8 8 6 6 4 4 2 2 day 1 day 1 10 10 8 8 6 6 4 4 2 2 day 1 day 1 day 1 day 1
Virus isolation at the age of +_20 m o n t h s
S u b g r o u p Neutralisingantibodies:~
Buffycoat m
m
A
B
no challenge
--
--
--
+
--
+
A+B
--
+
A+B
+
+
A+B
--
+
A+B
-
+
A+B
--
--
--
+
--
+
--
+
A+B
--
+
A+B
--
+
A+B
--
+
A+B
--
+
A+B
--
+
A+B
B
A+B B
Anti A
Anti B
>8192 512 8192 2048 2048 8192 1024 1024 38192 38192 >8192 128 1024 4096 4096
3
4096 >8192 256 >8192 128
3
2048 512
32 32 16 3 32 1024 < 2 32 3 32 < 2 1024 1024 < 2 16 256 512 32 32 1024 4096 1024
8
32
* Vaccination with 105 TCIDs0 of LL viruses RAV-CVI-1 and RAV-2. ~"Challenge-infectionwith 105 TCIDs0 of LL virus RAV-C VI-1 or RAV-2. :~390% reduction of 100 FFU RSV RAV-1 or RSV RAV-2.
of these birds is immunologically tolerant for L L viruses and is persistently viremic. The epidemiologically most important flow o f infection is interrupted when virus-free eggs are used to hatch the succeeding generation. Initially sera as well as pooled embryo extracts prepared from eggs collected during twoweek periods from individual hens were examined. Very little correlation was observed between the results obtained with both types o f material from the same hens. A l t h o u g h the time lapse between the samplings has to be considered, in our hands the examination o f sera
104
G. t7. DE BOEK,J. VAN VLOTEN,J. E. GROENENDAL,H. J. L. MAASand A. HOOGERBRUGGE
did not seem a reliable method for the elimination of hens which excrete virus congenitally. However Zander et al. [21] recently reported on a successful LL control programme in spf breeding flocks that was based on the elimination of viremic birds. In order to make the control method applicable under field conditions the chicks were immunised by a controlled exposure of LL viruses before being transferred to conventional chicken houses. Virus strains used in the "vaccine" will cause lymphoid leukosis in chickens when exposed shortly after hatching but due to the age-related resistance the disease incidence is significantly reduced in older birds. The number of cells susceptible for tumour formation decreases with time [ 1]. Burmester et al. [3] reported on the age-related resistance against infection with the R P L I 2 strain administered by various routes. Although these authors observed a greater resistance against infection via oral and intranasal avenues of infection, in our study the intramuscular route was chosen for practical reasons. In contrast with Brumester's results obtained with two month-old chickens, no clinical cases of LL were observed in the "vaccinated" generations of the four flocks under study. However one should always take into account that tumour formation may occur in an occasional chicken. (In another experiment, designed to study the age-related resistance at various ages, overt disease symptoms were observed in one chick of a group inoculated at eight weeks of age.) Presumably our virus strains used for "vaccination" are less virulent than the RPL 12 strain. It is also possible that Burmester's residual cases of LL after infection at older age are due to the extreme susceptibility of the WL line 151. The age-related resistance is not effective against LL virus infections acquired by horizontal exposure. The flock of 47 WL strain A chickens hatched from virus-free eggs (left side, Table 6) were transferred to the conventional chicken house at two months of age without being "vaccinated". Since these chickens were hatched from virus-negative eggs, they can only have been infected by horizontal route. Their exposure differs from that of their "vaccinated" sisters (Table 4) in that they were infected by natural route after two months of age and probably received less virus than had they been vaccinated. Presumably immunity is only acquired with a large antigenic stimulus. Furthermore, the inoculation results in non virusexcreting hens only when the "vaccination" is the first contact with the LL viruses involved. The treatment at two months of age did not affect shedding of previously infected hens of the WL strain A control flock. Intermittent virus-excretion was occasionally encountered in this study, e.g. one of the WL strain A hens had an interval of 4~ months between positive samples (Table 4). Such intermittent virus-shedders are easily missed when the hens are only examined for two 14-day periods of egg production. However, with large numbers of chickens more frequent testing becomes too laborious. The hens that excrete LL viruses intermittently represent the Achilles' heel of this control method. The data of Tables 6 and 7 indicate that the conventional environment of the three WL strains is highly contaminated with LL virus. These chickens were separated from virusinfected birds by a wire netting, but the same non-flowing drinking fountains were used in adjacent pens. Therefore ample opportunity for virus spreading was present. The incidence of virus-excreting birds may be less on farms with an all-in all-out type of management, but one cannot wish better circumstances for the purpose of this study. The relatively high percentage of virus-excreting hens in Table 6 indicates the role that horizontal transmission may play in the epizootiology of LL. The reported data emphasise the value of the FAPP isolation facilities and make it unlikely that a method of LL control based on maternal immunity
On epizootiology and control of lymphoid leukosis in chickens
105
o b t a i n e d via active i m m u n i s a t i o n o f the hens as p r o p o s e d by L61iger and V o n d e m H a g e n [ 10] will be effective u n d e r c o n v e n t i o n a l conditions. E x p e r i m e n t s h a v e been designed to d e t e r m i n e m o r e precisely the age-related resistance at v a r i o u s ages and to better define the m e c h a n i s m o f i m m u n i t y . N e u t r a l i s i n g antibodies are present after a b o u t t w o w e e k s p.i. and persist for the entire life o f the bird. A n t i b o d i e s m a y be e x p e c t e d to h a v e an adverse effect on virus r e c o v e r y . T h e t e c h n i q u e for virus detection was c o n s i d e r a b l y i m p r o v e d w h e n the samples were treated to a v o i d virus inhibitory activity. W a s h e d buffy c o a t cells p r o v e d to be v e r y useful and viruses could only be r e c o v e r e d f r o m o r g a n s by the use o f explant cultures. T h e controlled e x p o s u r e p r e s u m a b l y results in a persistent infection during the entire life o f the chicken. H o w e v e r , o f p a r a m o u n t i m p o r t a n c e is the o b s e r v a t i o n that the i n o c u l a t e d viruses are not excreted in the eggs. W h e n at the end of the egg-laying period " v a c c i n a t e d " hens were i n o c u l a t e d i n t r a t r a c h e a l l y or i n t r a m u s c u l a r l y with 105 T C I D s 0 o f R A V - C V I - 1 or R A V - 2 viruses, no egg-transmission o f these viruses could be d e m o n s t r a t e d . Acknowledgements--The authors wish to thank Mrs. H. Kappert, A. W. Manders, A. P. C. H. van Beek, H. M.
Boerrigter, C. van Engelen, L. Hartog, G. Petersen and J. IJzerman for their technical assistance. We thank Drs. B. R. Burmester and J. G. van Bekkum for helpful criticism of the manuscript. REFERENCES 1. Baluda, M. A. and Jamieson, P. P., In vivo infectivity studies with avian myeloblastosis virus. Virology 14, 33-45 (1961). 2. De Boer, G. F., Van Vloten, J., Groenendal, J. E., M ~ s , H. J. L., Borm, F., Hoogerbrugge, A. and Krasselt, M., The control of lymphoid leukosis in a commercial White Plymouth Rock line. Neth. J. Vet. Sci. (in press). 3. Burmester, B. R., Fontes, A. K. and Walter, W. G., Pathogenicity of a viral strain (RPL 12) causing avian visceral lymphomatosis and related neoplasms. III. Influence of host age and route of inoculation. J. hath. Cancerlnst. 24, 1423-1442 (1960). 4. Calnek, B. W., Lesions in young chickens induced by lymphoid leukosis virus. Avian Dis. 12, 111-129 (1968). 5. Calnek, B. W., Lymphoid leukosis virus: A survey of commercial breeding flocks for genetic resistance and incidence of embryo infection. Avian Dis. 12, 105-111 (1968). 6. Calnek, B. W., Bankowski, R. A., Beasly, J. N., Biggs, P. M., Burmester, B. R., Cho, B. R., Cole, R. K., Eidson, C. S., Furrow, R. D., Helmboldt, C. F., Hughes, W. F., Kenzy, S. C., Luginbuhl, R. E., Nazerian, K., Purchase, H. G., Rispens, B. H., Schmitfle, S. C., Siccardi, F. and Witter, R. L., Report of the AAAPsponsored leukosis workshop. Avian Dis. 11,694-702 (1967). 7. Chubb, R. C. and Biggs, P. M., The neutralization of Rous sarcoma virus. J. gen. ViroL 3, 87-96 (1968). 8. Di Stefano, H. S. and Dougherty, R. M., Mechanisms for congenital transmission of avian leukosis virus. J. hath. Cancer Inst. 37, 869-883 (1966). 9. Katz, D., Luft, A. and Kohn, A., Incidence of Marek's disease and lymphoid leukosis in poultry flocks in Israel. Refuah Vet. 25, 20-25 (1971). 10. L61iger, H. Ch. und Von dem Hagen, D., Versuche zur Immunisierung yon Legehennen zum Schutz vor Leukoseerkrankungen unter den Nachkommen. Arch. Geflfigelk. 5, 190-194 (1974). 11. Rispens, B. H., De Boer, G. F., Hoogerbrugge, A. and Van Vloten, J., A method for the control of lymphoid leukosis in chickens. Z hath. Cancer Inst. 57, 1151-1156 (1976). 12. Rispens, B. H. and Long, Ph. A., The non-producer cell activation test in avian leukosis virus assay. Comp. Leukemia Res., B ibl. haemat, no. 36, Dutcher, R. M. (Ed.), pp. 192-197. K arger, Basel (1970). 13. Rispens, B. H., Long, Ph. A., Okazaki, W. and Burmester, B. R., The NP activation test for assay of avian leukosis/sarcoma viruses. Avian Dis. 14, 738-751 (1970). 14. Rispens, B. H., Van Vloten, J. and Maas, H. J. L., Some virological and serological observations in Marek's disease. Br. vet. J. 125,445-453 (1969). 15. Rispens, B. H., Van Vloten, J., Mastenbroek, N., Maas, H. J. L. and Schat, K. A., Control of Marek's disease in The Netherlands. II. Field trials on vaccination with an avirulent strain (CVI 988) of Marek's disease virus. Avian Dis. 16, 126-138 (1972). 16. Rubin, H., A virus in chick embryos which induces resistance in vitro to infection with Rous sarcoma virus. Proc. hath. Aead. Sci., U.S.A. 46, 1105-1119 (1960).
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17. Rubin, H., Cornelius, A. and Fanshier, L., The pattern of congenital transmission of an avian leukosis virus. Proc. natn. Acad. Sci., U.S.A. 47, 1058-1060 (1961). 18. Rubin, H. and Vogt, P. K., An avian leukosis virus associated with stocks of Rous sarcoma virus. Virology 17, 184-194 (1962). 19. Sandelin, K. and Estola, T., Occurrence of different subgroups of avian leukosis virus in Finnish poultry. Avian Path. 3, 159-168 (1974). 20. Selenski, W. P., L6sungswege zu Fragen der Diagnostik und Bek~mpfung der Geflfigelleukose. Mhft. Vet. Med. 27, 647-651 (1972). 21. Zander, D. V., Raymond, R. G., McClary, C. F. and Goodwin, K., Eradication of subgroup A and B lymphoid leukosis virus from commercial poultry breeding flocks. Avian Dis. 19, 408-423 (1975).