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Genetic Response of the Fowl to Exposure to Marek’s Disease1
Genetic Response of the Fowl to Exposure to Marek's Disease1 GAD ZEITLIN, 2 J. ROBERT SMYTH, JR. AND MARTIN SEVOIAN Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01002 (Received for publication August 9, 1971)
POULTRY SCIENCE 51: 602-608,
T
HE elimination of Marek's disease as a health problem of chickens is an agreed goal of everyone concerned with the poultry industry. It is not possible at the present time to design a final blueprint for such an accomplishment, although recent information on the use of vaccines, genetic selection and environmental control techniques offers promising avenues for control. The use of nonpathogenic viruses, either attenuated Marek's disease herpesvirus or a herpesvirus of turkeys, for immunization against Marek's disease has proved to protect chickens effectively under both laboratory and field conditions (see Witter for review, 1971). Similarly, progress following genetic selection for resistance using the JM virus looks very promising (Cole, 1968). Ultimately these approaches possibly combined with environmental control procedures may all play important roles in accomplishing the complete elimination of 1
Contribution from the Massachusetts Experiment Station, Amherst, Massachusetts. 2 Present address: Poultry Breeders Union, Agricultural Cooperative Society, Ltd., 12 Bialik Street, Tel-Aviv, Israel.
1972
the causative agent from poultry populations. It is now well established that a bird's response to either leukosis-sarcoma (Type I) or Marek's disease (Type II) viruses is greatly influenced by genetic factors. A genetically simple mechanism has been shown to involve resistance to the leukosis-sarcoma viruses at the cellular level. Recessive genes at each of two autosomal loci confer resistance to subgroups A and B, respectively, probably by blocking viral penetration (Crittenden et al., 1967; Piraino, 1967). More recently, a similar system of resistance to subgroup C has been suggested by Motta et al. (1969). Genetic influence on the development of Marek's disease following exposure has been demonstrated for the JM (Cole, 1964, 1968; Han et al., 1969; and Han, 1970) and the HPRS-B14 (Payne and Biggs, 1964) isolates. Present evidence indicates that genetic control of resistance to the RIF and JM-type viruses results from separate genetic mechanisms (Payne and Biggs, 1964; Purchase, 1966; and Crittenden and Burmester, 1969). The present study was designed to ob-
602
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ABSTRACT Genetic differences in response of three genetic stocks and their reciprocal crossbred progeny were studied following inoculation at one day of age with the J M Marek's disease virus. There were marked differences among the pure breeds in respect to the general level of resistance as well as the response of specific tissues. A maternal effect was found to influence the incidence of individuals with one or more positive lesions regardless of location (P < 0.05) as well as specific gonadal lesions (P < 0.01). Differences in general combining ability were significant for the incidence of paralysis and lesions of the DRG (P < 0.01) as well as mortality (P < 0.0S). A significant reciprocal effect (P < 0.05) was found for the incidence of positive lesions regardless of location. Since the analysis had supposedly eliminated the variance associated with maternal effects and general combinability, a sex-linked effect is suggested, although its effect is not clear or consistent in the crossbred data.
GENETIC RESPONSE TO MAREK'S DISEASE
603
ml. Experimental chicks were inoculated at one day of age by injections of 0.25 ml. of the inoculum intraperitoneally. General Management. The injected birds were housed on the floor in one room containing approximately 800 square feet of floor space. Heat was provided by infrared bulbs and wood shavings were used for litMATERIALS AND METHODS ter. A similar uninjected population served Stocks Used. A total of 720 chicks repre- as controls and were reared on a separate senting three pure lines and the six possible farm. All chicks were fed a commercial reciprocal crosses among them were used in chick starter mash fed ad libitum. The incithis study. The pure lines included the dence of paralysis and mortality were reLight Brown Leghorn, Barred Plymouth corded daily. All dead birds from both the Rock and a synthetic recessive white line experimental and control groups were exdesignated as NE-6. The Brown Leghorn amined for the presence of gross leukotic and Barred Rock lines have been resident lesions. At the conclusion of the experiment at the University of Massachusetts Re- all survivors were sacrificed and examined search Farm for 12 and 6 years, respec- for leukotic lesions. Necropsy included an tively, and neither has been previously sub- examination of the viscera and the dorsal jected to artificial selection for response to root ganglia (D.R.G.) from the cervicoMarek's disease. The Brown Leghorn line thoracic region. A diagnosis of Marek's diswas used previously by Han et al. (1969) ease was made when one or more of the and found to be particularly susceptible to nerve ganglia were characteristically envisceral lesions and to be associated with a larged and/or when typical tumors were maternal effect for susceptibility. Prelimi- present in any of the visceral organs. The nary observations indicated the Barred few birds that died during the trial from Rock line to be relatively susceptible, while causes other than Marek's disease and the NE-6 line was relatively resistant to those that lost their bands were excluded the JM virus. The latter was synthesized in from the data. This represented 3.8% of 1960 and had been maintained as a random the original experimental group. breeding line without intentional selection Statistical analysis. The data were anasince that time. lyzed by use of the method of least-squares Inoculum. All genetic resistance trials analysis as outlined by Harvey (1960), utiwere conducted utilizing the JM virus iso- lizing a CDC-3600 computer. The method lated by Sevoian et al. (1962). The JM in- is applicable for computing data with unoculum was prepared from visceral tumors equal subclass numbers. The mathematical and blood taken from previously injected model used was as follows: susceptible Cornell S-line chicks. The cellular suspension was prepared by grinding Y\im = n+Sh+a,i+bijj+g2j(gSk)+jmij the tissues in Ten Broeck grinders, and sus+rijk+sahi+sbhijj+sghaj(sghsk) pending as a 10% concentration in phos+smhSj+srhSjk+ phate-buffered saline (NaCl 8.5 g., Na2 HP0 4 1.07 g., NaH 2 P0 4 -2H 2 0 0.39 g. per where: liter H 2 0, pH 7.1) containing 10,000 units of penicillin and 10 mg. of streptomycin per ykijH= the measurement on the Ith. tain more information on the response of various genetic combinations to exposure to Marek's disease. Reciprocal crossbred progeny were studied in order to obtain information on the inheritance of resistance and susceptibility, as well as the role played by maternal effects.
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604
G. ZEITLIN, J. R. SMYTH, JR. AND M. SEVOIAN
EXPERIMENTAL RESULTS The first sign of Marek's disease was paralysis on day 21 postinoculation, while the
first mortality occurred during the fourth week of the experiment. All deaths during the first six weeks occurred in birds showing paralysis. Although some of these had gonadal lesions, the other visceral organs were consistently negative during this period. During the last two weeks of the experiment, lesions of the liver, spleen, kidney, lung, proventriculus, as well as the gonads were common. The response of the three pure lines and their crossbred progeny to inoculation at one day of age with JM leukosis virus is shown in Table 1, while the results of the least-squares analysis of variance are presented in Table 2. This study verifies the greater susceptibility of females to Marek's disease previously reported by Payne and Biggs (1964), Purchase and Biggs (1967), Biggs et al. (1968), Han et al. (1969) and others. Although the sex effect was most pronounced for gonadal lesions, it was also apparent for all tissues analyzed with the exception of the liver. Breed differences were highly significant (P < 0.01) for the incidences of mortality, paralysis, positive D.R.G. and those with one or more positive lesions regardless of location (total positive lesions), and significant at the 0.0S probability level for liver lesions. The Barred Rock was the most susceptible for mortality, paralysis, lesions of the D.R.G. and total positive lesions, while the NE-6 line was the most resistant. The Brown Leghorn line had more liver and gonadal lesions, although the latter difference was not statistically significant. The Barred Rocks were extremely susceptible to paralysis. These data provide another example of genetic differences in response of specific tissues to exposure to a viral agent for a neoplastic disease. Differences in the general combining ability of the three breeds were statistically significant (P < 0.01) for the incidences of paralysis and lesions of the D.R.G. mor-
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progeny in the ith type of breeding of the Mh sex of a mating between a male of jth line and a female of the Mh line jU = the overall mean, an effect common to all birds Sh = an effect common to the Mh sex of offspring #,-= an effect common to the ith type of mating, purebred vs. crossbred Jiyy = an effect common to the progeny of a mating between a male of the jth line and a female of the jth line, thus measuring the effects of the breeds of purebreds gijigsk) = the general combining ability effect for the jth (Mh) line m2j = the maternal effect for the j t h line r?jk= the reciprocal effect resulting from the difference between using the jth line as a male and the Mh line as a female, rather than the Mh line as a male and the j t h line as a female sa%i= the interaction between sex and type of mating sbhin= the interaction between sex and breed of purebred sghsjisghsk) = the interaction between sex and general combining ability smusj— the interaction between sex and maternal ability srh31k = the interaction between sex and the reciprocal effects ehijki=& random error assumed to be NID (0, oy*)
605
GENETIC RESPONSE TO MAREK'S DISEASE TABLE 1.—Res pome of purebred and crossbred chickens during an eight week period following inoculation at one day of age with JM virus Matings 1,2
Sex
^T -&• A ulTas
Percent Incidence Positive Lesions Mortality Paralysis '
DRG
Gonads
Liver
Spleen
Total 3
M F Both
45 34 79
22.2 52.9 35.4
33.3 61.8 45.6
57.8 76.5 65.8
31.1 41.2 35.4
0.0 2.9 1.3
4.4 5.9 5.1
71.1 85.3 77.2
NE-6
M F Both
31 47 78
0.0 10.6 6.4
0.0 12.8 • 7.7
12.9 23.4 19.2
32.3 38.3 35.9
6.5 6.4 6.4
9.7 8.5 9.0
35.5 44.7 41.0
B.L.
M F Both
39 35 74
10.3 25.7 17.6
5.1 11.4 8.1
23.1 14.3 18.9
38.5 65.7 51.4
12.8 14.3 13.5
2.6 8.6 5.4
56.4 71.4 63.5
NE-6 X B.R.
M F Both
35 44 79
5.7 29.6 19.0
11.4 27.3 20.3
25.7 31.8 29.1
28.6 38.6 34.2
0.0 6.8 3.8
0.0 11.4 6.3
34.3 50.0 43.0
B.R. X NE-6
M F Both
38 40 78
7.9 32.5 20.5
10.5 32.5 21.8
26.3 60.0 43.6
42.1 52.5 47.4
7.9 7.5 7.7
2.6 7.5 5.1
55.3 72.5 64.1
B.L. X B.R.
M F Both
35 39 74
17.1 46.2 32.4
17.1 25.6 21.6
31.4 48.7 40.5
28.6 51.3 40.5
8.6 18.0 13.5
2.9 12.8 8.1
57.1 69.2 63.5
B.R. X. B.L.
M F Both
34 41 75
17.7 34.2 26.7
14.7 22.0 18.7
44.1 39.0 41.3
55.9 58.5 57.3
14.7 17.1 16.0
8.8 9.8 9.3
70.6 70.7 70.7
B.L. X NE-6
M F Both
40 39 79
5.0 12.8 8.9
2.5 0.0 1.3
12.5 15.4 13.9
45.0 51.3 48.1
7.5 10.3 8.9
7.5 10.3 8.9
55.0 53.9 54.4
NE-6 X B.L.
M F Both
39 38 77
7.7 23.7 15.6
5.1 2.6 3.9
18.0 26.3 22.1
48.7 73.7 61.0
7.7 18.4 13.0
0.0 15.8 7.8
59.0 81.6 70.1
1 2 3
B.R.=Barred Plymouth Rock; NE-6 = synthetic recessive white line; B.L.=Light Brown Leghorn. Male parent listed first. Total positive lesions—includes all birds with one or more positive lesions regardless of location.
tality differences were significant at the 0.05 probability level. The reciprocal crosses in the present experiment showed a higher degree of general combining ability than did those in the similar study by Han et al. (1969). The latter found general combining ability to be significant only for the incidence of gonadal lesions. This difference is probably due to differences in the genetic lines used in the two studies. These data suggest that the relative importance of specific versus general combining ability is a function of the particular genetic combinations with the latter becoming more im-
portant as the difference in resistance levels of the stocks increases. The response of the purebred and crossbred offspring to the JM virus differed only in respect to the incidence of paralysis, the purebreds being more susceptible (P < 0.05). Earlier Hutt and Cole (1952) noted that crossbreeding does not improve the incidence of natural occurring leukosis. In a study similar to the present one, Han et al. (1969) found that crossbreds were significantly more susceptible to paralysis, lesions of the D.R.G. and the incidence of one or more lesions irrespective of location. The
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B.R.
606
G. ZEITLIN, J. R. SMYTH, JR. AND M. SEVOIAN TABLE 2.—Results of analysis of variance Mean Squares Source of Variation
-.,
d.f.
Positive Lesions
. ,?tr" lallty
Paralvsis
Total DRG
Gonads
Liver
Spleen
1
0.001
0.630
0.141
0.685
0.157
0.011
0.000
Breed within Purebreds (B)
2
2.012**
4.118**
6.033**
0.675
0.279*
0.035
2.814**
Sex (S)
1
5.627**
2.153**
1.125*
2.837**
0.173
0.356*
2.168**
General combining Ability (G) 2
0.669*
1.162**
2.325**
0.052
0.181
0.035
0.436
Maternal Effects (M)
2
0.019
0.001
0.243
1.181**
0.059
0.000
0.900
Reciprocal Effects (A)
1
0.291
0.079
0.568
0.115
0.044
0.012
1.159*
2 Factor Interactions (A)X(S) (B)X(S) (S)X(G) (S)X(M) (S)X(R)
1 2 2 2 1
0.002 0.209 0.075 0.047 0.001
0.225 0.246 0.354 0.068 0.171
0.054 0.374 0.112 0.010 0.041
0.910 0.236 0.080 0.109 0.008
0.055 0.004 0.032 0.010 0.042
0.115 0.024 0.069 0.138 0.088
0.011 0.018 0.187 0.028 0.008
675 0.146
0.120
0.197
0.240
0.083
0.067
0.226
Error
* Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.
latter utilized three breeds that were all relatively susceptible. This suggests the possibility that a negative heterotic response to Marek's disease exposure may be most often associated with lines with similar levels of susceptibility and/or with relatively high levels of susceptibility. Maternal effects were found to be significant (P < 0.01) for the incidence of gonadal lesions and also for total positive lesions (P < 0.0S). The incidence of gonadal lesions and those with one or more positive lesions was approximately 17% and 14% greater respectively when the Brown Leghorn was used as the female rather than the male. On the other hand, the incidence of offspring with gonadal lesions and those with one or more positive lesions was approximately 13% and 14% lower respectively when the Barred Rock was used as the female parent. A significant (P < 0.05) difference was found among reciprocal crosses for the inci-
dence of individuals with one or more lesions regardless of location. Since the variance associated with maternal effects and general combining abilities had been eliminated, sex-linked genes may be involved, although such an effect is not readily apparent in these data. The interpretation is confused further as none of the analyses concerned with any of the specific lesions or responses showed such an effect. The control group showed no clinical signs of Marek's disease. Following necropsy at the end of the experimental period, seven birds were found to have enlarged dorsal root ganglia and three had gonadal lesions. This indicates that the incidence of natural occurring Marek's disease was very low in the stocks studied at the time of this investigation. DISCUSSION The limited amount of available data concerning the inheritance of resistance
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Purebreds vs. Crossbreds (A)
GENETIC RESPONSE TO MAREK'S DISEASE
sumably result from a sex-linked effect. Since there is no consistent relationship between sex of offspring and response to the virus in the reciprocal cross progeny, sexlinkage does not appear to explain these data satisfactorily. No maternal effects were associated directly with the NE-6 female. That the relative importance of maternal effects depends on the specific mating is also suggested by the observations of Han et al. (1969) and Cole (1971). ACKNOWLED GMENTS
The authors wish to thank Dr. Richard A. Damon, Jr. for his assistance with the statistical analysis. Further appreciation is expressed to Drs. T. W. Fox and Peter F.S. Han for their advice and counsel during the course of this work. REFERENCES Biggs, P. M., R. J. Thorpe and L. N. Payne, 1968. Studies on genetic resistance to Marek's disease in the domestic chicken. British Poultry Sci. 9 : 37-52. Cole, R. K., 1964. Strain difference in response to the J M leukosis virus. Poultry Sci. 4 3 : 13081309. Cole, R. K., 1968. Studies on genetic resistance to Marek's disease. Avian Diseases, 12: 9-28. Cole, R. K., 1971. The genetic resistance to Marek's disease. Paper presented at Symposium on Oncogenesis and Herpes-type Viruses, Cambridge, England, June, 1971. Abstract: 16. Crittenden, L. B., and B. R. Burmester, 1969. Influence of host genotype on mortality from lymphoid leukosis and Marek's disease after artificial and natural exposure. Poultry Sci. 48: 197-204. 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. Han, F.-S., 1970. The influence of growth rate on the response of chickens to the JM-leukosis virus. Ph.D. Thesis, University of Massachusetts. Han, F.-S., J. R. Smyth, Jr., M. Sevoian and F. N. Dickinson, 1969. Genetic resistance to leukosis caused by the J M virus in the fowl. Poultry Sci. 48: 76-87. Harvey, W. R., 1960. Least-squares analysis of
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and/or susceptibility to Marek's disease suggests the involvement of a polygenic system, although future clarification may reveal the presence of major single genes that are individually identifiable. At the present time there is disagreement on the degree of dominance for resistance, information that is of importance for planning the course of future breed, strain and inbred cross mating programs. Han et al. (1969) studied the offspring of crosses between relatively susceptible breeds and observed certain combinations to be more susceptible than the parent stocks, resulting in an interesting example of negative heterosis. On the other hand, crosses between the extremely resistant Cornell JM-N line and a number of other genetic stocks indicate that resistance can show essentially complete dominance (Cole, 1971). In the present study dominance appears to be incomplete with the Fi progeny showing a degree of susceptibility intermediate to the response of the parent lines. At the present time one can only conclude that the degree of dominance for response to Marek's disease exposure varies with the particular stocks crossed. The significant maternal effect (P < 0.01) associated with the incidence of gonadal lesions and total positive lesions is of particular interest. Similar evidence for a maternal effect associated with susceptibility to the JM virus was demonstrated previously for this Brown Leghorn strain by Han et al. (1969). In contrast, Cole (1971) did not observe maternal effects to significantly influence the response to Marek's disease exposure of progeny from crosses between his selected resistant (JMN) and more susceptible lines. The present study also suggests that the Barred Rock females contributed a maternal factor for resistance to their offspring. This hypothesis fits the data better than one involving a specific sire contribution, that would pre-
607
608
G. ZEITLIN, J. R. SMYTH, JR. AND M. SEVOIAN Rous sarcoma virus-Bryan strain (BS-RSV). Virology, 32: 700-707. Purchase, H. G., 1966. The epizootiology of the avian leukosis complex. Proc. International Wenner-Gren Symposium. Comparative Leukamias, Stockholm, Pergamon Press 37. Purchase, H. G., and P. M. Biggs, 1967. Characterization of five isolates of Marek's disease. Res. Vet. Sci. 8 : 440-449. Sevoian, M., D. M. Chamberlain and F. T. Counter, 1962. Avian lymphomatosis. 1. Experimental reproduction of the neural and visceral forms. Vet. Med. 57 : 500-501. Witter, R. L., 1971. Marek's disease research-history and perspectives. Poultry Sci. 50: 333342.
Sulfate Metabolism and Taurine Synthesis in the Chick1 W I L L I A M G. M A R T I N 2
Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, West Virginia 26506 (Received for publication August 9, 1971) ABSTRACT Sulfate has been suggested as an essential nutrient in the diet of chickens. One-day-old cockerels were fed a purified diet supplemented with varying levels of methionine and at each methionine level four levels of sulfate were added. In a second experiment methionine, sulfate and taurine were supplemented to the diet. A significant growth response was observed with supplemental methionine and sulfate. A portion of the dietary sulfate was used in the synthesis of phosphoadenosinephosphosulfate (PAPS) which is required for the synthesis of taurine by this pathway. Methionine was shown to enhance the synthesis of taurine in the liver while supplemental sulfate enhanced the activity of the sulfate activating enzymes and the in vitro synthesis of PAP3:;S. The data of these experiments support the theory that in the chick at least a part of the growth response obtained by sulfate supplementation is due to the reactions whereby activated sulfate is converted to taurine. POULTRY SCIENCE 51: 608-612, 1972
C
YSTEINE and methionine constitute about 90 percent of the total sulfur of most plants (Allaway and Thompson, 1966), thus the availability of sulfate-sulfur to poultry fed a natural feedstuffs diet is limited. Further, when corn-soybean meal diets are fed to poultry, DL-methio-
'This manuscript is published with the approval of the Director of the West Virginia University Agricultural Experiment Station, Morgantown, as Scientific Paper No. 1185. 2 Associate Agricultural Biochemist.
nine is usually added to increase the sulfur amino acid concentration to about 0.8 percent, or 3.5 percent of dietary protein (Nelson et al., 1960). Inorganic sulfur per se has been ascribed a role in the metabolism of the chicken (Machlin, 1955). Gordon and Sizer (1955) shared this view and Miraglia et al. (1966) observed a growth response in chicks to sulfate added to a purified diet adequate in methionine. Recently Ross and Harms (1970) have substantiated the earlier reports of the benefi-
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data with unequal subclass numbers. A.R.S. 208, U.S.D.A. 1-156. Hutt, F. B., and R. K. Cole, 1952. Heterosis in an interstrain cross of White Leghorns. Poultry Sci. 3 1 : 365-374. Motta, J. V., L. B. Crittenden and E. F. Godfrey, 1969. The inheritance of resistance to subgroup C Ieukosis-sarcoma viruses in New Hampshires. Proc. Assn. Agric. Workers, Inc., Memphis, Tennessee, Feb. 1-4, p. 189. Payne, L. N., and P. M. Biggs, 1964. Transmission experiments with Marek's disease (fowl paralysis) and lymphoid leukosis. World's Poultry Sci. J. 20: 284-297. Piraino, F., 1967. The mechanisms of genetic resistance of chick embryo cells to infection by
POULTRY SCIENCE 51: 602-608,
T
HE elimination of Marek's disease as a health problem of chickens is an agreed goal of everyone concerned with the poultry industry. It is not possible at the present time to design a final blueprint for such an accomplishment, although recent information on the use of vaccines, genetic selection and environmental control techniques offers promising avenues for control. The use of nonpathogenic viruses, either attenuated Marek's disease herpesvirus or a herpesvirus of turkeys, for immunization against Marek's disease has proved to protect chickens effectively under both laboratory and field conditions (see Witter for review, 1971). Similarly, progress following genetic selection for resistance using the JM virus looks very promising (Cole, 1968). Ultimately these approaches possibly combined with environmental control procedures may all play important roles in accomplishing the complete elimination of 1
Contribution from the Massachusetts Experiment Station, Amherst, Massachusetts. 2 Present address: Poultry Breeders Union, Agricultural Cooperative Society, Ltd., 12 Bialik Street, Tel-Aviv, Israel.
1972
the causative agent from poultry populations. It is now well established that a bird's response to either leukosis-sarcoma (Type I) or Marek's disease (Type II) viruses is greatly influenced by genetic factors. A genetically simple mechanism has been shown to involve resistance to the leukosis-sarcoma viruses at the cellular level. Recessive genes at each of two autosomal loci confer resistance to subgroups A and B, respectively, probably by blocking viral penetration (Crittenden et al., 1967; Piraino, 1967). More recently, a similar system of resistance to subgroup C has been suggested by Motta et al. (1969). Genetic influence on the development of Marek's disease following exposure has been demonstrated for the JM (Cole, 1964, 1968; Han et al., 1969; and Han, 1970) and the HPRS-B14 (Payne and Biggs, 1964) isolates. Present evidence indicates that genetic control of resistance to the RIF and JM-type viruses results from separate genetic mechanisms (Payne and Biggs, 1964; Purchase, 1966; and Crittenden and Burmester, 1969). The present study was designed to ob-
602
Downloaded from http://ps.oxfordjournals.org/ at University of Arizona on June 6, 2016
ABSTRACT Genetic differences in response of three genetic stocks and their reciprocal crossbred progeny were studied following inoculation at one day of age with the J M Marek's disease virus. There were marked differences among the pure breeds in respect to the general level of resistance as well as the response of specific tissues. A maternal effect was found to influence the incidence of individuals with one or more positive lesions regardless of location (P < 0.05) as well as specific gonadal lesions (P < 0.01). Differences in general combining ability were significant for the incidence of paralysis and lesions of the DRG (P < 0.01) as well as mortality (P < 0.0S). A significant reciprocal effect (P < 0.05) was found for the incidence of positive lesions regardless of location. Since the analysis had supposedly eliminated the variance associated with maternal effects and general combinability, a sex-linked effect is suggested, although its effect is not clear or consistent in the crossbred data.
GENETIC RESPONSE TO MAREK'S DISEASE
603
ml. Experimental chicks were inoculated at one day of age by injections of 0.25 ml. of the inoculum intraperitoneally. General Management. The injected birds were housed on the floor in one room containing approximately 800 square feet of floor space. Heat was provided by infrared bulbs and wood shavings were used for litMATERIALS AND METHODS ter. A similar uninjected population served Stocks Used. A total of 720 chicks repre- as controls and were reared on a separate senting three pure lines and the six possible farm. All chicks were fed a commercial reciprocal crosses among them were used in chick starter mash fed ad libitum. The incithis study. The pure lines included the dence of paralysis and mortality were reLight Brown Leghorn, Barred Plymouth corded daily. All dead birds from both the Rock and a synthetic recessive white line experimental and control groups were exdesignated as NE-6. The Brown Leghorn amined for the presence of gross leukotic and Barred Rock lines have been resident lesions. At the conclusion of the experiment at the University of Massachusetts Re- all survivors were sacrificed and examined search Farm for 12 and 6 years, respec- for leukotic lesions. Necropsy included an tively, and neither has been previously sub- examination of the viscera and the dorsal jected to artificial selection for response to root ganglia (D.R.G.) from the cervicoMarek's disease. The Brown Leghorn line thoracic region. A diagnosis of Marek's diswas used previously by Han et al. (1969) ease was made when one or more of the and found to be particularly susceptible to nerve ganglia were characteristically envisceral lesions and to be associated with a larged and/or when typical tumors were maternal effect for susceptibility. Prelimi- present in any of the visceral organs. The nary observations indicated the Barred few birds that died during the trial from Rock line to be relatively susceptible, while causes other than Marek's disease and the NE-6 line was relatively resistant to those that lost their bands were excluded the JM virus. The latter was synthesized in from the data. This represented 3.8% of 1960 and had been maintained as a random the original experimental group. breeding line without intentional selection Statistical analysis. The data were anasince that time. lyzed by use of the method of least-squares Inoculum. All genetic resistance trials analysis as outlined by Harvey (1960), utiwere conducted utilizing the JM virus iso- lizing a CDC-3600 computer. The method lated by Sevoian et al. (1962). The JM in- is applicable for computing data with unoculum was prepared from visceral tumors equal subclass numbers. The mathematical and blood taken from previously injected model used was as follows: susceptible Cornell S-line chicks. The cellular suspension was prepared by grinding Y\im = n+Sh+a,i+bijj+g2j(gSk)+jmij the tissues in Ten Broeck grinders, and sus+rijk+sahi+sbhijj+sghaj(sghsk) pending as a 10% concentration in phos+smhSj+srhSjk+ phate-buffered saline (NaCl 8.5 g., Na2 HP0 4 1.07 g., NaH 2 P0 4 -2H 2 0 0.39 g. per where: liter H 2 0, pH 7.1) containing 10,000 units of penicillin and 10 mg. of streptomycin per ykijH= the measurement on the Ith. tain more information on the response of various genetic combinations to exposure to Marek's disease. Reciprocal crossbred progeny were studied in order to obtain information on the inheritance of resistance and susceptibility, as well as the role played by maternal effects.
Downloaded from http://ps.oxfordjournals.org/ at University of Arizona on June 6, 2016
604
G. ZEITLIN, J. R. SMYTH, JR. AND M. SEVOIAN
EXPERIMENTAL RESULTS The first sign of Marek's disease was paralysis on day 21 postinoculation, while the
first mortality occurred during the fourth week of the experiment. All deaths during the first six weeks occurred in birds showing paralysis. Although some of these had gonadal lesions, the other visceral organs were consistently negative during this period. During the last two weeks of the experiment, lesions of the liver, spleen, kidney, lung, proventriculus, as well as the gonads were common. The response of the three pure lines and their crossbred progeny to inoculation at one day of age with JM leukosis virus is shown in Table 1, while the results of the least-squares analysis of variance are presented in Table 2. This study verifies the greater susceptibility of females to Marek's disease previously reported by Payne and Biggs (1964), Purchase and Biggs (1967), Biggs et al. (1968), Han et al. (1969) and others. Although the sex effect was most pronounced for gonadal lesions, it was also apparent for all tissues analyzed with the exception of the liver. Breed differences were highly significant (P < 0.01) for the incidences of mortality, paralysis, positive D.R.G. and those with one or more positive lesions regardless of location (total positive lesions), and significant at the 0.0S probability level for liver lesions. The Barred Rock was the most susceptible for mortality, paralysis, lesions of the D.R.G. and total positive lesions, while the NE-6 line was the most resistant. The Brown Leghorn line had more liver and gonadal lesions, although the latter difference was not statistically significant. The Barred Rocks were extremely susceptible to paralysis. These data provide another example of genetic differences in response of specific tissues to exposure to a viral agent for a neoplastic disease. Differences in the general combining ability of the three breeds were statistically significant (P < 0.01) for the incidences of paralysis and lesions of the D.R.G. mor-
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progeny in the ith type of breeding of the Mh sex of a mating between a male of jth line and a female of the Mh line jU = the overall mean, an effect common to all birds Sh = an effect common to the Mh sex of offspring #,-= an effect common to the ith type of mating, purebred vs. crossbred Jiyy = an effect common to the progeny of a mating between a male of the jth line and a female of the jth line, thus measuring the effects of the breeds of purebreds gijigsk) = the general combining ability effect for the jth (Mh) line m2j = the maternal effect for the j t h line r?jk= the reciprocal effect resulting from the difference between using the jth line as a male and the Mh line as a female, rather than the Mh line as a male and the j t h line as a female sa%i= the interaction between sex and type of mating sbhin= the interaction between sex and breed of purebred sghsjisghsk) = the interaction between sex and general combining ability smusj— the interaction between sex and maternal ability srh31k = the interaction between sex and the reciprocal effects ehijki=& random error assumed to be NID (0, oy*)
605
GENETIC RESPONSE TO MAREK'S DISEASE TABLE 1.—Res pome of purebred and crossbred chickens during an eight week period following inoculation at one day of age with JM virus Matings 1,2
Sex
^T -&• A ulTas
Percent Incidence Positive Lesions Mortality Paralysis '
DRG
Gonads
Liver
Spleen
Total 3
M F Both
45 34 79
22.2 52.9 35.4
33.3 61.8 45.6
57.8 76.5 65.8
31.1 41.2 35.4
0.0 2.9 1.3
4.4 5.9 5.1
71.1 85.3 77.2
NE-6
M F Both
31 47 78
0.0 10.6 6.4
0.0 12.8 • 7.7
12.9 23.4 19.2
32.3 38.3 35.9
6.5 6.4 6.4
9.7 8.5 9.0
35.5 44.7 41.0
B.L.
M F Both
39 35 74
10.3 25.7 17.6
5.1 11.4 8.1
23.1 14.3 18.9
38.5 65.7 51.4
12.8 14.3 13.5
2.6 8.6 5.4
56.4 71.4 63.5
NE-6 X B.R.
M F Both
35 44 79
5.7 29.6 19.0
11.4 27.3 20.3
25.7 31.8 29.1
28.6 38.6 34.2
0.0 6.8 3.8
0.0 11.4 6.3
34.3 50.0 43.0
B.R. X NE-6
M F Both
38 40 78
7.9 32.5 20.5
10.5 32.5 21.8
26.3 60.0 43.6
42.1 52.5 47.4
7.9 7.5 7.7
2.6 7.5 5.1
55.3 72.5 64.1
B.L. X B.R.
M F Both
35 39 74
17.1 46.2 32.4
17.1 25.6 21.6
31.4 48.7 40.5
28.6 51.3 40.5
8.6 18.0 13.5
2.9 12.8 8.1
57.1 69.2 63.5
B.R. X. B.L.
M F Both
34 41 75
17.7 34.2 26.7
14.7 22.0 18.7
44.1 39.0 41.3
55.9 58.5 57.3
14.7 17.1 16.0
8.8 9.8 9.3
70.6 70.7 70.7
B.L. X NE-6
M F Both
40 39 79
5.0 12.8 8.9
2.5 0.0 1.3
12.5 15.4 13.9
45.0 51.3 48.1
7.5 10.3 8.9
7.5 10.3 8.9
55.0 53.9 54.4
NE-6 X B.L.
M F Both
39 38 77
7.7 23.7 15.6
5.1 2.6 3.9
18.0 26.3 22.1
48.7 73.7 61.0
7.7 18.4 13.0
0.0 15.8 7.8
59.0 81.6 70.1
1 2 3
B.R.=Barred Plymouth Rock; NE-6 = synthetic recessive white line; B.L.=Light Brown Leghorn. Male parent listed first. Total positive lesions—includes all birds with one or more positive lesions regardless of location.
tality differences were significant at the 0.05 probability level. The reciprocal crosses in the present experiment showed a higher degree of general combining ability than did those in the similar study by Han et al. (1969). The latter found general combining ability to be significant only for the incidence of gonadal lesions. This difference is probably due to differences in the genetic lines used in the two studies. These data suggest that the relative importance of specific versus general combining ability is a function of the particular genetic combinations with the latter becoming more im-
portant as the difference in resistance levels of the stocks increases. The response of the purebred and crossbred offspring to the JM virus differed only in respect to the incidence of paralysis, the purebreds being more susceptible (P < 0.05). Earlier Hutt and Cole (1952) noted that crossbreeding does not improve the incidence of natural occurring leukosis. In a study similar to the present one, Han et al. (1969) found that crossbreds were significantly more susceptible to paralysis, lesions of the D.R.G. and the incidence of one or more lesions irrespective of location. The
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B.R.
606
G. ZEITLIN, J. R. SMYTH, JR. AND M. SEVOIAN TABLE 2.—Results of analysis of variance Mean Squares Source of Variation
-.,
d.f.
Positive Lesions
. ,?tr" lallty
Paralvsis
Total DRG
Gonads
Liver
Spleen
1
0.001
0.630
0.141
0.685
0.157
0.011
0.000
Breed within Purebreds (B)
2
2.012**
4.118**
6.033**
0.675
0.279*
0.035
2.814**
Sex (S)
1
5.627**
2.153**
1.125*
2.837**
0.173
0.356*
2.168**
General combining Ability (G) 2
0.669*
1.162**
2.325**
0.052
0.181
0.035
0.436
Maternal Effects (M)
2
0.019
0.001
0.243
1.181**
0.059
0.000
0.900
Reciprocal Effects (A)
1
0.291
0.079
0.568
0.115
0.044
0.012
1.159*
2 Factor Interactions (A)X(S) (B)X(S) (S)X(G) (S)X(M) (S)X(R)
1 2 2 2 1
0.002 0.209 0.075 0.047 0.001
0.225 0.246 0.354 0.068 0.171
0.054 0.374 0.112 0.010 0.041
0.910 0.236 0.080 0.109 0.008
0.055 0.004 0.032 0.010 0.042
0.115 0.024 0.069 0.138 0.088
0.011 0.018 0.187 0.028 0.008
675 0.146
0.120
0.197
0.240
0.083
0.067
0.226
Error
* Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.
latter utilized three breeds that were all relatively susceptible. This suggests the possibility that a negative heterotic response to Marek's disease exposure may be most often associated with lines with similar levels of susceptibility and/or with relatively high levels of susceptibility. Maternal effects were found to be significant (P < 0.01) for the incidence of gonadal lesions and also for total positive lesions (P < 0.0S). The incidence of gonadal lesions and those with one or more positive lesions was approximately 17% and 14% greater respectively when the Brown Leghorn was used as the female rather than the male. On the other hand, the incidence of offspring with gonadal lesions and those with one or more positive lesions was approximately 13% and 14% lower respectively when the Barred Rock was used as the female parent. A significant (P < 0.05) difference was found among reciprocal crosses for the inci-
dence of individuals with one or more lesions regardless of location. Since the variance associated with maternal effects and general combining abilities had been eliminated, sex-linked genes may be involved, although such an effect is not readily apparent in these data. The interpretation is confused further as none of the analyses concerned with any of the specific lesions or responses showed such an effect. The control group showed no clinical signs of Marek's disease. Following necropsy at the end of the experimental period, seven birds were found to have enlarged dorsal root ganglia and three had gonadal lesions. This indicates that the incidence of natural occurring Marek's disease was very low in the stocks studied at the time of this investigation. DISCUSSION The limited amount of available data concerning the inheritance of resistance
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Purebreds vs. Crossbreds (A)
GENETIC RESPONSE TO MAREK'S DISEASE
sumably result from a sex-linked effect. Since there is no consistent relationship between sex of offspring and response to the virus in the reciprocal cross progeny, sexlinkage does not appear to explain these data satisfactorily. No maternal effects were associated directly with the NE-6 female. That the relative importance of maternal effects depends on the specific mating is also suggested by the observations of Han et al. (1969) and Cole (1971). ACKNOWLED GMENTS
The authors wish to thank Dr. Richard A. Damon, Jr. for his assistance with the statistical analysis. Further appreciation is expressed to Drs. T. W. Fox and Peter F.S. Han for their advice and counsel during the course of this work. REFERENCES Biggs, P. M., R. J. Thorpe and L. N. Payne, 1968. Studies on genetic resistance to Marek's disease in the domestic chicken. British Poultry Sci. 9 : 37-52. Cole, R. K., 1964. Strain difference in response to the J M leukosis virus. Poultry Sci. 4 3 : 13081309. Cole, R. K., 1968. Studies on genetic resistance to Marek's disease. Avian Diseases, 12: 9-28. Cole, R. K., 1971. The genetic resistance to Marek's disease. Paper presented at Symposium on Oncogenesis and Herpes-type Viruses, Cambridge, England, June, 1971. Abstract: 16. Crittenden, L. B., and B. R. Burmester, 1969. Influence of host genotype on mortality from lymphoid leukosis and Marek's disease after artificial and natural exposure. Poultry Sci. 48: 197-204. 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. Han, F.-S., 1970. The influence of growth rate on the response of chickens to the JM-leukosis virus. Ph.D. Thesis, University of Massachusetts. Han, F.-S., J. R. Smyth, Jr., M. Sevoian and F. N. Dickinson, 1969. Genetic resistance to leukosis caused by the J M virus in the fowl. Poultry Sci. 48: 76-87. Harvey, W. R., 1960. Least-squares analysis of
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and/or susceptibility to Marek's disease suggests the involvement of a polygenic system, although future clarification may reveal the presence of major single genes that are individually identifiable. At the present time there is disagreement on the degree of dominance for resistance, information that is of importance for planning the course of future breed, strain and inbred cross mating programs. Han et al. (1969) studied the offspring of crosses between relatively susceptible breeds and observed certain combinations to be more susceptible than the parent stocks, resulting in an interesting example of negative heterosis. On the other hand, crosses between the extremely resistant Cornell JM-N line and a number of other genetic stocks indicate that resistance can show essentially complete dominance (Cole, 1971). In the present study dominance appears to be incomplete with the Fi progeny showing a degree of susceptibility intermediate to the response of the parent lines. At the present time one can only conclude that the degree of dominance for response to Marek's disease exposure varies with the particular stocks crossed. The significant maternal effect (P < 0.01) associated with the incidence of gonadal lesions and total positive lesions is of particular interest. Similar evidence for a maternal effect associated with susceptibility to the JM virus was demonstrated previously for this Brown Leghorn strain by Han et al. (1969). In contrast, Cole (1971) did not observe maternal effects to significantly influence the response to Marek's disease exposure of progeny from crosses between his selected resistant (JMN) and more susceptible lines. The present study also suggests that the Barred Rock females contributed a maternal factor for resistance to their offspring. This hypothesis fits the data better than one involving a specific sire contribution, that would pre-
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G. ZEITLIN, J. R. SMYTH, JR. AND M. SEVOIAN Rous sarcoma virus-Bryan strain (BS-RSV). Virology, 32: 700-707. Purchase, H. G., 1966. The epizootiology of the avian leukosis complex. Proc. International Wenner-Gren Symposium. Comparative Leukamias, Stockholm, Pergamon Press 37. Purchase, H. G., and P. M. Biggs, 1967. Characterization of five isolates of Marek's disease. Res. Vet. Sci. 8 : 440-449. Sevoian, M., D. M. Chamberlain and F. T. Counter, 1962. Avian lymphomatosis. 1. Experimental reproduction of the neural and visceral forms. Vet. Med. 57 : 500-501. Witter, R. L., 1971. Marek's disease research-history and perspectives. Poultry Sci. 50: 333342.
Sulfate Metabolism and Taurine Synthesis in the Chick1 W I L L I A M G. M A R T I N 2
Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, West Virginia 26506 (Received for publication August 9, 1971) ABSTRACT Sulfate has been suggested as an essential nutrient in the diet of chickens. One-day-old cockerels were fed a purified diet supplemented with varying levels of methionine and at each methionine level four levels of sulfate were added. In a second experiment methionine, sulfate and taurine were supplemented to the diet. A significant growth response was observed with supplemental methionine and sulfate. A portion of the dietary sulfate was used in the synthesis of phosphoadenosinephosphosulfate (PAPS) which is required for the synthesis of taurine by this pathway. Methionine was shown to enhance the synthesis of taurine in the liver while supplemental sulfate enhanced the activity of the sulfate activating enzymes and the in vitro synthesis of PAP3:;S. The data of these experiments support the theory that in the chick at least a part of the growth response obtained by sulfate supplementation is due to the reactions whereby activated sulfate is converted to taurine. POULTRY SCIENCE 51: 608-612, 1972
C
YSTEINE and methionine constitute about 90 percent of the total sulfur of most plants (Allaway and Thompson, 1966), thus the availability of sulfate-sulfur to poultry fed a natural feedstuffs diet is limited. Further, when corn-soybean meal diets are fed to poultry, DL-methio-
'This manuscript is published with the approval of the Director of the West Virginia University Agricultural Experiment Station, Morgantown, as Scientific Paper No. 1185. 2 Associate Agricultural Biochemist.
nine is usually added to increase the sulfur amino acid concentration to about 0.8 percent, or 3.5 percent of dietary protein (Nelson et al., 1960). Inorganic sulfur per se has been ascribed a role in the metabolism of the chicken (Machlin, 1955). Gordon and Sizer (1955) shared this view and Miraglia et al. (1966) observed a growth response in chicks to sulfate added to a purified diet adequate in methionine. Recently Ross and Harms (1970) have substantiated the earlier reports of the benefi-
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data with unequal subclass numbers. A.R.S. 208, U.S.D.A. 1-156. Hutt, F. B., and R. K. Cole, 1952. Heterosis in an interstrain cross of White Leghorns. Poultry Sci. 3 1 : 365-374. Motta, J. V., L. B. Crittenden and E. F. Godfrey, 1969. The inheritance of resistance to subgroup C Ieukosis-sarcoma viruses in New Hampshires. Proc. Assn. Agric. Workers, Inc., Memphis, Tennessee, Feb. 1-4, p. 189. Payne, L. N., and P. M. Biggs, 1964. Transmission experiments with Marek's disease (fowl paralysis) and lymphoid leukosis. World's Poultry Sci. J. 20: 284-297. Piraino, F., 1967. The mechanisms of genetic resistance of chick embryo cells to infection by