Immunoassay. hens was separated from the main flock in 1978 and. served as the source of all samples.

Transcription

1 INFECTION AND IMMUNITY, May 1981, p Vol. 32, No /81/ $02.00/0 Analysis of Avian Leukosis Virus Infections with an Enzyme Immunoassay DONALD P. CLARK,' ROBERT F. BALL,2 AND ROBERT M. DOUGHERTY' * Department of Microbiology, State University of New York, Upstate Medical Center, Syracuse, New York 13210' and Babcock Poultry Farm, Inc., Ithaca, New York An enzyme-linked immunosorbent assay (ELISA) for avian leukosis virus group-specific antigen was used to study infections with and shedding of avian leukosis virus in a commercial flock of chickens with a known high incidence of infection. Avian leukosis virus group-specific antigen was detected in serum or cloacal washings from 76% of a group of week-old hens. With eggs collected during the next 3, antigen was detected in the albumen of 88% of the eggs from ELISA-posi hens and 2% of the eggs from ELISA-nega hens. Results of assays for infectivity correlated closely with the ELISA results. Serum and cloacal specimens were almost equally sensi in detecting infection; however, a higher proportion of cloaca-posi hens (97%) than serum-posi hens (91%) shed virus in their eggs. Similar results were obtained from a second sampling of eggs taken when the hens were 84 old, after an intervening molt. Avian leukosis virus infection was correlated with reduced egg production and increased mortality. Eggs were produced by 100% of the ELISA-nega birds during both sampling periods, whereas only 69% of the ELISA-posi birds produced eggs at 61 and 76% produced eggs at 84. All birds that were ELISA nega at 61 survived the experiment. Of 14 ELISA-posi birds that died and were examined postmortem between 61 and 84, 8 had malignancies, and the remainder died of a variety of nonmalignant diseases. With the exception of a few isolated flocks, avian oncovirus infections occur in all chicken flocks, and most sexually mature birds show virological or serological evidence of exposure to avian leukosis virus (ALV) (9). ALV infection may be spread either vertically or horizontally (12, 13). Congenitally infected birds shed virus copiously, and hens regularly transmit infection to their progeny through the eggs. Contact-infected birds only occasionally transmit virus congenitally and shed less virus, less regularly (17). Although sporadic cases of neoplastic disease occur in most flocks, heavy losses from avian leukosis are uncommon (10). Recent reports suggest that ALV infection may be associated with increased mortality from causes other than neoplasia and with physiological dysfunctions, such as delayed maturity and reduced fertility (J. S. Gravora, J. L. Spencer, R. S. Gowe, and D. L. Harris, Genetics 91[Suppl.]:S38, 1979). Although obviously desirable, control of ALV infections has been hampered by the difficulty and expense of laboratory tests for detection of ALV. The purpose of this investigation was to evaluate the use of an enzyme-linked immunosorbent assay (ELISA) for ALV group-specific (gs) antigen (1) for detection of ALV infections in large groups of birds. The resulting data showed a marked increase in mortality and de- 716 creased egg production in hens infected with ALV. MATERIALS AND METHODS Experimental chickens. A research line of White Leghorn chickens, which was bred for egg production by Babcock Poultry Farms, Inc., Ithaca, N.Y., was selected for testing because of an unusually high incidence of leukosis virus infection detectable with complement fixation assays. A group of week-old hens was separated from the main flock in 1978 and served as the source of all samples. Collection and processing of samples. Serum and cloacal specimens were collected from all hens when they were 61 old. Each cloacal sample was taken with a sterile cotton swab, which was transferred to a tube containing 0.5 ml of sterile phosphatebuffered saline. Albumen specimens were obtained from eggs of hens that had been trap-nested when they were 61 to 64 old. A small hole was punched in the large end of each egg, and 0.5 ml of albumen was withdrawn under sterile conditions. Each egg was then incubated and hatched, and the meconium was harvested from the newly hatched chick. After production of the first group of eggs, the birds underwent a molting period, and when they were 84 old, albumen was collected from eggs produced by the survivors. All samples were stored at -60 C and subsequently tested for the presence of ALV gs antigen by the ELISA. Each cloacal and serum specimen and representa albumen samples were tested for the presence of infectious ALV. For compara

2 VOL. 32, 1981 purposes, selected serum, cloacal, meconium, and albumen specimens were tested for ALV gs antigen by the complement fixation assay. Infectivity assays. Chicken embryo fibroblasts were prepared and grown as described previously (6) from c/o, gs antigen-nega embryos that were free of ALV, as determined by the COFAL test (14). At passage 3 or 4, 8 x 105 cells were transferred onto a 60-mm plastic cell culture dish and inoculated with 0.1 ml of sample. The medium was removed after 1 day, and, if necessary, the cells were washed; 3 ml of fresh medium was added, and the cells were incubated until confluent (usually 2 to 3 more days). The medium was then tested for the presence of the ALV gs antigen by the ELISA. A posi ELISA response with passage 1 fluid was taken to indicate the presence of infectious ALV. Fluids from ELISA-nega cultures were passaged three times in cell culture, and the fluids were again tested for ALV gs antigen by the ELISA. If the supernatant fluid of the passage 3 culture was nega for gs antigen, the original sample was considered free of infectious ALV. Antiserum. Antisera against ALV gs antigen for the ELISA and the complement fixation test were obtained from hamsters with tumors that were induced by injection of newborn animals with the Schmidt-Ruppin B strain of Rous sarcoma virus (11). The immunoglobulin G fraction obtained by chromatography with Sephadex G-200 was employed for the ELISA. Complement fixation assay. The procedure of Kabat and Mayer (7) was used, with modification of the volumes for microtiter plates (11); 5 50% hemolytic complement units were used for the test, whereas anticomplementary controls were performed with 3 and 5 50% hemolytic complement units. Virus antigen. Control virus antigen was prepared from purified avian myeloblastosis virus, as described previously (1). Elisa. The ALV gs antigen ELISA which is based on the double-antibody sandwich procedure (16) was performed as described by Clark and Dougherty (1), with minor modifications. The details of the procedure and the preparation of reagents were as described by Clark and Dougherty. Briefly, the plastic solid phase was sensitized with hamster anti-gs antigen immunoglobulin G. Then control antigen or sample was added and allowed to react with the immobilized antibody. A conjugate of horseradish peroxidase covalently coupled to hamster anti-gs antigen immunoglobulin G by the periodate method (8) was added and incubated so that it could bind to any antigen-antibody complexes on the solid phase. The enzyme was then reacted with its substrate, and color development was measured spectrophotometrically. Specifically, each well of polystyrene microtiter plates (type IS-MRC-96; Linbro Plastics) was coated with 50 1A of a solution containing 20 ng of hamster anti-gs antigen immunoglobulin G per ml in 0.02 M tris(hydroxymethyl)aminomethanehydrochloride buffer (ph 9.0). The plates were sealed and stored at 4 C for 18 h or more. Sensitized plates were stable at 4 C for at least 3, but were generally used within a few days. To test for ALV gs antigen, the wells of sensitized plates were washed three times for 3 min with buffer containing 0.02 M ANALYSIS OF ALV INFECTIONS 717 phosphate, 0.15 M NaCl, and 0.05% Tween-80, ph 6.8 (PBS-Tween). Untreated test or control samples were added in 25-pl portions to the wells, plates were incubated for 30 min at room temperature, and the wells were washed as described above with PBS-Tween. The hamster anti-gs antigen immunoglobulin G- horseradish peroxidase conjugate was diluted to a concentration of 40 ng/ml in PBS-Tween containing 5% pig serum (to inhibit nonspecific binding), 25 pd was added to each well, and the plates were incubated for 30 min at room temperature. The wells were washed four times for 3 min, and 100!l of peroxidase substrate was added. The substrate solution contained M H202 and M o-dianisidine in 0.1 M citrate buffer (ph 5.4). The peroxidase reaction was allowed to proceed for 45 min and then stopped by adding 10G 1Id of 0.5 N NaOH. The absorbance of the product was measured spectrophotometrically at 460 nm. Routinely, a set of controls was included with the samples. The posi control was ether-extracted avian myeloblastosis virus, which was tested in serial 10-fold dilutions from 3 x 104 to 3 x 10-' ng of protein per ml. The nonspecific activity of the conjugate was monitored by the following set of elimination reactions: coat plus conjugate (no antigen), antigen plus conjugate (no coat), and conjugate alone. RESULTS Table 1 shows representa data for the ELISA. Posi controls (in this case, avian myeloblastosis virus antigen) were detectable at concentrations as low as 3 x 10-1 ng of protein per ml or 8 pg of total avian myeloblastosis virus protein. This table also shows the ELISA values for a series of clinical specimens pretested for infectivity. Absorbance values for test materials ranged from to U for nega materials and were more than U for posi materials. The controls performed to monitor possible nonspecific reactivity were uniformly low. Table 2 shows the results obtained by the ELISA and infectivity tests on serum and cloacal samples from the original population of week-old hens. This group was categorized in terms of egg production, shedding characteristics, and survival. Birds were considered posi if antigen or infectious virus. was detected in either a serum sample or a cloacal swab or both. A bird was defined as a shedder if antigen or infectious ALV was present in the albumen from its eggs. The original population was 76% posi for antigen in serum samples or cloacal samples or both. Of the 100 birds, 76 were layers (i.e., laid eggs in trap nests), and 68% of these were antigen posi. It is striking that all 24 birds that failed to produce eggs were posi for antigen. Of the 76 layers, 47 regularly shed antigen into albumen, and all of these were posi by the combined serum-cloaca criteria.

3 718 CLARK, BALL, AND DOUGHERTY Prepn TABLE 1. Representa ELISA results ELISA results Type'( ofconcn 460 (ng of protein/mi) Range of absorbance at 460 nm A nc (U/h) (U/h)b Infectivity nega Infectivity posi AMV antigen 3 x x x x x x 10' x Controls No AMV (coat + POG) No coat (AMV + POG) POG alone Test materials Serum c Cloacal swab Albumen Meconium a AMV, Avian myeloblastosis virus; POG, peroxidase-labeled immunoglobulin G. Average of three wells. ' Range of five samples. TABLE 2. Presence ofalvgs antigens or infectious ALV in serum and cloacal swabs of hens No. antigen No. infectivity Sample posi/total posi/total no. no. Original population 76/100 (76)a 72/100 (72) Layers, 61 52/76 (68) 49/76 (64) Nonlayers, 61 24/24 (100) 23/24 (96) Shedders, 61 47/47 (100) 47/47 (100) Layers, 84 35/59 (59) 32/59 (54) Nonlayers, 84 18/18 (100) 17/18 (94) Shedders, 84 31/32 (97) 31/32 (97) a Numbers in parentheses are percentages. During the second sampling period 5 months later, 59 survivors produced eggs, and the shedding rate declined to 60%. Again, it is remarkable that all 18 birds which did not produce eggs were antigen posi. All but 1 shedder of 32 during the second sampling were posi for antigen. The infectivity data did not differ significantly from the ELISA values for antigen. In Table 3 the hens are subdivided into four categories based on the presence of gs antigen in serum or cloacal samples. The two types of specimens were equally effec for detecting antigen, with each identifying 69% of the original group as antigen posi (91% of the posi birds). The majority of antigen-posi birds (82%) were posi in both serum and cloaca samples, whereas 9% were nega in one sample or the other. Because of mortality of infected birds, the proportion of layers originally measured as antigen posi fell from 68% in the first sampling period to 59% in the second sam- TABLE 3. INFECT. IMMUN. Distribution ofgs antigen in hens No. of birds in the following groups: Serum Serum Serum Sample posi, nega- posi,, cloaca, cloaca loaca posi.. cloaca nega- posi nega Original population, 61 Layers, Nonlayers, Shedders, Layers, Nonlayers, Shedders, pling period, and the proportion of antigen-nega birds in the total laying group rose from 32 to 41% during the 5-month period. It is important that all 24 birds that were nega for antigen in both serum and cloacal samples at 61 survived for the entire experiment and produced eggs during both collection periods. Table 4 summarizes the incidence of gs antigen found in the albumen samples of eggs from hens classified according to the presence of antigen in cloacal or serum samples. The totals for eggs from both time periods showed that 88% of all albumen samples from hens classified as an-

4 VOL. 32, 1981 tigen posi were posi in the gs antigen ELISA, whereas 2% of the albumen samples from the eggs of serum-nega cloaca-nega birds contained detectable gs antigen. Table 5 shows the distribution of viral gs antigen in albumen samples according to the site of antigen in hens and the sampling period. The presence of antigen in both serum and cloaca was a highly reliable indication that the animal was a shedder. In the serum-posi cloaca-posi class, only 2 eggs were nega out of a combined total of 607 obtained during the two collection periods. Antigen in the cloacal sample alone (serum-nega cloaca-posi class) was a less reliable predictor, with 73% posi albumen samples during the first collection period and 85% during the second collection period. However, a single hen was responsible for most of the variation. This bird, which was serum nega and cloaca posi for both antigen and infectious ALV, produced 16 eggs during the first collection period, none ofwhich was antigen posi. Three eggs from this collection period were tested for infectivity, and two were posi. This animal gave similar results in the second sampling period. Of three eggs collected, one was posi for virus, and none was posi for antigen. It is clear that the results from this hen significantly altered the shedding percentages for the serum-nega cloaca-posi class, but it is important that the posi cloacal ELISA accurately predicted virus shedding. If cloacal sampling alone was used, 572 of 590 (97%) eggs TABLE 4. Assay of albumen for gs antigen Albumen samples Source of albumen No. anti- No. anti- p gen posi- gen nega- Antigen-posi hensa Antigen-nega hensb a Antigen was present in serum or cloaca or both. b No antigen in serum or cloaca. TABLE 5. Assay of albumen samples for gs antigen according to the serum-cloaca status of hens Presence of gs No. antigen in: antigen posi/total no. in: June albumen sam- November albuples men samples /531 (99)a 76/76 (100) /59 (73) 11/13 (85) + 15/64 (23) 5/11 (45) - - 8/353 (2) 0/65 (0) a Numbers in parentheses are percentages. ANALYSIS OF ALV INFECTIONS 719 from cloaca-posi hens were posi, whereas serum alone gave a value of 91% (544 of 595) posi. The serum-posi, cloaca-nega class had the poorest correlation with shedding status, since for the 61- and 84-week sampling periods together 20 of 75 (27%) albumen samples were posi for antigen. The final category, the serum-nega, cloaca-nega class, contained eight antigenposi albumen specimens, five of which were from a single bird. In this instance, infectious ALV was not demonstrated in the albumen. It is important that the shedding characteristics of individual hens remained constant, as determined by infectivity and antigen testing; i.e., birds either shed consistently or remained nega consistently (data not shown). Furthermore, molting had no apparent influence on shedding. The samplings taken at 61 and 84 (before and after a strenuous molting regime) indicated that posi hens continued to shed, whereas nega individuals remained nega. Table 6 shows infectivity assays in the same terms as Table 5. When possible, three albumen samples taken at 61 and two samples taken at 84 were selected from each hen. The distribution of infectious ALV among the serum and cloacal classes correlated well with results obtained by the direct ELISA test for gs antigen. Samples of meconia were collected only from chicks hatching from eggs sampled at 61 (Table 7). The incidence of antigen-posi meconia from hens displaying virus antigen in either serum or cloaca or both was much lower than the incidence of antigen-posi albumen samples (Table 4). Unexpectedly, a higher percentage of posi samples was found in meconia from antigen-nega hens than in the corresponding albumen samples. Infectivity data were more erratic, with no apparent correlation between serum and cloacal status and the presence of ALV. When the serum-cloacal subdivision was made TABLE 6. Subset of albumen samples tested for infectivity Presence of in- No infectivity posi/total no. in: fectious ALV in: June albumen sam- November albu- Serum Cloaca ples men samples /121 (97)a 63/64 (98) /17 (76) 9/10 (90) + - 4/14 (29) 4/10 (40) - - 1/64 (2) 1/43 (2) a Numbers in parentheses are percentages.

5 720 CLARK, BALL, AND DOUGHERTY (Table 8), the highest incidence of antigen was found in the serum-posi cloaca-posi class. The serum-nega cloaca-posi and serumposi cloaca-nega classes had lower percentages and little predic value. Meconium values were also inconsistent if the offspring from individual hens were examined. Posi meconia appeared sporadically in chicks from antigen-posi hens, although a few instances were encountered in which hatchlings from a hen were all posi or all nega. During the first collection period, albumen was removed from each egg before it was incubated for hatching. Thus, it was possible to determine the influence of virus-containing albumen on embryonic infection. Of a total of 253 albumen-meconium pairs from antigen-posi hens, 227 had posi albumen samples; from these 108 chicks developed with posi meconia. In the serum-posi cloaca-posi class, 202 pairs were available, with 201 and 104 posi in albumen and meconium, respecly. The data from antigen-nega hens included 118 pairs, in which there were no posi albumen specimens and 7 posi meconium specimens. Mortality data were also collected when possible. A total of 14 birds were given postmortem examinations. Eight of these had malignancies, seven of which were osteopetrosis; there was one case of adenocarcinoma. All eight of these hens were serum posi and cloaca posi as determined by both the ELISA and the infectivity TABLE 7. Assay of meconia for gs antigen Meconium samples Source of meconia No. anti- No. anti- % Posigen posi- gen nega- Antigen-posi hensa Antigen-nega hensb a Antigen was present in serum or cloaca or both. bno antigen in serum or cloaca. TABLE 8. Assay of meconia for gs antigen according to the serum-cloaca status of hens Presence of gs Meconium samples antigen in: Cloaca No. antigen No. infectivity posi/total no. posi/total no /204 (51)a 2/27 (7) - + 4/32 (13) 2/8 (25) + - 0/21 (0) 1/8 (13) - - 8/122 (7) 7/23 (30) a Numbers in parentheses are percentages. Serum INFECT. IMMUN. assay. The remaining six hens had a variety of nonmalignant diseases (predominately reproduc disorders), and half were serum posi and cloaca posi. As mentioned above, none of the birds that died during the course of the experiment were from the serum-nega cloaca-nega class. DISCUSSION The ELISA procedure proved to be a highly efficient means for the detection of ALV antigen in a variety of specimens (1). Because of its simplicity, this technique was particularly adaptable to large-scale screening of flocks for shedding birds. The most sensi method for identifying infected hens was to examine both serum and cloacal samples, as about 7% of the posi hens would have escaped detection if only a single sample had been taken. Of the two sample types, cloacal samples were more sensi and, in addition, were more accurate predictors of shedding. As Table 5 shows 99% of the albumen samples from serum-posi cloaca-posi hens and 70% of the samples from serum-nega cloaca-posi hens were posi, whereas only 25% of the albumen samples from serumposi cloaca-nega hens were posi. This may represent partitioning of virus within the hens due to different routes of infection. Although young contact-infected birds may become persistently infected, the process is erratic and may include periods of transient viremia without accompanying extensive shedding (17). In contrast, virus continually replicates in the tissues of congenitally infected birds, with the exception of neural tissue (4). ALV replication is abundant in the reproduc organs of hens, particularly the ovary, oviduct, and albumensecreting glands of the magnum (2), and ALV is shed copiously in albumen. Thus, it may be that the serum-posi cloaca-posi hens were infected congenitally, whereas the serum-nega cloaca-posi and serum-posi cloaca-nega hens were infected horizontally. The most effec type of sample for identifying persistent shedders, cloacal swabs, is also the simplest to collect. Surveillance of egg albumen was also an effec test for ALV infection. The shedding of gs antigen into albumen was consistent in individual birds. Of 42 serum-positivp cloaca-posi hens, 39 shed viral protein detectable by the ELISA into at least 95% of their eggs. The concentration of antigen in albumin from an individual hen tended to be uneven, but the layers could be divided into two groups on this basis. The larger group was the group with easily detectable antigen, and the smaller group was

6 VOL. 32, 1981 the group with consistently low concentrations of antigen. Samples from copious shedders always had values of more than 0.2 absorbance unit, and usually the range was 0.4 to 0.8 U. The four borderline shedders were usually in the range of 0.05 to 0.10 U, values that would not be detected by complement fixation tests. Molting had no apparent effect on the incidence of shedding. As noted above, the 61- and 84-week rates were virtually identical, which indicates that the intervening molt was of little consequence. During this study, it became evident that congenitally infected birds were adversely affected in terms of overall mortality not necessarily related to malignant disease. The effect of ALV infection on the perforinance of chickens has been examined recently (15; Gavora et al., Genetics 91[Suppl.]:S38, 1979). It was found that the average incidence of ALV infection in flocks which were not selected for egg production was approximately 20%. Infected birds generally weighed less, produced fewer eggs of poorer quality, and displayed a lag in sexual maturation. Mortality rates from seemingly unrelated syndromes, such as reproduc disorders, were also significantly higher in infected individuals. This paper confirms those conclusions with respect to increased mortality and reduced egg production in ALV-infected hens. The hens for our experiment were selected randomly from a flock with an unusually high prevalence of lymphoid leukosis virus infection and shedding. Of the original 100 birds, 76 excreted viral antigen. This incidence is rarely encountered in natural or commercial settings; however, because we examined a group of birds with a high rate of infection, the effects of persistent ALV infections were magnified. During the original sampling period, nearly one-quarter of the population failed to lay eggs, and all of the nonlaying hens were posi for gs antigen in serum or cloacal specimens. By the second sampling period, a further decline had occurred, with the same correlation; all 18 nonlaying hens had evidence of viral protein in cloacal swabs and sera. Conversely, none of the 24 hens that were nega for viral antigen during the initial sampling period died during the experiment, and all 24 produced eggs at 61 and 84. The rela increase during this period of antigennega hens from 24 to 40% of the population may reflect a posi selection for healthy, nonshedding hens. This study was terminated before we could establish whether an equilibrium was attained between the shedding and nonshedding groups and whether the rela proportion of nonshedders continued to increase substantially. This is pertinent in view of the ANALYSIS OF ALV INFECTIONS 721 findings of Gavora et al. (Genetics 91- [Suppl.]:S38, 1979) and Spencer et al. (15), who reported that the rate of ALV infection dropped in strains that were selected for high egg productivity. In those studies genetic resistance to the virus was eliminated as an inadvertently coselected trait. The effect of genetic resistance could not be evaluated in our study, which was limited to a single generation. Nevertheless, it appears that there was a natural pressure against persistently infected, shedding birds, presumably due to the physiological effects of infection. If this was the case, then, paradoxically, the leukosis virus itself was the agent which selected for a continually higher percentage of nonshedding individuals in the closed group. No physiological data are available to suggest the mechanisms of the apparent debilitating effects of ALV infection. Virtually all cells of congenitally infected chickens support ALV replication (12), including those of the major endocrine organs (5), but morphological studies have not revealed impairmnent of the differentiated functions of these cells (3). Obviously, morphological observations alone would not reveal subtle quantita alterations in endocrine function. This study suggests that careful physiological examination of ALV-infected birds might be fruitful. LITERATRJE CITED 1. Clark, D. P., and R. M. Dougherty Detection of avian oncovirus group-specific antigens by the enzymelinked immunosorbent assay. J. Gen. Virol. 47: DiStefano, I. S., and R. M. Dougherty Mechanisms for congenital transmission of avian leukosis virus. J. Natl. Cancer Inst. 37: DiStefano, H. S., and R. M. Dougherty Multiplication of avian leukosis virus in endocrine organs of congenitally infected chickens. J. Natl. Cancer Inst. 42: Dougherty, R. M., and H. S. DiStefano Sites of avian leukosis virus multiplication in congenitally infected chickens. Cancer Res. 27: Dougherty, R. M., and H. S. DiStefano Cytotropism of leukemia viruses. Prog. Med. Virol. 11: Dougherty, R. M., P. J. Simons, and F. C. Chesterman Biological properties of three variants of Rous sarcoma virus. J. Natl. Cancer Inst. 31: Kabat, E., and M. Mayer Experimental immunochemistry. Charles C Thomas, Publisher, Springfield, m. 8. Nakane, P. K., and A. Kawaoi Peroxidase-labeled antibody-a new method of conjugation. J. Histochem. Cytochem. 22: Purchase, H. G., and B. R. Burmester Avian leukosis/sarcoma group, p In M. S. Hofstadt et al. (ed.), Diseases of poultry, 7th ed., Iowa State University Press, Ames. 10. Purchase, H. G., W. Okazaki, and B. R. Burmester Long-term field trials with the herpes virus of turkeys vaccine against Marek's disease. Avian Dis. 16: Roth, F. K., and R. M. Dougherty Multiple antigenic components of the group-specific antigen of

7 722 CLARK, BALL, AND DOUGHERTY INFECT. IMMUN. the avian leukosis-sarcoma viruses. Virology 38: Rubin, H., A. Cornelius, and L. Fanshier The pattern of congenital transmission of an avian leukosis virus. Proc. Natl. Acad. Sci. U.S.A. 47: Rubin, H., L. Fanshier, A. Cornelius, and W. F. Hughes Tolerance and immunity in chickens after congenital and contact infection with an avian leukosis virus. Virology 17: Sarma, P. S., H. C. Turner, and R. J. Huebner An avian leucosis group-specific complement fixation reaction. Application for the detection and assay of non- cytopathogenic leucosis viruses. Virology 23: Spencer, J. L., J. S. Gavora, and R. S. Gowe Effect of selection for high egg production in chickens on shedding of lymphoid leukosis virus and gs antigen into eggs. Poult. Sci. 58: Voller, A., A. Bartlett, D. E. Bidwell, M. F. Clark, and A. N. Adams Detection of viruses by enzymelinked immunosorbent assay (ELISA). J. Gen. Virol. 33: Weyl, K. G., and R. M. Dougherty The contact transmission of avian leukosis virus. J. Natl. Cancer Inst. 58: