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Influence of Host Genotype on Mortality from Lymphoid Leukosis and Marek’s Disease after Artificial and Natural Exposure
196
L. D. ANDREWS AND T. L. GOODWIN
Moreng, R. E., H. L. Enos, E. G. Buss and T. E. Hartung, 1961. The relationship of floor space to factors influencing broiler growth. Poultry Sci. 40: 1039-1044. Siegel, P. B., and R. H. Coles, 1958. Effects of floor space on broiler performance. Poultry Sci. 37: 1243. Sunde, M. L., 1956. A relationship between protein level and energy level in chicks rations. Poultry
Sci. 35: 350-354. Tomhave, A. E., and K. C. Seegar, 1945. Floor space requirements of broilers. Delaware Agr. Exp. Sta. Bull. 255. Turk, D. E., W. G. Hoekstra, H. R. Bird and M. L. Sunde, 1961. The effect of dietary protein and energy levels on the growth of replacement pullets. Poultry Sci. 40: 708-716.
L. B. CRITTENDEN 1 AND B. R. BURMESTER U. S. Department of Agriculture1 (Received for publication June 24, 1968)
T
WO independent-autosomal-recessive genes (tva r , tvb r ) control resistance to subgroups A and B of the avian leukosis-sarcoma group of viruses, respectively (Crittenden et al., 1967). Resistance to subgroup A serves to exclude the viral genome from the host cell (Piraino, 1967; Crittenden, 1968), and resistance to subgroup B appears to operate by a similar mechanism (Vogt and Ishizaki, 1965). The excluded genome can induce neither viral replication nor neoplastic development in a host whose cells do not allow penetration. Therefore, breeding for resistance controlled by these genes should not only reduce disease but also reduce the prevalence of the causal virus. Payne et al. (1968) showed that resistance at the tva locus did, indeed, reduce the incidence of acute neoplasms and 1 Present Address: Poultry Research Branch, Animal Husbandry Research Division, ARS, Beltsville, Maryland 20705. 2 Poultry Research Branch, Animal Husbandry Research Division, ARS, Regional Poultry Research Laboratory, East Lansing, Michigan 48823.
lymphoid leukosis after inoculation of embryos with subgroup A leukosis viruses, but had little or no effect on the response to mixtures of subgroups A and B. The results presented here extended these observations to contact and natural exposure to lymphoid leukosis viruses and to Marek's disease. MATERIALS AND METHODS
Matings: The C/O (susceptible to subgroups A and B of the avian tumor viruses) and C/A (selectively resistant to subgroups A) progeny used were obtained from the matings described by Payne et al. (1968). Twenty-one males from families of line 15 known to be segregating at the tva locus were progeny-tested as described by Crittenden and Okazaki (1965). Four homozygous susceptible (a8as) and four homozygous resistant (a r a r ) males were chosen and mated with randomly selected line 7 females which were known to be homozygous a r a r . Line 15 is now considered to be homozygous susceptible to subgroup B (Crittenden and Okazaki, 1965; Stone, 1968). Thus,
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Influence of Host Genotype on Mortality from Lymphoid Leukosis and Marek's Disease after Artificial and Natural Exposure
GENOTYPE AND LEUKOSIS
all progeny were assumed to be susceptible to subgroup B viruses.
197
Exposure Methods: Two viruses of the avian leukosis-sarcoma virus group were used. RPL-12, L-37 prepared as described by Burmester and Gentry (1956) and used previously by us (Crittenden and Okazaki, 1965) appears to be largely of subgroup A. Stated doses of this virus represent the proportion of a gram of original erythroblastotic liver injected. The BAI-A strain of avian myeloblastosis virus (AMV) was originally obtained from J. W. Beard and was passaged in line 151 chickens (Purchase and Okazaki, 1966). The dose used represents the proportion of 1.0 ml. of plasma from leukemic chickens inoculated. The subgroup composition of this stock is unknown, but other stocks of this material have been shown to contain both subgroups A and B (Vogt and Diagnosis: All birds that died during the Ishizaki, 1965). Both viruses were inocu- experiments were necropsied. In chicks lated intravenously into embryos on the inoculated as embryos with RPL-12 and 12th day of incubation (Piraino et at., 1963). AMV, a diagnosis was made on gross Marek's disease material was injected examination alone. Doubtful cases were into chicks intraperitoneally at 1 day of confirmed histopathologically (Gross et age. The inoculum was 0.2 ml. of un- al., 1959). The following set of tissues diluted heparinized whole blood obtained were taken for histopathology from all from line 7 chickens inoculated with the birds in the contact and naturally exposed 10th serial passage of the JM isolate groups which died: liver, spleen, gonad, proventriculus, brachial and sciatic plex(Witter and Burmester, 1967). For contact exposure, test chicks were uses and the vagus nerve. Differentiation exposed to RPL-12 by hatching and between lymphoid leukosis and Marek's brooding them with susceptible 151X6 disease was made according to the followchicks inoculated as embryos. The ratio ing criteria. All birds with lymphoid of experimental chicks to inoculated lesions which died before 101 days of age chicks was approximately 2:1 in all were classified as deaths with Marek's hatching groups. The 151X6 embryos disease. Deaths between 101 days and were inoculated intravenously at 12 days termination with nerve lesions alone or of incubation with a 10 -4 dose of RPL-12, with gonadal and nerve lesions were and then at 18 days of incubation, food classified as Marek's disease, whereas coloring was injected into the fluids those with visceral lesions alone were around the embryo through the small end classified as lymphoid leukosis. Birds with of the egg so that the chicks could be both nerve and visceral lesions in organs identified at hatching by the color of their other than the gonads were assumed to
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down. These chicks were banded and remained intermingled with the test chicks for approximately six weeks. Exposure to Marek's disease was obtained by inoculating one half of the chicks of each mating and then intermingling all chicks at random for the entire experimental period. Natural exposure was obtained at the Regional Poultry Research Laboratory by hatching and brooding at a location separate from the inoculation facility, but in the same building as the regular breeding stock of the farm. Chicks exposed at a commercial farm were incubated and hatched at that farm in separate trays, in the same incubator as four separate sources of egg-production chickens. They were then intermingled with these birds for the full experimental period.
198
L. B. CRITTENDEN AND B. R.
BURMESTER
TABLE 1.—Mortality from erythroblastosis and related neoplasms from 1 through 100 days of age in C/0 and C/A chickens after intravenous inoculation with a 10~i dose of RPL-12 as 12-day embryos Mating
Progeny Male
Phenotype
Subline of
1 1 2 2
C/A*
1 1 1 2
1 2 3 4
Total 5 6 7 8
Total
Female
Percent dead
Percent dead
Percent survivors
Number
Erythroblastosis
Other
Percent survivors
0.0 0.0 0.0 0.0
14 20 7 16
100.0 95.0 100.0 100.0
0.0 5.0 0.0 0.0
0.0 0.0 0.0 0.0
Number
Erythroblastosis
Other
15 15 9 12
100.0 100.0 100.0 100.0
0.0 0.0 0.0 0.0
51
100.0
0.0
0.0
57
98.2
1.8
0.0
10 19 12 14
0.0 0.0 0.0 7.1
30.0 15.8
10 19 14 13
0.0 0.0 0.0 7.7
20.0 10.5
14.3
70.0 84.2 91.7 78.6
0.0 7.7
80.0 89.5 100.0 84i6
55
1.8
16.4
81.8
56
1.8
8.9
89.3
8.3
* Difference in mortality from erythroblastosis is statistically significant. P <0.01.
intravenous inoculation of a 10 - 4 dose of RPL-12 virus as 12 day embryos are given in Table 1. The extreme resistance Statistical Analysis: A chi-square analysis of C/A chickens to induction of erythrodeveloped by Mantel and Haenszel blastosis by RPL-12 virus reflects the (1959) was used to compare the mortality cellular resistance of C/A chickens to this of the C / 0 and C/A groups. This method virus composed primarily of subgroup A, allowed pooling information from suband confirms previous results (Crittenden comparisons, giving a test with one degree and Okazaki, 1965). On the other hand, of freedom. Paired comparisons within inoculation of embryos from the same each subline and sex were pooled for each matings with a 10~6-3 dose of AMV proover-all test. duced no significant differences between RESULTS the phenotypes in mortality from myeloblastosis (Table 2). These results are Erythroblastosis and Myeloblastosis: The consistent with the conclusion that this responses of C/O and C/A chickens to have both lymphoid leukosis and Marek's disease.
TABLE 2.—Mortality from myeloblastosis and related neoplasms from 1 through 100 days of age in C/O and C/A chickens after intravenous inoculation with a 10~h-3 dose of AMV as 12-day embryos Mating
Progeny Male
Su
Phenotype
C/O*
Mine sire
Number
1 2 3 4
1 1 2 2
Total C/A*
1 1 1 2
5 6 7 8
Total
Female
Percent dead
Male number
Percent dead - Percent survivors
Myeloblastosis
Other causes
9 12 6 13
100.0 91.7 50.0 69.2
0.0 0.0
0.0 8.3
33.3 15.4
16.7 15.4
40
80.0
10.0
16 16 5 4
81.2 68.8 100.0 50.0
41
75.6
Number
- Percent survivors
Myeloblastosis
Other Causes
11 10 9 16
63.6 70.0 77.8 81.2
36.4
0.0
0.0
30.0 11.1
10.0
46
73.9
17.4
8.7
6.2 0.0 0.0
12.5 31.2 25.0
44.4 66.7 40.0 71.4
11.1 13.3
25.0
9 15 5 14
21.4
44.4 20.0 60.0
4.9
19.5
43
60.5
13.9
0.0
* Difference in mortality from myeloblastosis is not statistically significant. P>0.05.
11.1 18.8
0.0
0.0
7.1
25.6
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c/o*
Male number
199
GENOTYPE AND LEUKOSIS
TABLE 3.—Mortality from lymphoid leukosis from 101 through 328 days of age in C/O and C/A chickens after exposure to chicks inoculated as 12-day embryos with a ID-* dose of RPL-12 Progeny
Matings Subline Phenotype
1 1 2 2
C/A*
1 1 1 2
1 2 3 4 Total 5 6 7 8 Total
Female
Percent dead
Percent dead
Number 101 days Lymphoid leukosis
Other causes
Number 101 days Lymphoid leukosis
- Percent survivors
Other causes
- Percent survivors
6 18 10 21
16.7 11.1 20.0 9.5
50.0 38.9 0.0 14.3
33.3 50.0 80.0 76.2
16 20 13 21
31.2 5.0 23.1 38.1
55
12.7
23.6
63.6
70
24.3
4.3
71.4
18 25 15 13
0.0 0.0 0.0 0.0
33.3 44.0 40.0 7.7
66.7 56.0 60.0 92.3
7 20 17 13
0.0 3.4 0.0 0.0
14.3 3.4 17.6 0.0
85.7 93.1 82.4 100.0
71
0.0
33.8
66.2
66
1.5
7.6
90.9
0.0 5.0 7.7 4.8
68.8 90.0 69.2 57.1
' Difference in mortality from lymphoid leukosis is statistically significant. P<0.01.
virus stock is composed primarily of subgroup B or that the induction of typical myeloblastic leukemia is a property of a component of the inoculum other than subgroup A. These results and those presented by Payne el al. (1968) suggest that the resistance controlled at the tva locus is specific for subgroup A virus. Lymphoid Leukosis: The C/O and C/A chickens were raised in several locations where lymphoid leukosis was expected to occur. Differences in incidence of lymphoid leukosis between these groups would be expected if resistance at the tva locus
were effective under natural exposure conditions. This difference was observed when these matings were exposed to RPL-12 virus by hatching and brooding them in contact with embryo-inoculated chicks, and mortality was tabulated from 101 through 328 days of age (Table 3). Another exposure trial was conducted at the Regional Poultry Research Laboratory where the chicks were hatched and reared with the non-isolated stocks of the laboratory. Table 4 shows that the incidence of this disease was very low. The level of mortality with lymphoid leukosis was
TABLE 4.—Mortality from lymphoid leukosis from 101 through 328 days of age in C/O and C/A chickens after natural exposure at the Regional Poultry Research Laboratory Mating
Progeny
Subline of sire
Male number
C/O*
1 1 2 2
1 2 3 4
C/A*
1 1 1 2
Phenotype
Total 5 6 7 8 Total
Male
Female
Percent dead
Percent dead
Number 101 days Lymphoid leukosis
Other causes
Percent survivors
Number 101 days Lymphoid leukosis
Other causes
• Percent survivors
71.4 60.0 83.3 94.4
15 22 11 27
18.5
78.5
75
6.6
14.7
78.7
50.0 21.1 40.0 16.7
50.0 78.9 60.0 77.8
13 27 19 15
0.0 0.0 0.0 0.0
23.1 11.1 47.4 6.7
76.9 88.9 52.6 93.3
70.9
74
0.0
21.6
78.4
14 15 18 18
0.0 0.0 11.1 0.0
28.6 40.0 5.6 5.6
65
3.1
8 19 10 18
0.0 0.0 0.0 5.6
55
1.8
27.3
' Difference in mortality from lymphoid leukosis is not statistically significant. P>0.05.
6.7 0.0 27.3 3.7
13.3 22.7 9.1 11.1
80.0 77.3 63.6 85.2
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C/O*
Male number
Male
200
L. B. CRITTENDEN AND B. R. BURMESTER TABLE 5.—Mortality from lymphoid leukosis from 100 through 583 days of age in C/O and C/A chickens after hatching and rearing in contact with commercial chickens Mating
Phenotype
C/O*
Female progeny Percent dead
Subline of sire
Male number
Number 101 days
1 1 2 2
1 2 3 4
Total 1 1 1 2 Total :
5 6 7 8
Other causes
survivors
21 31 37 55
0.0 0.0 8.1 14.5
9.5 9.7 21.6 14.5
90.5 90.3 70.3 70.9
144
7.6
14.6
77.8
49 45 46 46
0.0 0.0 0.0 0.0
6.1 4.4 8.7 6.5
93.9 95.6 91.3 93.5
186
0.0
6.5
93.5
Difference in mortality from lymphoid leukosis is statistically significant. P<0.05.
higher in the C/O group, but the slight difference observed was not statistically significant. Exposure was also attempted at two commercial farms. On one of these farms the incidence of lymphoid leukosis was so low that no valid comparison could be made and the histopathological diagnoses of all the birds was not completed. The incidence was higher in the other trial and the mortality among the females observed from 101 through 583 days of age was significantly higher in the C/O group (Table 5). These results suggest that resistance at the tva locus may be of practical value in reducing losses from lymphoid leukosis, but similar trials should be run in commercial stock under a number of different exposure conditions. Marek's Disease: C/O and C/A matings were used in experiments to assess the influence of the tva locus on mortality after severe artificial and natural exposure to Marek's disease. The data of Tables 6 and 7 were collected in an experiment in which half of each mating group was
inoculated with infective blood of the JM strain of Marek's disease at one day of age. Lesions in birds dying from the 11th through the 117th day of age were observed. No significant differences between the C/O and C/A groups were found even though a high proportion died with neoplasms. Tables 8 and 9 present mortality from naturally occurring Marek's disease from 11 through 328 days of age. These birds were raised for the purpose of investigating lymphoid leukosis mortality (Tables 3 and 4), but a number of birds died with lesions of Marek's disease contracted by chance exposure to the agent. Again no significant differences between the C/O and C/A progeny were found in either experiment. However, it can be seen that the mortality from Marek's disease in progeny of subline 1 males was considerably higher than that observed in the progeny of subline 2 males, suggesting a subline difference in susceptibility within line 15. DISCUSSION
The data presented here confirm the
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C/A*
Lymphoid leukosis
201
GENOTYPE AND LEUKOSIS TABLE 6.—Mortality from Marek's disease from 11 through 117 days of age in C/O and C/A chickens after inoculation with JM infected blood Progeny
Mating
Female
Male Su
Phenotype
Hine sire
number
1 1 2 2
C/O*
1 2 3 4
Total
Total
Number
Marek's disease
Other causes
7 9 3 9
100.0 100.0 66.7 88.9
0.0 0.0 0.0 0.0
28
92.8
6 17 7 15 45
- Percent survivors
Number
- Percent survivors
Marek's disease
Other causes
33.3 11.1
11 10 6 20
100.0 90.0 83.3 100.0
10.0
0.0
7.2
47
95.7
2.1
2.2
100.0 100.0 85.7 93.3
0.0 0.0 0.0 0.0
0.0 0.0 6.7
7 14 15 9
100.0 100.0 100.0 100.0
0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0
96.6
0.0
5.4
45
100.0
0.0
0.0
0.0 0.0
14.3
0.0 O.O 0.0
0.0 0.0
16.7 0.0
* Difference in mortality from Marek's disease is not statistically significant. P>0.05.
subgroups may be more efficient in reducing field occurrence of this disease. The specificity of this cellular resistance which selectively prevents infection with one subgroup of the avian leukosissarcoma viruses, raises important questions (Crittenden, 1968). First, will it be necessary to select for resistance to several viral subgroups at once to reduce disease effectively? Secondly, will cellular resistance drastically change the population of viruses by selection of mutant types which will grow in resistant chickens? The potential disadvantages of utilizing a system of genes controlling genetic cellular resis-
extreme specificity of genetic cellular resistance to avian leukosis-sarcoma group of viruses. They also demonstrate the influence of the tva locus on naturally occurring lymphoid leukosis, and thus strengthen the results of Payne et al. (1968) which suggests that genetic cellular resistance may be useful for reducing the occurrence of lymphoid leukosis in the field. This locus alone would achieve this purpose only if subgroup A were the predominant subgroup in the field. Most isolations have in fact been subgroup A or mixtures of A and B (Calnek, 1968). However, multiple resistance to several
TABLE 7 - -Mortality from Marek's disease from 11 through 117 days of age \ • C/O and C/A chickens after contact exposure to JM inoculated chicks Mating
Progeny
Subline of sire
Male number
Number
C/O*
1 1 2 2
1 2 3 4
9 9 6 16
C/A*
1 1 1 2
Phenotype
Total 5 6 7 8
Total
Male
Female
Percent dead
Percent dead
Marek's disease
Other causes
100.0 66.7 50.0 75.0
0.0 0.0 0.0 0.0
40
75.0
8 17 10 12
75.0 76.5 70.0 83.3
47
76.6
- Percent survivors
Number
- Percent survivors
Marek's disease
Other causes 11.1
0.0
0.0 0.0 8.3
33.3 33.3 16.7
33.3 50.0 25.0
9 12 3 12
88.9 66.7 66.7 75.0
0.0
25.0
36
75.0
5.6
19.4
0.0 0.0
7 15 12 14
71.4 80.0 100.0 71.4
0.0 6.7 0.0 0.0
28.6 13.3
0.0
25.0 23.5 20.0 16.7
28.6
2.1
21.3
48
81.2
2.1
16.7
10.0
0.0
* Difference in mortality from Marek's disease is not statistically significant. P >0.05.
0.0
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5 6 7 8
1 1 1 2
C/A*
Percent dead
Male
202
L. B. CRITTENDEN AND B. R.
BURMESTER
TABLE 8.—Mortality from Marek's disease from 11 through 328 days of age in C/O and CI A chickens after contact hatching and rearing with RPL-12 inoculated chicks Progeny
Mating
Male number
C/O*
1 1 2 2
1 2 3 4
C/A*
1 1 1 2
Phenotype
Total 5 6 7 8 Total
Number
Male
Female
Percent dead
Percent dead - Percent survivors
Number
Marek's disease
Other causes
53.3 17.4 0.0 4.5
33.3 43.5 20.0 22.7
13.3 39.1 80.0 72.7
17 22 13 22
70
18.6
31.4
50.0
19 27 20 14
31.4 29.6 25.0 0.0
5.3 18.5 30.0 14.3
63.2 51.8 45.0 85.7
80
23.8
17.5
58.8
15 23 10 22
- Percent survivors
Marek's disease
Other causes
5.9 9.1 0.0 9.1
29.4 9.1 30.8 36.4
64.7 81.8 69.2 54.5
74
6.8
25.7
67.6
9 32 18 13
22.2 3.1 11.1 0.0
11.1 12.5 11.1 0.0
66.7 84.4 77.8 100.0
72
6.9
9.7
83.3
* Difference in mortality from Marek's disease is not statistically significant. P >0.05.
tance may be outweighed by the advantages in a program for eradication of the leukosis-sarcoma group of viruses from chicken flocks. Data published so far are severely limited because they represent comparisons in a narrow genetic background and under a restricted set of environmental conditions. Before widespread introduction of genes for cellular resistance can be recommended, testing of other stocks under a variety of exposure conditions should be conducted. The lack of influence of this locus on the incidence of Marek's disease is not sur-
prising in the light of recent results which suggest that the Marek's disease agent belongs to a group of viruses totally unrelated to the avian leukosis sarcoma group (Churchill and Biggs, 1967; Nazerian et al., 1968). It is evident, therefore, that selection for resistance to lymphoid leukosis is independent of the problem of selecting for resistance to Marek's disease. A comparison of Tables 3 and 8 which present lymphoid leukosis and Marek's disease mortality of birds in the same experiment is interesting. Table 3 shows a striking influence of the tva locus on lymphoid leukosis while Table 8 shows
TABLE 9.—Mortality from Marek's disease from 11 through 328 days of age in C/O and C/A chickens not purposefully exposed to RPL-12 or Marek's disease Progeny
Mating Subline of sire
Male number
C/O*
1 1 2 2
1 2 3 4
C/A*
1 1 1 2
Phenotype
Total 5 6 7 8 Total
Number
Male
Female
Percent dead
Percent dead - Percent survivors
Number
Marek's disease
Other causes
19 17 19 20
42.1 35.3 5.3 10.0
5.3 11.8 15.8 5.0
52.6 52.9 78.9 85.0
21 26 11 29
75
22.7
9.3
68.0
12 22 13 19
66.7 27.3 23.1 15.8
0.0 4.5 30.8 10.5
33.3 68.2 46.2 73.7
66
30.3
10.6
59.1
* Difference in mortality from Marek's disease is not statistically significant. P>0.05.
Marek's disease
Other causes
survivors
38.1 11.5 9.1 13.8
4.8 23.1 27.3 6.9
57.1 65.4 63.6 79.3
87
18.4
13.8
67.8
18 32 20 15
44.4 15.6 30.0 6.7
0.0 9.4 20.0 0.0
55.5 75.0 50.0 93.3
85
23.5
8.2
68.2
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Subliii e of sire
GENOTYPE AND LEUKOSIS
that this locus has no influence on Marek's disease. These data suggest that our arbitrary criteria for distinguishing between these pathologically similar neoplasms in the same flock are accurate. Thus, carefully selected groups of genetically resistant and susceptible stock may aid in the evaluation of histopathological criteria for distinguishing between these two lymphoid neoplasms.
Paired matings of chickens known to be susceptible (C/O) or resistant (C/A) to subgroup A Rous sarcoma virus were exposed by injection, contact or natural means to leukosis viruses and the Marek's disease agent. Differences between these phenotypes in mortality from erythroblastosis and lymphoid leukosis were highly significant after exposure to the subgroup A RPL-12 virus. A similar, though smaller difference, was also found with exposure on a commercial poultry farm. No significant difference in mortality was observed after exposure to a sample of mixed subgroup avian myeloblastosis virus, the JM strain of Marek's disease or naturally occurring Marek's disease. These results indicate that resistance controlled by the tva locus is specific for the diseases caused by subgroup A of the leukosis-sarcoma group of viruses, but has no influence on mortality induced by other viruses of the avian leukosis complex. ACKNOWLEDGEMENTS
The authors acknowledge the expert assistance of Drs. D. C. Johnson, P. A. Long, R. Muhm, L. N. Payne, H. G. Purchase and W. Okazaki who provided inocula, inoculated chicks and made the diagnoses. The very competent technical assistance of Myrtle Bartlett and Curtis Bartlett is gratefully acknowledged.
REFERENCES Burmester, B. R., and R. F. Gentry, 1956. The response of susceptible chickens to graded doses of the virus of visceral lymphomatosis. Poultry Sci. 35: 17-26. Calnek, B. W. 1968 Lymphoid leukosis virus: A survey of commercial breeding flocks for genetic resistance and incidence of embryo infection. Avian Dis. 12: 104-111. Churchill, A. E., and P. M. Biggs, 1967. Agent of Marek's disease in tissue culture. Nature, 215: 528-530. Crittenden, L. B., 1968. Observations on the nature of a genetic cellular resistance to avian tumor viruses. J. Nat. Cancer Inst. 41: 145-153. Crittenden, L. B., and W. Okazaki, 1965. Genetic influence of the Rs locus on susceptibility to avian tumor viruses. I. Neoplasms induced by RPL 12 and three strains of Rous sarcoma virus. J. Nat. Cancer Inst. 35:857-863. Crittenden, L. B., H. A. Stone, R. H. Reamer and W. Okazaki, 1967. Two loci controlling genetic cellular resistance to avian leukosis-sarcoma viruses. J. Virology, 1: 898-904. Gross, M. A., B. R. Burmester and W. G. Walter, 1959. Pathogenicity of a viral strain (RPL 12) causing avian visceral lymphomatosis and related neoplasms. I. Nature of the lesions. J. Nat. Cancer Inst. 22:83-101. Mantel, N., and W. Haenszel, 1959. Statistical aspects of the analysis of data from retrospective studies of disease. J. Nat. Cancer Inst. 22: 719— 748. Nazerian, K. N., J. J. Solomon, R. L. Witter and B. R. Burmester, 1968. Etiological studies of Marek's disease. I I . Finding of a herpesvirus in cell culture. Proc. Soc. Expt. Biol. Med. 127:116119. Payne, L. N., L. B. Crittenden and W. Okazaki, 1968. Influence of host genotype on responses to four strains of avian leukosis virus. J. Nat. Cancer Inst. 40:907-916. Piraino, F., W. Okazaki, B. R. Burmester and T. N. Fredrickson, 1963. Bioassay of fowl leukosis virus in chickens by the inoculation of 11-day-old embryos. Virology, 21: 396-401. Piraino, F., 1967. The mechanism of genetic resistance of chick embryo cells to infection by Rous sarcoma virus-Bryan strain (BS-RSV). Virology, 32: 700-707. Purchase, H. G., and W. Okazaki, 1966. In vivo replacement of the helper virus in Bryan's strain of Rous sarcoma virus by AMV and RPL 12 viruses. J. Nat. Cancer Inst. 37: 563-571. Stone, H. A., 1968. Unpublished.
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SUMMARY
203
204
L. B. CRITTENDEN AND B. R. BURMESTER
Witter, R. W., and B. R. Burmester, 1967. Transmission of Marek's disease with oral washings and feces from infected chickens. Proc. Soc. Expt. Biol. Med. 124: 59-62.
Vogt, P. K., and R. Ishizaki, 1965. Reciprocal patterns of genetic resistance to avian tumor viruses in two lines of chickens. Virology, 26: 664-672.
Hematological Response of Different Stocks of Chickens to Iron-Copper Deficient Diet Department of Poultry Science, University of Georgia, Athens, Georgia 30601 (Received for publication June 26, 1968)
T
INDELL et al. (1967) reported that genotype-environmental interactions for body weight, although statistically significant, were relatively unimportant when genetically different stocks were reared under different management and geographical environments. This type of study enables one to obtain information about differences in the effects of the average environment of one location compared to that of another location. Another approach is to create a definable stress environment and then compare the stocks in stress and non-stress environments. Genetic differences between stocks and lines of chickens have been reported for packed erythrocyte volume (Washburn and Smyth, 1968). Slight genetic differences in packed erythrocyte volume values may have little or no effect on a stock as long as certain environmental conditions are not present. However, a bird with slightly decreased packed erythrocyte volume values might be more severely affected by marginal deficiencies University of Goergia, College of Agriculture Experiment Stations, College Station, Athens Journal Paper number 300.
of nutritients necessary for hematopoiesis. If this is true, genotype—environmental interactions involving anemia and diet could become important. The purpose of this study was to evaluate this hypothesis utilizing the response to a marginal deficiency of iron and copper of stocks used in the Southern Regional genotypeenvironment study reported by Tindell etal. (1967). METHODS
Eggs from four stocks; Athens-Canadian randombred (ACRB), Cornell randombred (Cornell), North Carolina Rhode Island Red (NCRIR) and Texas-50Leghorn (Texas-50) were obtained from the Southern Regional Poultry Genetics Laboratory. At hatching the chicks were wing-banded, weighed and randomized into two groups of 40 chicks per stock. All chicks, for a two week period following hatching, were fed the basal skim milk diet (7.0 p.p.m. Fe, 2.5 p.p.m. Cu)to which 100 p.p.m. Fe and 10 p.p.m. Cu was added. The skim milk diet was similar to that described by Hill and Matrone (1961). At the conclusion of this two-week period one group of each stock was con-
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K. W. WASHBURN
L. D. ANDREWS AND T. L. GOODWIN
Moreng, R. E., H. L. Enos, E. G. Buss and T. E. Hartung, 1961. The relationship of floor space to factors influencing broiler growth. Poultry Sci. 40: 1039-1044. Siegel, P. B., and R. H. Coles, 1958. Effects of floor space on broiler performance. Poultry Sci. 37: 1243. Sunde, M. L., 1956. A relationship between protein level and energy level in chicks rations. Poultry
Sci. 35: 350-354. Tomhave, A. E., and K. C. Seegar, 1945. Floor space requirements of broilers. Delaware Agr. Exp. Sta. Bull. 255. Turk, D. E., W. G. Hoekstra, H. R. Bird and M. L. Sunde, 1961. The effect of dietary protein and energy levels on the growth of replacement pullets. Poultry Sci. 40: 708-716.
L. B. CRITTENDEN 1 AND B. R. BURMESTER U. S. Department of Agriculture1 (Received for publication June 24, 1968)
T
WO independent-autosomal-recessive genes (tva r , tvb r ) control resistance to subgroups A and B of the avian leukosis-sarcoma group of viruses, respectively (Crittenden et al., 1967). Resistance to subgroup A serves to exclude the viral genome from the host cell (Piraino, 1967; Crittenden, 1968), and resistance to subgroup B appears to operate by a similar mechanism (Vogt and Ishizaki, 1965). The excluded genome can induce neither viral replication nor neoplastic development in a host whose cells do not allow penetration. Therefore, breeding for resistance controlled by these genes should not only reduce disease but also reduce the prevalence of the causal virus. Payne et al. (1968) showed that resistance at the tva locus did, indeed, reduce the incidence of acute neoplasms and 1 Present Address: Poultry Research Branch, Animal Husbandry Research Division, ARS, Beltsville, Maryland 20705. 2 Poultry Research Branch, Animal Husbandry Research Division, ARS, Regional Poultry Research Laboratory, East Lansing, Michigan 48823.
lymphoid leukosis after inoculation of embryos with subgroup A leukosis viruses, but had little or no effect on the response to mixtures of subgroups A and B. The results presented here extended these observations to contact and natural exposure to lymphoid leukosis viruses and to Marek's disease. MATERIALS AND METHODS
Matings: The C/O (susceptible to subgroups A and B of the avian tumor viruses) and C/A (selectively resistant to subgroups A) progeny used were obtained from the matings described by Payne et al. (1968). Twenty-one males from families of line 15 known to be segregating at the tva locus were progeny-tested as described by Crittenden and Okazaki (1965). Four homozygous susceptible (a8as) and four homozygous resistant (a r a r ) males were chosen and mated with randomly selected line 7 females which were known to be homozygous a r a r . Line 15 is now considered to be homozygous susceptible to subgroup B (Crittenden and Okazaki, 1965; Stone, 1968). Thus,
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Influence of Host Genotype on Mortality from Lymphoid Leukosis and Marek's Disease after Artificial and Natural Exposure
GENOTYPE AND LEUKOSIS
all progeny were assumed to be susceptible to subgroup B viruses.
197
Exposure Methods: Two viruses of the avian leukosis-sarcoma virus group were used. RPL-12, L-37 prepared as described by Burmester and Gentry (1956) and used previously by us (Crittenden and Okazaki, 1965) appears to be largely of subgroup A. Stated doses of this virus represent the proportion of a gram of original erythroblastotic liver injected. The BAI-A strain of avian myeloblastosis virus (AMV) was originally obtained from J. W. Beard and was passaged in line 151 chickens (Purchase and Okazaki, 1966). The dose used represents the proportion of 1.0 ml. of plasma from leukemic chickens inoculated. The subgroup composition of this stock is unknown, but other stocks of this material have been shown to contain both subgroups A and B (Vogt and Diagnosis: All birds that died during the Ishizaki, 1965). Both viruses were inocu- experiments were necropsied. In chicks lated intravenously into embryos on the inoculated as embryos with RPL-12 and 12th day of incubation (Piraino et at., 1963). AMV, a diagnosis was made on gross Marek's disease material was injected examination alone. Doubtful cases were into chicks intraperitoneally at 1 day of confirmed histopathologically (Gross et age. The inoculum was 0.2 ml. of un- al., 1959). The following set of tissues diluted heparinized whole blood obtained were taken for histopathology from all from line 7 chickens inoculated with the birds in the contact and naturally exposed 10th serial passage of the JM isolate groups which died: liver, spleen, gonad, proventriculus, brachial and sciatic plex(Witter and Burmester, 1967). For contact exposure, test chicks were uses and the vagus nerve. Differentiation exposed to RPL-12 by hatching and between lymphoid leukosis and Marek's brooding them with susceptible 151X6 disease was made according to the followchicks inoculated as embryos. The ratio ing criteria. All birds with lymphoid of experimental chicks to inoculated lesions which died before 101 days of age chicks was approximately 2:1 in all were classified as deaths with Marek's hatching groups. The 151X6 embryos disease. Deaths between 101 days and were inoculated intravenously at 12 days termination with nerve lesions alone or of incubation with a 10 -4 dose of RPL-12, with gonadal and nerve lesions were and then at 18 days of incubation, food classified as Marek's disease, whereas coloring was injected into the fluids those with visceral lesions alone were around the embryo through the small end classified as lymphoid leukosis. Birds with of the egg so that the chicks could be both nerve and visceral lesions in organs identified at hatching by the color of their other than the gonads were assumed to
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down. These chicks were banded and remained intermingled with the test chicks for approximately six weeks. Exposure to Marek's disease was obtained by inoculating one half of the chicks of each mating and then intermingling all chicks at random for the entire experimental period. Natural exposure was obtained at the Regional Poultry Research Laboratory by hatching and brooding at a location separate from the inoculation facility, but in the same building as the regular breeding stock of the farm. Chicks exposed at a commercial farm were incubated and hatched at that farm in separate trays, in the same incubator as four separate sources of egg-production chickens. They were then intermingled with these birds for the full experimental period.
198
L. B. CRITTENDEN AND B. R.
BURMESTER
TABLE 1.—Mortality from erythroblastosis and related neoplasms from 1 through 100 days of age in C/0 and C/A chickens after intravenous inoculation with a 10~i dose of RPL-12 as 12-day embryos Mating
Progeny Male
Phenotype
Subline of
1 1 2 2
C/A*
1 1 1 2
1 2 3 4
Total 5 6 7 8
Total
Female
Percent dead
Percent dead
Percent survivors
Number
Erythroblastosis
Other
Percent survivors
0.0 0.0 0.0 0.0
14 20 7 16
100.0 95.0 100.0 100.0
0.0 5.0 0.0 0.0
0.0 0.0 0.0 0.0
Number
Erythroblastosis
Other
15 15 9 12
100.0 100.0 100.0 100.0
0.0 0.0 0.0 0.0
51
100.0
0.0
0.0
57
98.2
1.8
0.0
10 19 12 14
0.0 0.0 0.0 7.1
30.0 15.8
10 19 14 13
0.0 0.0 0.0 7.7
20.0 10.5
14.3
70.0 84.2 91.7 78.6
0.0 7.7
80.0 89.5 100.0 84i6
55
1.8
16.4
81.8
56
1.8
8.9
89.3
8.3
* Difference in mortality from erythroblastosis is statistically significant. P <0.01.
intravenous inoculation of a 10 - 4 dose of RPL-12 virus as 12 day embryos are given in Table 1. The extreme resistance Statistical Analysis: A chi-square analysis of C/A chickens to induction of erythrodeveloped by Mantel and Haenszel blastosis by RPL-12 virus reflects the (1959) was used to compare the mortality cellular resistance of C/A chickens to this of the C / 0 and C/A groups. This method virus composed primarily of subgroup A, allowed pooling information from suband confirms previous results (Crittenden comparisons, giving a test with one degree and Okazaki, 1965). On the other hand, of freedom. Paired comparisons within inoculation of embryos from the same each subline and sex were pooled for each matings with a 10~6-3 dose of AMV proover-all test. duced no significant differences between RESULTS the phenotypes in mortality from myeloblastosis (Table 2). These results are Erythroblastosis and Myeloblastosis: The consistent with the conclusion that this responses of C/O and C/A chickens to have both lymphoid leukosis and Marek's disease.
TABLE 2.—Mortality from myeloblastosis and related neoplasms from 1 through 100 days of age in C/O and C/A chickens after intravenous inoculation with a 10~h-3 dose of AMV as 12-day embryos Mating
Progeny Male
Su
Phenotype
C/O*
Mine sire
Number
1 2 3 4
1 1 2 2
Total C/A*
1 1 1 2
5 6 7 8
Total
Female
Percent dead
Male number
Percent dead - Percent survivors
Myeloblastosis
Other causes
9 12 6 13
100.0 91.7 50.0 69.2
0.0 0.0
0.0 8.3
33.3 15.4
16.7 15.4
40
80.0
10.0
16 16 5 4
81.2 68.8 100.0 50.0
41
75.6
Number
- Percent survivors
Myeloblastosis
Other Causes
11 10 9 16
63.6 70.0 77.8 81.2
36.4
0.0
0.0
30.0 11.1
10.0
46
73.9
17.4
8.7
6.2 0.0 0.0
12.5 31.2 25.0
44.4 66.7 40.0 71.4
11.1 13.3
25.0
9 15 5 14
21.4
44.4 20.0 60.0
4.9
19.5
43
60.5
13.9
0.0
* Difference in mortality from myeloblastosis is not statistically significant. P>0.05.
11.1 18.8
0.0
0.0
7.1
25.6
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c/o*
Male number
199
GENOTYPE AND LEUKOSIS
TABLE 3.—Mortality from lymphoid leukosis from 101 through 328 days of age in C/O and C/A chickens after exposure to chicks inoculated as 12-day embryos with a ID-* dose of RPL-12 Progeny
Matings Subline Phenotype
1 1 2 2
C/A*
1 1 1 2
1 2 3 4 Total 5 6 7 8 Total
Female
Percent dead
Percent dead
Number 101 days Lymphoid leukosis
Other causes
Number 101 days Lymphoid leukosis
- Percent survivors
Other causes
- Percent survivors
6 18 10 21
16.7 11.1 20.0 9.5
50.0 38.9 0.0 14.3
33.3 50.0 80.0 76.2
16 20 13 21
31.2 5.0 23.1 38.1
55
12.7
23.6
63.6
70
24.3
4.3
71.4
18 25 15 13
0.0 0.0 0.0 0.0
33.3 44.0 40.0 7.7
66.7 56.0 60.0 92.3
7 20 17 13
0.0 3.4 0.0 0.0
14.3 3.4 17.6 0.0
85.7 93.1 82.4 100.0
71
0.0
33.8
66.2
66
1.5
7.6
90.9
0.0 5.0 7.7 4.8
68.8 90.0 69.2 57.1
' Difference in mortality from lymphoid leukosis is statistically significant. P<0.01.
virus stock is composed primarily of subgroup B or that the induction of typical myeloblastic leukemia is a property of a component of the inoculum other than subgroup A. These results and those presented by Payne el al. (1968) suggest that the resistance controlled at the tva locus is specific for subgroup A virus. Lymphoid Leukosis: The C/O and C/A chickens were raised in several locations where lymphoid leukosis was expected to occur. Differences in incidence of lymphoid leukosis between these groups would be expected if resistance at the tva locus
were effective under natural exposure conditions. This difference was observed when these matings were exposed to RPL-12 virus by hatching and brooding them in contact with embryo-inoculated chicks, and mortality was tabulated from 101 through 328 days of age (Table 3). Another exposure trial was conducted at the Regional Poultry Research Laboratory where the chicks were hatched and reared with the non-isolated stocks of the laboratory. Table 4 shows that the incidence of this disease was very low. The level of mortality with lymphoid leukosis was
TABLE 4.—Mortality from lymphoid leukosis from 101 through 328 days of age in C/O and C/A chickens after natural exposure at the Regional Poultry Research Laboratory Mating
Progeny
Subline of sire
Male number
C/O*
1 1 2 2
1 2 3 4
C/A*
1 1 1 2
Phenotype
Total 5 6 7 8 Total
Male
Female
Percent dead
Percent dead
Number 101 days Lymphoid leukosis
Other causes
Percent survivors
Number 101 days Lymphoid leukosis
Other causes
• Percent survivors
71.4 60.0 83.3 94.4
15 22 11 27
18.5
78.5
75
6.6
14.7
78.7
50.0 21.1 40.0 16.7
50.0 78.9 60.0 77.8
13 27 19 15
0.0 0.0 0.0 0.0
23.1 11.1 47.4 6.7
76.9 88.9 52.6 93.3
70.9
74
0.0
21.6
78.4
14 15 18 18
0.0 0.0 11.1 0.0
28.6 40.0 5.6 5.6
65
3.1
8 19 10 18
0.0 0.0 0.0 5.6
55
1.8
27.3
' Difference in mortality from lymphoid leukosis is not statistically significant. P>0.05.
6.7 0.0 27.3 3.7
13.3 22.7 9.1 11.1
80.0 77.3 63.6 85.2
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C/O*
Male number
Male
200
L. B. CRITTENDEN AND B. R. BURMESTER TABLE 5.—Mortality from lymphoid leukosis from 100 through 583 days of age in C/O and C/A chickens after hatching and rearing in contact with commercial chickens Mating
Phenotype
C/O*
Female progeny Percent dead
Subline of sire
Male number
Number 101 days
1 1 2 2
1 2 3 4
Total 1 1 1 2 Total :
5 6 7 8
Other causes
survivors
21 31 37 55
0.0 0.0 8.1 14.5
9.5 9.7 21.6 14.5
90.5 90.3 70.3 70.9
144
7.6
14.6
77.8
49 45 46 46
0.0 0.0 0.0 0.0
6.1 4.4 8.7 6.5
93.9 95.6 91.3 93.5
186
0.0
6.5
93.5
Difference in mortality from lymphoid leukosis is statistically significant. P<0.05.
higher in the C/O group, but the slight difference observed was not statistically significant. Exposure was also attempted at two commercial farms. On one of these farms the incidence of lymphoid leukosis was so low that no valid comparison could be made and the histopathological diagnoses of all the birds was not completed. The incidence was higher in the other trial and the mortality among the females observed from 101 through 583 days of age was significantly higher in the C/O group (Table 5). These results suggest that resistance at the tva locus may be of practical value in reducing losses from lymphoid leukosis, but similar trials should be run in commercial stock under a number of different exposure conditions. Marek's Disease: C/O and C/A matings were used in experiments to assess the influence of the tva locus on mortality after severe artificial and natural exposure to Marek's disease. The data of Tables 6 and 7 were collected in an experiment in which half of each mating group was
inoculated with infective blood of the JM strain of Marek's disease at one day of age. Lesions in birds dying from the 11th through the 117th day of age were observed. No significant differences between the C/O and C/A groups were found even though a high proportion died with neoplasms. Tables 8 and 9 present mortality from naturally occurring Marek's disease from 11 through 328 days of age. These birds were raised for the purpose of investigating lymphoid leukosis mortality (Tables 3 and 4), but a number of birds died with lesions of Marek's disease contracted by chance exposure to the agent. Again no significant differences between the C/O and C/A progeny were found in either experiment. However, it can be seen that the mortality from Marek's disease in progeny of subline 1 males was considerably higher than that observed in the progeny of subline 2 males, suggesting a subline difference in susceptibility within line 15. DISCUSSION
The data presented here confirm the
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C/A*
Lymphoid leukosis
201
GENOTYPE AND LEUKOSIS TABLE 6.—Mortality from Marek's disease from 11 through 117 days of age in C/O and C/A chickens after inoculation with JM infected blood Progeny
Mating
Female
Male Su
Phenotype
Hine sire
number
1 1 2 2
C/O*
1 2 3 4
Total
Total
Number
Marek's disease
Other causes
7 9 3 9
100.0 100.0 66.7 88.9
0.0 0.0 0.0 0.0
28
92.8
6 17 7 15 45
- Percent survivors
Number
- Percent survivors
Marek's disease
Other causes
33.3 11.1
11 10 6 20
100.0 90.0 83.3 100.0
10.0
0.0
7.2
47
95.7
2.1
2.2
100.0 100.0 85.7 93.3
0.0 0.0 0.0 0.0
0.0 0.0 6.7
7 14 15 9
100.0 100.0 100.0 100.0
0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0
96.6
0.0
5.4
45
100.0
0.0
0.0
0.0 0.0
14.3
0.0 O.O 0.0
0.0 0.0
16.7 0.0
* Difference in mortality from Marek's disease is not statistically significant. P>0.05.
subgroups may be more efficient in reducing field occurrence of this disease. The specificity of this cellular resistance which selectively prevents infection with one subgroup of the avian leukosissarcoma viruses, raises important questions (Crittenden, 1968). First, will it be necessary to select for resistance to several viral subgroups at once to reduce disease effectively? Secondly, will cellular resistance drastically change the population of viruses by selection of mutant types which will grow in resistant chickens? The potential disadvantages of utilizing a system of genes controlling genetic cellular resis-
extreme specificity of genetic cellular resistance to avian leukosis-sarcoma group of viruses. They also demonstrate the influence of the tva locus on naturally occurring lymphoid leukosis, and thus strengthen the results of Payne et al. (1968) which suggests that genetic cellular resistance may be useful for reducing the occurrence of lymphoid leukosis in the field. This locus alone would achieve this purpose only if subgroup A were the predominant subgroup in the field. Most isolations have in fact been subgroup A or mixtures of A and B (Calnek, 1968). However, multiple resistance to several
TABLE 7 - -Mortality from Marek's disease from 11 through 117 days of age \ • C/O and C/A chickens after contact exposure to JM inoculated chicks Mating
Progeny
Subline of sire
Male number
Number
C/O*
1 1 2 2
1 2 3 4
9 9 6 16
C/A*
1 1 1 2
Phenotype
Total 5 6 7 8
Total
Male
Female
Percent dead
Percent dead
Marek's disease
Other causes
100.0 66.7 50.0 75.0
0.0 0.0 0.0 0.0
40
75.0
8 17 10 12
75.0 76.5 70.0 83.3
47
76.6
- Percent survivors
Number
- Percent survivors
Marek's disease
Other causes 11.1
0.0
0.0 0.0 8.3
33.3 33.3 16.7
33.3 50.0 25.0
9 12 3 12
88.9 66.7 66.7 75.0
0.0
25.0
36
75.0
5.6
19.4
0.0 0.0
7 15 12 14
71.4 80.0 100.0 71.4
0.0 6.7 0.0 0.0
28.6 13.3
0.0
25.0 23.5 20.0 16.7
28.6
2.1
21.3
48
81.2
2.1
16.7
10.0
0.0
* Difference in mortality from Marek's disease is not statistically significant. P >0.05.
0.0
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5 6 7 8
1 1 1 2
C/A*
Percent dead
Male
202
L. B. CRITTENDEN AND B. R.
BURMESTER
TABLE 8.—Mortality from Marek's disease from 11 through 328 days of age in C/O and CI A chickens after contact hatching and rearing with RPL-12 inoculated chicks Progeny
Mating
Male number
C/O*
1 1 2 2
1 2 3 4
C/A*
1 1 1 2
Phenotype
Total 5 6 7 8 Total
Number
Male
Female
Percent dead
Percent dead - Percent survivors
Number
Marek's disease
Other causes
53.3 17.4 0.0 4.5
33.3 43.5 20.0 22.7
13.3 39.1 80.0 72.7
17 22 13 22
70
18.6
31.4
50.0
19 27 20 14
31.4 29.6 25.0 0.0
5.3 18.5 30.0 14.3
63.2 51.8 45.0 85.7
80
23.8
17.5
58.8
15 23 10 22
- Percent survivors
Marek's disease
Other causes
5.9 9.1 0.0 9.1
29.4 9.1 30.8 36.4
64.7 81.8 69.2 54.5
74
6.8
25.7
67.6
9 32 18 13
22.2 3.1 11.1 0.0
11.1 12.5 11.1 0.0
66.7 84.4 77.8 100.0
72
6.9
9.7
83.3
* Difference in mortality from Marek's disease is not statistically significant. P >0.05.
tance may be outweighed by the advantages in a program for eradication of the leukosis-sarcoma group of viruses from chicken flocks. Data published so far are severely limited because they represent comparisons in a narrow genetic background and under a restricted set of environmental conditions. Before widespread introduction of genes for cellular resistance can be recommended, testing of other stocks under a variety of exposure conditions should be conducted. The lack of influence of this locus on the incidence of Marek's disease is not sur-
prising in the light of recent results which suggest that the Marek's disease agent belongs to a group of viruses totally unrelated to the avian leukosis sarcoma group (Churchill and Biggs, 1967; Nazerian et al., 1968). It is evident, therefore, that selection for resistance to lymphoid leukosis is independent of the problem of selecting for resistance to Marek's disease. A comparison of Tables 3 and 8 which present lymphoid leukosis and Marek's disease mortality of birds in the same experiment is interesting. Table 3 shows a striking influence of the tva locus on lymphoid leukosis while Table 8 shows
TABLE 9.—Mortality from Marek's disease from 11 through 328 days of age in C/O and C/A chickens not purposefully exposed to RPL-12 or Marek's disease Progeny
Mating Subline of sire
Male number
C/O*
1 1 2 2
1 2 3 4
C/A*
1 1 1 2
Phenotype
Total 5 6 7 8 Total
Number
Male
Female
Percent dead
Percent dead - Percent survivors
Number
Marek's disease
Other causes
19 17 19 20
42.1 35.3 5.3 10.0
5.3 11.8 15.8 5.0
52.6 52.9 78.9 85.0
21 26 11 29
75
22.7
9.3
68.0
12 22 13 19
66.7 27.3 23.1 15.8
0.0 4.5 30.8 10.5
33.3 68.2 46.2 73.7
66
30.3
10.6
59.1
* Difference in mortality from Marek's disease is not statistically significant. P>0.05.
Marek's disease
Other causes
survivors
38.1 11.5 9.1 13.8
4.8 23.1 27.3 6.9
57.1 65.4 63.6 79.3
87
18.4
13.8
67.8
18 32 20 15
44.4 15.6 30.0 6.7
0.0 9.4 20.0 0.0
55.5 75.0 50.0 93.3
85
23.5
8.2
68.2
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Subliii e of sire
GENOTYPE AND LEUKOSIS
that this locus has no influence on Marek's disease. These data suggest that our arbitrary criteria for distinguishing between these pathologically similar neoplasms in the same flock are accurate. Thus, carefully selected groups of genetically resistant and susceptible stock may aid in the evaluation of histopathological criteria for distinguishing between these two lymphoid neoplasms.
Paired matings of chickens known to be susceptible (C/O) or resistant (C/A) to subgroup A Rous sarcoma virus were exposed by injection, contact or natural means to leukosis viruses and the Marek's disease agent. Differences between these phenotypes in mortality from erythroblastosis and lymphoid leukosis were highly significant after exposure to the subgroup A RPL-12 virus. A similar, though smaller difference, was also found with exposure on a commercial poultry farm. No significant difference in mortality was observed after exposure to a sample of mixed subgroup avian myeloblastosis virus, the JM strain of Marek's disease or naturally occurring Marek's disease. These results indicate that resistance controlled by the tva locus is specific for the diseases caused by subgroup A of the leukosis-sarcoma group of viruses, but has no influence on mortality induced by other viruses of the avian leukosis complex. ACKNOWLEDGEMENTS
The authors acknowledge the expert assistance of Drs. D. C. Johnson, P. A. Long, R. Muhm, L. N. Payne, H. G. Purchase and W. Okazaki who provided inocula, inoculated chicks and made the diagnoses. The very competent technical assistance of Myrtle Bartlett and Curtis Bartlett is gratefully acknowledged.
REFERENCES Burmester, B. R., and R. F. Gentry, 1956. The response of susceptible chickens to graded doses of the virus of visceral lymphomatosis. Poultry Sci. 35: 17-26. Calnek, B. W. 1968 Lymphoid leukosis virus: A survey of commercial breeding flocks for genetic resistance and incidence of embryo infection. Avian Dis. 12: 104-111. Churchill, A. E., and P. M. Biggs, 1967. Agent of Marek's disease in tissue culture. Nature, 215: 528-530. Crittenden, L. B., 1968. Observations on the nature of a genetic cellular resistance to avian tumor viruses. J. Nat. Cancer Inst. 41: 145-153. Crittenden, L. B., and W. Okazaki, 1965. Genetic influence of the Rs locus on susceptibility to avian tumor viruses. I. Neoplasms induced by RPL 12 and three strains of Rous sarcoma virus. J. Nat. Cancer Inst. 35:857-863. Crittenden, L. B., H. A. Stone, R. H. Reamer and W. Okazaki, 1967. Two loci controlling genetic cellular resistance to avian leukosis-sarcoma viruses. J. Virology, 1: 898-904. Gross, M. A., B. R. Burmester and W. G. Walter, 1959. Pathogenicity of a viral strain (RPL 12) causing avian visceral lymphomatosis and related neoplasms. I. Nature of the lesions. J. Nat. Cancer Inst. 22:83-101. Mantel, N., and W. Haenszel, 1959. Statistical aspects of the analysis of data from retrospective studies of disease. J. Nat. Cancer Inst. 22: 719— 748. Nazerian, K. N., J. J. Solomon, R. L. Witter and B. R. Burmester, 1968. Etiological studies of Marek's disease. I I . Finding of a herpesvirus in cell culture. Proc. Soc. Expt. Biol. Med. 127:116119. Payne, L. N., L. B. Crittenden and W. Okazaki, 1968. Influence of host genotype on responses to four strains of avian leukosis virus. J. Nat. Cancer Inst. 40:907-916. Piraino, F., W. Okazaki, B. R. Burmester and T. N. Fredrickson, 1963. Bioassay of fowl leukosis virus in chickens by the inoculation of 11-day-old embryos. Virology, 21: 396-401. Piraino, F., 1967. The mechanism of genetic resistance of chick embryo cells to infection by Rous sarcoma virus-Bryan strain (BS-RSV). Virology, 32: 700-707. Purchase, H. G., and W. Okazaki, 1966. In vivo replacement of the helper virus in Bryan's strain of Rous sarcoma virus by AMV and RPL 12 viruses. J. Nat. Cancer Inst. 37: 563-571. Stone, H. A., 1968. Unpublished.
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SUMMARY
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L. B. CRITTENDEN AND B. R. BURMESTER
Witter, R. W., and B. R. Burmester, 1967. Transmission of Marek's disease with oral washings and feces from infected chickens. Proc. Soc. Expt. Biol. Med. 124: 59-62.
Vogt, P. K., and R. Ishizaki, 1965. Reciprocal patterns of genetic resistance to avian tumor viruses in two lines of chickens. Virology, 26: 664-672.
Hematological Response of Different Stocks of Chickens to Iron-Copper Deficient Diet Department of Poultry Science, University of Georgia, Athens, Georgia 30601 (Received for publication June 26, 1968)
T
INDELL et al. (1967) reported that genotype-environmental interactions for body weight, although statistically significant, were relatively unimportant when genetically different stocks were reared under different management and geographical environments. This type of study enables one to obtain information about differences in the effects of the average environment of one location compared to that of another location. Another approach is to create a definable stress environment and then compare the stocks in stress and non-stress environments. Genetic differences between stocks and lines of chickens have been reported for packed erythrocyte volume (Washburn and Smyth, 1968). Slight genetic differences in packed erythrocyte volume values may have little or no effect on a stock as long as certain environmental conditions are not present. However, a bird with slightly decreased packed erythrocyte volume values might be more severely affected by marginal deficiencies University of Goergia, College of Agriculture Experiment Stations, College Station, Athens Journal Paper number 300.
of nutritients necessary for hematopoiesis. If this is true, genotype—environmental interactions involving anemia and diet could become important. The purpose of this study was to evaluate this hypothesis utilizing the response to a marginal deficiency of iron and copper of stocks used in the Southern Regional genotypeenvironment study reported by Tindell etal. (1967). METHODS
Eggs from four stocks; Athens-Canadian randombred (ACRB), Cornell randombred (Cornell), North Carolina Rhode Island Red (NCRIR) and Texas-50Leghorn (Texas-50) were obtained from the Southern Regional Poultry Genetics Laboratory. At hatching the chicks were wing-banded, weighed and randomized into two groups of 40 chicks per stock. All chicks, for a two week period following hatching, were fed the basal skim milk diet (7.0 p.p.m. Fe, 2.5 p.p.m. Cu)to which 100 p.p.m. Fe and 10 p.p.m. Cu was added. The skim milk diet was similar to that described by Hill and Matrone (1961). At the conclusion of this two-week period one group of each stock was con-
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K. W. WASHBURN