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Variation in the Presence of the Virus of Visceral Lymphomatosis in the Eggs of the Same Hens
Variation in the Presence of the Virus of Visceral Lymphomatosis in the Eggs of the Same Hens B . R . BURMESTER AND NELSON F . WATERS United States Department of Agriculture, Agricultural Research Service, Regional Poultry Research Laboratory, East Lansing, Michigan (Received for publication March 28, 1956)
P
939
1955). Similar tests of eggs laid by the same hens at different times over a 2-year period, and tests of eggs laid over a V/*year period by a part of the progeny of these hens, have been conducted. The results of these tests will be summarized in this paper. MATERIALS AND METHODS
The procedure used for determining the deposition of virus in the egg was the same for all tests, and similar to that employed in the initial test, previously described (Burmester et al., 1955). The recipient chicks for all tests were from the same inbred line 15 used in previous tests. The inoculated chicks and non-inoculated controls were maintained in isolated cubicles and in pens, according to the procedure previously described (Burmester and Gentry, 1954a). The initial tests on the infectivity of embryonating eggs of the original 22 dams were made on eggs laid during March of 1953. Subsequent tests, on eggs laid by a part of the same group of hens, were made on eggs laid during June 1953, September 1953, April 1954, and January 1955. Similar tests were made on eggs laid by the progeny of a part of the original 22 dams. The first of these was made on eggs laid during September and October 1953. Subsequent tests on this group of hens were made on eggs laid during March 1954, and January 1955. Choice of hens after the first test of each dam or progeny series, was determined by the number of fertile eggs laid in the 2 weeks' period just
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REVIOUS studies have demonstrated the presence of the causative agent of visceral lymphomatosis in the eggs of certain hens. This was done by incubating eggs to the fifteenth day, and preparing an inoculum from the pooled livers of 3 to 6 embroys of each hen. This inoculum was tested by intraperitoneal injection into susceptible day-old chicks. Deaths to 270 days of age were almost entirely due to visceral lymphomatosis (Cottral et al., 1954; and Burmester et al., 1955). Although Burmester and co-workers (1955) found that most hens of a susceptible and infected flock deposited a significant amount of virus in their eggs, further work (Burmester and Waters, 1955) showed that such virus did not appear to be responsible for much, if any, lymphomatosis in the progeny of such dams. They found no significant difference with respect to the incidence of lymphomaflfcis between families of dams that under test were known to shed the virus, and families of dams that did not shed detectable virus. However, Burmester and Waters (1955), showed that maternally infected chicks may well be the source of infection that may spread in the hatcher or brooder units to chicks of non-infected parents, resulting in visceral lymphomatosis. Families reared together and families reared in isolation showed similar responses. Results of initial tests on the infectivity of the embryonating eggs of 22 dams, distributed among inbred lines 6, 9, and 15 of the Regional Poultry Research Laboratory, have been reported (Burmester et al.,
940
B . R . BUEMESTEE AND N . F . WATEES
RESULTS AND DISCUSSION The results of the infectivity tests of 15day embryos of eggs laid at different periods by the various dams are summarized in Table 1. Similar data on embryos of eggs laid by daughters of a part of these dams are presented in Table 2. The principal response, resulting from inoculation, was death with tumors typical of visceral lymphomatosis. The incidence
of such cases within each lot that occurred within a 270-day period is given in the tables, as percentage of the corrrected number per lot. Data on test number 1, Table 1, were reported previously (Burmester et al., 1955), and are given here only for ease of comparison with results on embryonating eggs laid by the same chickens during later periods. The occurrence of other forms of lymphomatosis was insignificant. In all tests (Tables 1 and 2,) except number 1 of Table 1, the average incidence of neurolymphomatosis was less than 0.5 percent, and no cases of ocular lymphomatosis or of osteopetrosis occurred. The second test was made on 11 of the original 22 dams. The eggs were laid during June, about 3 months after the eggs for the first test had been laid. The percentage of visceral lymphomatosis resulting from inoculation of the embryo liver, was much greater in the second test of 3 dams (C, I, V) and lower in 2 dams (F, J) in comparison to the result of the first test. On the basis of previous interpreta-
TABLE 1.—Occurrence of visceral lymphomatosis {VL) in groups of chickens inoculated with embryonic liver of eggs laid at different periods T e s t ]n u m b e r ^ K D a t e eggs laid: Age of he:ns:
1
2
3
4
5
3/10-3/31/53 335-425 days
6/24-7/1/53 441-517 days
9/10-10/1/53 519-609 days
4/8-4/15/54 729-805 days
1/20-1/27/55 1,016-1,092 days
%
%
Inbred line
Dam tested
Number*
6
B C D E
40.7 41.9 44.7 38.2
44.2 2.4 0.0 5.2
46.9 44.8
F G H I
63.5 25.3 35.7 29.3 60.2 49.4 16.5 59.5 56.5
44.9 43.2 46.0 41.5 40.7
2.2 23.1 43.5 55.4 22.1
36.2
M N
42.5 43.4 36.4 44.3 38.2 46.6 42.4 35.3 46.0
46.3
0 Q U V
42.6 39.1 36.0 41.2
63.4 40.9 36.1 12.2
38.7
0.0
9
J
K
IS
Controls
VLt
%
VL
16.6 7.8
40.7 38.0 33.7 46.0 43.7
7.4 7.9 8.9 10.7 9.1
39.0
2.6
43.0
4.7
80.5
16.0
84.0
6.0
41.0
4.8
38.0
5.2
39.8 44.5
5.0 0.0
47.0
41.0
7.3
40.5
46.9
78.5 38.7
58.3
44.7
73.8
42.7 44.3 40.6
53.9 45.1 36.9
34.9
47.7
42.5
0.0
41.6
7.2
VL
49.0 20.0
%
Number
VL
VL
Number
%
Number
Number
40.2
0.0
40.0
0.0
* Adjusted ::or the number that died of other causes. f The proportion that died of other forms of lymphomatosis was less than J of 1 percent in all except thefirstdam test.
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prior to the incubation of the eggs needed for the preparation of the inoculum. Since a minimum of 4 embryonating eggs was used to prepare each inoculum, only those hens that had 4 or more eggs available at the time of a particular test were included. A total of 4,420 chicks were either inoculated or used as controls for the infectivity tests reported here. A few of these chicks died of diseases or conditions other than the various forms of lymphomatosis. Such cases were subtracted from the number assigned to each lot in a manner described elsewhere (Burmester and Gentry, 1954b).
VISCERAL LYMPHOMATOSIS VIRUS IN EGGS TABLE 2.—Occurrence of visceral lymphomatosis (VL)
in groups of chickens inoculated with embryonic liver of eggs laid at different intervals by the progeny of the dams in Table 1 Test number Bate eggs laid: Age of hen:
1
2
3
9/10-10/15/53 3/10-3/17/54 1/17-1/24/55 211-287 392-440 705-753
Identification ber*
VLt
ber
VL
Fl F2
41.2 42.6
60.7 54.0
44.1 49.0
22.6 0.0
J
Jl J2
34.4 30.2
72.6 69.5
45.7 46.0
6.6 13.0
K
Kl K2
41.4 41.8
72.4 50.3
46.8
12.8
Dam
Progeny
F
Ml
34.1
64.5
40.5
4.9
Nl N2
3t.9 33.4
47.0 65.8
46.6 43.0
19.3 11.6
0
Ol 02
38.2 37.0
18.3 5.4
49.6
2.0
Q
Ql Q2
34.9 39.4
2.9 7.6
46.5
2.2
38.7
2.6
48.0
0.0
Controls
* Adjusted for the number that died of other causes, t The proportion that died of other forms of lymphomatosis was less than \ of 1 percent.
tions (Burmester et al., 1955) the second test results indicate a definite shift in the shedding characteristic of 2 of the dams— dam C from non-shedder to shedder, and dam F the reverse. The third test was made on eggs laid during September, or about 3 months after eggs of the second test were laid. Only four of the hens were laying at this time, thus data are available for only a small part of the original 22 dams. The embryo liver inoculum of these 4 dams produced a high incidence of visceral hymphomatosis in the recipient chicks. These dams had been tested twice previously. Three of them (dams J, N, and Q) gave evidence of consistent significant shedding for the 3 tests on eggs laid in March, June, and September. The fourth dam (F) showed a lack of significant shedding in eggs laid in June; whereas, inoculums of eggs laid in March and September produced high rates of transmission.
The fourth test was made on eggs from 10 of the original 22 dams, laid about one year after the first test, which was during April of the second laying year. Inoculums from embryonating eggs of dams I, N, and O, were tested in replicate lots. The percentages of visceral lymphomatosis obtained for dam I, were 15.7 and 17.4; for dam N, 14.4 and 18.6; and for dam O, 2.4 and 9.5. The percentage transmission obtained, ranged from a low of 0.0, for eggs of dams E and V, to a high of 16.0 and 16.6, for dams I and N. Five dams were in the intermediate range of 5.0 to 7.5 percent. Although no cases of visceral lymphomatosis were obtained in the control lot, and in two inoculated lots, transmission obtained with inoculums of only dams I and N can be considered significant.1 Although positive evidence of shedding of the virus into the eggs was obtained for 2 of the hens, the transmission data indicate a comparatively low rate of shedding. Furthermore, such evidence was obtained in only 2 of 8 hens tested, all of which had showed evidence of a high shedding rate 7 to 13 months previously. Another test was run on 6 of the hens when they were 3 years old. The results were similar to those obtained on eggs laid when the hens were 2 years of age (fourth test), except that none could be interpreted to indicate that significant virus was shed. Similar experiments were conducted on 2 daughters (with one exception) of each of 7 dams, for the purpose of obtaining some information on the relation between the shedding of virus by the dams and by their offspring. The 7 dams, F, J, K, M, N, O, and Q, were selected on the basis of high transmission rate induced by the embryo liver inoculum. Daughters were selected at random from among about 10 per dam ' Method of Brandt and Snedecor, see "Statistical Methods" p. 206, G. W. Snedecor, 4th Ed. Iowa State College Press, Ames.
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M N
941
942
B . R. BUEMESTER AND N . F . WATERS
Although the number of individuals is too small to estimate probability, it would appear that hens having a latent infection, usually, but may not always, produce offspring with a similar latent infection as measured by the shedding of virus into their eggs. The absence of such an infection, in some of the progeny, may be due to several possible factors; among them are the cessation of shedding which may be
permanent or temporary, as in dam F, or the abortion of infection in the offspring even though the virus was present in the egg. The second test on the progeny was conducted on eggs laid during March of the first laying year. The transmission rates, generally, were low. The embryoliver inoculum of only progeny Fl and Nl produced significant (see footnote 1) transmission, and that of 3 others (J2, Kl, and N2) produced transmission of doubtful significance. When the progeny were 2 years old, eggs from S of them were again tested. The results were similar to those of the second progeny test, in that transmission rates were low, with questionable significance in 3 cases. The procedure of breeding only from 2-year-old birds, for obtaining replacement laying stock, has often appeared in recommendations for the control of lymphomatosis. Recently, Carr (1952) emphasized the desirability of this practice. One of the purposes of this study was to obtain direct data on the comparative rates of shedding of the virus of visceral lymphomatosis into eggs laid by hens at progressive ages. Data were obtained on eggs laid at 5 different periods during 2 years by hens of 3 inbred lines, and on eggs laid at 3 different periods during V/2 years by the progeny of some of the hens employed in the first series. The data on the dams (Table 1) show that transmission indicative of general, high shedding rates was obtained for eggs of the first three tests, that is, for eggs laid when the hens were about 12, 15, and 18 months of age; whereas, low or insignificant transmission was obtained with eggs laid when the hens were about 2, and also when about 3 years of age. Thus,* these data indicate a distinct drop in the shedding of this virus by these hens during the age period of 18 to 24 months. The data on the progeny of line 9 (Table 2) also
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family. These progeny were hatched in January 1953 and eggs for the first test, indicating a shedding of the virus by the dams were laid during March of the same year. Data on the shedding of the virus into eggs by the progeny are presented in Table 2. The first test on the progeny was made on eggs laid when they were only 8-10 months of age. High rates of transmission of visceral lymphomatosis were obtained with embryo liver inoculum prepared from all 9 of the line 9 progeny tested, and one (01) of the 4, line IS progeny. Dams, F, J, N, and Q, had been tested by analysis of their eggs laid during the same month (September) in which their daughters laid the eggs for the first test, and also of their eggs laid 3 and 6 months previously. Similar tests on the remaining 3 dams (K, M, and O) were made on eggs laid 6 months previously. In all these tests on the dams, with one exception (dam F, test No. 2), high transmission rates indicative of much shedding of virus were obtained. Since similar results were obtained with all of the line 9 progeny tested, there is some indication, at least within the line, of a correlation between shedding by the dams and shedding by their daughters. Such a positive relation was not obtained with the 2 families of line IS. Although both dams indicated high rates of shedding, in one case, as recent as the same month in which the daughters' test eggs were laid, only one of 2 daughters of one of the dams gave indication of significant shedding of the virus.
VISCERAL LYMPHOMATOSIS VIRUS IN EGGS
Waters (1954a, b) has reported a marked reduction in the incidence of lymphomatosis (neural and visceral) occurring under conditions of natural exposure among all the inbred lines of the Laboratory. The averages for all hens of all 6 inbred lines hatched during 1950, 1951, 1952, 1953, and 1954, were 24.6, 6.8, 18.4, 9.3,* and
5.5,* percent respectively. It is apparent that there was a decided drop between the population hatches in 1950 with some recovery in 1952 and again a drop in 1953 and another in 19S4. Thus the first major reduction in lymphomatosis mortality among the breeding population occurred 3 years prior to the apparent decrease in the shedding of the virus in eggs laid by the chickens of this report. If these two factors were associated one would expect a more nearly coincident time relation. Another possible explanation for the apparent reduction in shedding is a possible shift in susceptibility of the test chickens. Estimation of the virus shed into eggs is an indirect one, depending on the response of a group of test animals to inoculation with liver preparations of embryonating eggs. The response, therefore, is the result of the amount of virus injected, and the susceptibility of the host. The chicks employed for all inoculations were obtained from the same source and varied from year to year only in generation. They were of inbred line 15, which has been selected for susceptibility to lymphomatosis during the past fifteen years, and the inbreeding coefficient is now about 75.0 percent. A part of the population of this line, which supplied the chicks for these experiments, has been maintained in strict isolation generation after generation since 1942, for the purpose of reducing naturally occurring lymphomatosis. This was generally effective (Waters and Prickett, 1944) and data for recent years indicate no material change. In fact, an average of only 1.71 percent visceral lymphomatosis and 0.73 percent neural lymphomatosis occurred in populations hatched during 1951 to 1954, inclusive. In view of the constancy of test chicks with respect to breeding, management practices, and amount of natural infection, it * Unpublished data.
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show a distinct drop in general level of virus shed; however, with this group the drop occurred between the first and second tests, which were on eggs laid at about 9 and IS months of age. Thus, shedding of the virus appeared to decrease with the age of the hens. In the first series it occurred after 18 months of age, and in the progeny series after only 9 months of age. This is a rather important variation inasmuch as the first occurred after the first laying year and before the second; whereas, in the progeny series, the decrease occurred shortly after the birds were one year of age. Further examination of the data shows that the first generally low transmission results, in both the dam and the progeny series, occurred with eggs laid at about the same period, i.e., March 10 to April IS, 1954. It may be suggested, therefore, that the general, apparent decrease, in shedding observed, may not be so much related to age of the hens laying the eggs as to other factors generally affecting latent infections with the virus. The dams were maintained in one pen of a mating house beginning with the laying of eggs for the second test. The progeny were in several other pens of the same mating house, during the periods of the first and second tests, and were placed in the pen with the dams shortly thereafter. Thus, during the period of apparent reduction in shedding, the two groups of hens were in the same house, but in different pens. Therefore, the general level of infection in the mating house may have decreased during the same period, which may account for the change in the amount of virus shed.
943
944
B. R. BURMESTER AND N. F. WATERS
SUMMARY
The deposition of the virus of visceral lymphomatosis in the eggs of 17 normal appearing hens was determined at specific intervals over a period of 2 years, and similar determinations were made on 13 progeny over a 1J4 year period. Detection of the virus was based on reproducing the disease in susceptible chickens inoculated with a preparation of a pool of livers from four 15-day embryos of each hen to be tested. All of 9 daughters from 5 inbred line 9 hens tested, shed as much or more virus in their eggs when 9 months of age as did their mothers at 12 months of age. However, only 1 of 4 daughters of two line-15 hens gave indication of significant shedding of virus into their eggs, even though both of the dams had shed the virus. Eggs laid by the hens when 12, 15, and
18 months of age gave transmission indicative of a fairly consistent, high level of shedding of the virus. Eggs laid by the same hens when 2 and 3 years of age, gave transmissions indicative of a generally low level of deposition of virus into the eggs. The progeny showed a high level of shedding at 9 months of age, but tests on eggs laid at 15 months and 2 years of age indicated a low level of shedding. Results obtained indicated a reduction in the shedding of the virus of visceral lymphomatosis into eggs as hens became older. However, such reduction may not occur at a uniform age, and may be influenced by other, yet unknown, factors. REFERENCES Burmester, B. R., and R. F. Gentry, 1954a. The presence of the virus causing visceral lymphomatosis in the secretions and excretions of chickens. Poultry Sci. 3 3 : 836-842. Burmester, B. R., and R. F. Gentry, 1954b. A study of possible avenues of infection with the virus of avian visceral lymphomatosis. Proc. 91st Annual Meeting Am. Vet. Med. Assoc: 311-316. Burmester, B. R., R. F. Gentry and N. F. Waters, 1955. The presence of the virus of visceral lymphomatosis in embryonated eggs of normal appearing hens. Poultry Sci. 34: 609-617. Burmester, B. R., and N. F. Waters, 1955. The role of the infected egg in the transmission of visceral lymphomatosis. Poultry Sci. 34: 14151429. Carr, J. G., 1952. A survey of the leucosis complex. Modern Poultry Keeping, Oct. 8, 1952: 360-361. Cottral, G. E., B. R. Burmester and N. F. Waters, 1954. Egg transmission of avian lymphomatosis. Poultry Sci. 33 : 1174-1184. Waters, N. F., and C. O. Prickett, 1944. The development of families of chickens free of lymphomatosis. Poultry Sci. 23: 321-333. Waters, N. F., 1954a. Etiological relationship of visceral and neural lymphomatosis. Poultry Sci. 33:365-373. Waters, N. F., 1954b. Avian lymphomatosis mortality among inbred line crosses. Proc. 10th World's Poultry Congress, Article 61.
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seems unlikely that the test chicks were a source of significant variation during the period in question. With few exceptions, the 15 dams and 10 progeny which were tested more than once, and which were involved in a total of 73 infectivity tests, showed a general consistency in the shedding of the virus of visceral lymphomatosis into their eggs. In each generation there appeared to be a generally high level of shedding which was followed by a much lower level. Age may have been a factor in this marked decrease; however, it does not appear to be of overriding importance, since the decrease occurred at distinctly different ages in the two generations tested. Moreover, the decrease appeared to have taken place in both generations at about the same time, indicating that a possible common environmental factor or factors were involved. Possible identity of the latter was not detected.
P
939
1955). Similar tests of eggs laid by the same hens at different times over a 2-year period, and tests of eggs laid over a V/*year period by a part of the progeny of these hens, have been conducted. The results of these tests will be summarized in this paper. MATERIALS AND METHODS
The procedure used for determining the deposition of virus in the egg was the same for all tests, and similar to that employed in the initial test, previously described (Burmester et al., 1955). The recipient chicks for all tests were from the same inbred line 15 used in previous tests. The inoculated chicks and non-inoculated controls were maintained in isolated cubicles and in pens, according to the procedure previously described (Burmester and Gentry, 1954a). The initial tests on the infectivity of embryonating eggs of the original 22 dams were made on eggs laid during March of 1953. Subsequent tests, on eggs laid by a part of the same group of hens, were made on eggs laid during June 1953, September 1953, April 1954, and January 1955. Similar tests were made on eggs laid by the progeny of a part of the original 22 dams. The first of these was made on eggs laid during September and October 1953. Subsequent tests on this group of hens were made on eggs laid during March 1954, and January 1955. Choice of hens after the first test of each dam or progeny series, was determined by the number of fertile eggs laid in the 2 weeks' period just
Downloaded from http://ps.oxfordjournals.org/ by guest on April 8, 2015
REVIOUS studies have demonstrated the presence of the causative agent of visceral lymphomatosis in the eggs of certain hens. This was done by incubating eggs to the fifteenth day, and preparing an inoculum from the pooled livers of 3 to 6 embroys of each hen. This inoculum was tested by intraperitoneal injection into susceptible day-old chicks. Deaths to 270 days of age were almost entirely due to visceral lymphomatosis (Cottral et al., 1954; and Burmester et al., 1955). Although Burmester and co-workers (1955) found that most hens of a susceptible and infected flock deposited a significant amount of virus in their eggs, further work (Burmester and Waters, 1955) showed that such virus did not appear to be responsible for much, if any, lymphomatosis in the progeny of such dams. They found no significant difference with respect to the incidence of lymphomaflfcis between families of dams that under test were known to shed the virus, and families of dams that did not shed detectable virus. However, Burmester and Waters (1955), showed that maternally infected chicks may well be the source of infection that may spread in the hatcher or brooder units to chicks of non-infected parents, resulting in visceral lymphomatosis. Families reared together and families reared in isolation showed similar responses. Results of initial tests on the infectivity of the embryonating eggs of 22 dams, distributed among inbred lines 6, 9, and 15 of the Regional Poultry Research Laboratory, have been reported (Burmester et al.,
940
B . R . BUEMESTEE AND N . F . WATEES
RESULTS AND DISCUSSION The results of the infectivity tests of 15day embryos of eggs laid at different periods by the various dams are summarized in Table 1. Similar data on embryos of eggs laid by daughters of a part of these dams are presented in Table 2. The principal response, resulting from inoculation, was death with tumors typical of visceral lymphomatosis. The incidence
of such cases within each lot that occurred within a 270-day period is given in the tables, as percentage of the corrrected number per lot. Data on test number 1, Table 1, were reported previously (Burmester et al., 1955), and are given here only for ease of comparison with results on embryonating eggs laid by the same chickens during later periods. The occurrence of other forms of lymphomatosis was insignificant. In all tests (Tables 1 and 2,) except number 1 of Table 1, the average incidence of neurolymphomatosis was less than 0.5 percent, and no cases of ocular lymphomatosis or of osteopetrosis occurred. The second test was made on 11 of the original 22 dams. The eggs were laid during June, about 3 months after the eggs for the first test had been laid. The percentage of visceral lymphomatosis resulting from inoculation of the embryo liver, was much greater in the second test of 3 dams (C, I, V) and lower in 2 dams (F, J) in comparison to the result of the first test. On the basis of previous interpreta-
TABLE 1.—Occurrence of visceral lymphomatosis {VL) in groups of chickens inoculated with embryonic liver of eggs laid at different periods T e s t ]n u m b e r ^ K D a t e eggs laid: Age of he:ns:
1
2
3
4
5
3/10-3/31/53 335-425 days
6/24-7/1/53 441-517 days
9/10-10/1/53 519-609 days
4/8-4/15/54 729-805 days
1/20-1/27/55 1,016-1,092 days
%
%
Inbred line
Dam tested
Number*
6
B C D E
40.7 41.9 44.7 38.2
44.2 2.4 0.0 5.2
46.9 44.8
F G H I
63.5 25.3 35.7 29.3 60.2 49.4 16.5 59.5 56.5
44.9 43.2 46.0 41.5 40.7
2.2 23.1 43.5 55.4 22.1
36.2
M N
42.5 43.4 36.4 44.3 38.2 46.6 42.4 35.3 46.0
46.3
0 Q U V
42.6 39.1 36.0 41.2
63.4 40.9 36.1 12.2
38.7
0.0
9
J
K
IS
Controls
VLt
%
VL
16.6 7.8
40.7 38.0 33.7 46.0 43.7
7.4 7.9 8.9 10.7 9.1
39.0
2.6
43.0
4.7
80.5
16.0
84.0
6.0
41.0
4.8
38.0
5.2
39.8 44.5
5.0 0.0
47.0
41.0
7.3
40.5
46.9
78.5 38.7
58.3
44.7
73.8
42.7 44.3 40.6
53.9 45.1 36.9
34.9
47.7
42.5
0.0
41.6
7.2
VL
49.0 20.0
%
Number
VL
VL
Number
%
Number
Number
40.2
0.0
40.0
0.0
* Adjusted ::or the number that died of other causes. f The proportion that died of other forms of lymphomatosis was less than J of 1 percent in all except thefirstdam test.
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prior to the incubation of the eggs needed for the preparation of the inoculum. Since a minimum of 4 embryonating eggs was used to prepare each inoculum, only those hens that had 4 or more eggs available at the time of a particular test were included. A total of 4,420 chicks were either inoculated or used as controls for the infectivity tests reported here. A few of these chicks died of diseases or conditions other than the various forms of lymphomatosis. Such cases were subtracted from the number assigned to each lot in a manner described elsewhere (Burmester and Gentry, 1954b).
VISCERAL LYMPHOMATOSIS VIRUS IN EGGS TABLE 2.—Occurrence of visceral lymphomatosis (VL)
in groups of chickens inoculated with embryonic liver of eggs laid at different intervals by the progeny of the dams in Table 1 Test number Bate eggs laid: Age of hen:
1
2
3
9/10-10/15/53 3/10-3/17/54 1/17-1/24/55 211-287 392-440 705-753
Identification ber*
VLt
ber
VL
Fl F2
41.2 42.6
60.7 54.0
44.1 49.0
22.6 0.0
J
Jl J2
34.4 30.2
72.6 69.5
45.7 46.0
6.6 13.0
K
Kl K2
41.4 41.8
72.4 50.3
46.8
12.8
Dam
Progeny
F
Ml
34.1
64.5
40.5
4.9
Nl N2
3t.9 33.4
47.0 65.8
46.6 43.0
19.3 11.6
0
Ol 02
38.2 37.0
18.3 5.4
49.6
2.0
Q
Ql Q2
34.9 39.4
2.9 7.6
46.5
2.2
38.7
2.6
48.0
0.0
Controls
* Adjusted for the number that died of other causes, t The proportion that died of other forms of lymphomatosis was less than \ of 1 percent.
tions (Burmester et al., 1955) the second test results indicate a definite shift in the shedding characteristic of 2 of the dams— dam C from non-shedder to shedder, and dam F the reverse. The third test was made on eggs laid during September, or about 3 months after eggs of the second test were laid. Only four of the hens were laying at this time, thus data are available for only a small part of the original 22 dams. The embryo liver inoculum of these 4 dams produced a high incidence of visceral hymphomatosis in the recipient chicks. These dams had been tested twice previously. Three of them (dams J, N, and Q) gave evidence of consistent significant shedding for the 3 tests on eggs laid in March, June, and September. The fourth dam (F) showed a lack of significant shedding in eggs laid in June; whereas, inoculums of eggs laid in March and September produced high rates of transmission.
The fourth test was made on eggs from 10 of the original 22 dams, laid about one year after the first test, which was during April of the second laying year. Inoculums from embryonating eggs of dams I, N, and O, were tested in replicate lots. The percentages of visceral lymphomatosis obtained for dam I, were 15.7 and 17.4; for dam N, 14.4 and 18.6; and for dam O, 2.4 and 9.5. The percentage transmission obtained, ranged from a low of 0.0, for eggs of dams E and V, to a high of 16.0 and 16.6, for dams I and N. Five dams were in the intermediate range of 5.0 to 7.5 percent. Although no cases of visceral lymphomatosis were obtained in the control lot, and in two inoculated lots, transmission obtained with inoculums of only dams I and N can be considered significant.1 Although positive evidence of shedding of the virus into the eggs was obtained for 2 of the hens, the transmission data indicate a comparatively low rate of shedding. Furthermore, such evidence was obtained in only 2 of 8 hens tested, all of which had showed evidence of a high shedding rate 7 to 13 months previously. Another test was run on 6 of the hens when they were 3 years old. The results were similar to those obtained on eggs laid when the hens were 2 years of age (fourth test), except that none could be interpreted to indicate that significant virus was shed. Similar experiments were conducted on 2 daughters (with one exception) of each of 7 dams, for the purpose of obtaining some information on the relation between the shedding of virus by the dams and by their offspring. The 7 dams, F, J, K, M, N, O, and Q, were selected on the basis of high transmission rate induced by the embryo liver inoculum. Daughters were selected at random from among about 10 per dam ' Method of Brandt and Snedecor, see "Statistical Methods" p. 206, G. W. Snedecor, 4th Ed. Iowa State College Press, Ames.
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M N
941
942
B . R. BUEMESTER AND N . F . WATERS
Although the number of individuals is too small to estimate probability, it would appear that hens having a latent infection, usually, but may not always, produce offspring with a similar latent infection as measured by the shedding of virus into their eggs. The absence of such an infection, in some of the progeny, may be due to several possible factors; among them are the cessation of shedding which may be
permanent or temporary, as in dam F, or the abortion of infection in the offspring even though the virus was present in the egg. The second test on the progeny was conducted on eggs laid during March of the first laying year. The transmission rates, generally, were low. The embryoliver inoculum of only progeny Fl and Nl produced significant (see footnote 1) transmission, and that of 3 others (J2, Kl, and N2) produced transmission of doubtful significance. When the progeny were 2 years old, eggs from S of them were again tested. The results were similar to those of the second progeny test, in that transmission rates were low, with questionable significance in 3 cases. The procedure of breeding only from 2-year-old birds, for obtaining replacement laying stock, has often appeared in recommendations for the control of lymphomatosis. Recently, Carr (1952) emphasized the desirability of this practice. One of the purposes of this study was to obtain direct data on the comparative rates of shedding of the virus of visceral lymphomatosis into eggs laid by hens at progressive ages. Data were obtained on eggs laid at 5 different periods during 2 years by hens of 3 inbred lines, and on eggs laid at 3 different periods during V/2 years by the progeny of some of the hens employed in the first series. The data on the dams (Table 1) show that transmission indicative of general, high shedding rates was obtained for eggs of the first three tests, that is, for eggs laid when the hens were about 12, 15, and 18 months of age; whereas, low or insignificant transmission was obtained with eggs laid when the hens were about 2, and also when about 3 years of age. Thus,* these data indicate a distinct drop in the shedding of this virus by these hens during the age period of 18 to 24 months. The data on the progeny of line 9 (Table 2) also
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family. These progeny were hatched in January 1953 and eggs for the first test, indicating a shedding of the virus by the dams were laid during March of the same year. Data on the shedding of the virus into eggs by the progeny are presented in Table 2. The first test on the progeny was made on eggs laid when they were only 8-10 months of age. High rates of transmission of visceral lymphomatosis were obtained with embryo liver inoculum prepared from all 9 of the line 9 progeny tested, and one (01) of the 4, line IS progeny. Dams, F, J, N, and Q, had been tested by analysis of their eggs laid during the same month (September) in which their daughters laid the eggs for the first test, and also of their eggs laid 3 and 6 months previously. Similar tests on the remaining 3 dams (K, M, and O) were made on eggs laid 6 months previously. In all these tests on the dams, with one exception (dam F, test No. 2), high transmission rates indicative of much shedding of virus were obtained. Since similar results were obtained with all of the line 9 progeny tested, there is some indication, at least within the line, of a correlation between shedding by the dams and shedding by their daughters. Such a positive relation was not obtained with the 2 families of line IS. Although both dams indicated high rates of shedding, in one case, as recent as the same month in which the daughters' test eggs were laid, only one of 2 daughters of one of the dams gave indication of significant shedding of the virus.
VISCERAL LYMPHOMATOSIS VIRUS IN EGGS
Waters (1954a, b) has reported a marked reduction in the incidence of lymphomatosis (neural and visceral) occurring under conditions of natural exposure among all the inbred lines of the Laboratory. The averages for all hens of all 6 inbred lines hatched during 1950, 1951, 1952, 1953, and 1954, were 24.6, 6.8, 18.4, 9.3,* and
5.5,* percent respectively. It is apparent that there was a decided drop between the population hatches in 1950 with some recovery in 1952 and again a drop in 1953 and another in 19S4. Thus the first major reduction in lymphomatosis mortality among the breeding population occurred 3 years prior to the apparent decrease in the shedding of the virus in eggs laid by the chickens of this report. If these two factors were associated one would expect a more nearly coincident time relation. Another possible explanation for the apparent reduction in shedding is a possible shift in susceptibility of the test chickens. Estimation of the virus shed into eggs is an indirect one, depending on the response of a group of test animals to inoculation with liver preparations of embryonating eggs. The response, therefore, is the result of the amount of virus injected, and the susceptibility of the host. The chicks employed for all inoculations were obtained from the same source and varied from year to year only in generation. They were of inbred line 15, which has been selected for susceptibility to lymphomatosis during the past fifteen years, and the inbreeding coefficient is now about 75.0 percent. A part of the population of this line, which supplied the chicks for these experiments, has been maintained in strict isolation generation after generation since 1942, for the purpose of reducing naturally occurring lymphomatosis. This was generally effective (Waters and Prickett, 1944) and data for recent years indicate no material change. In fact, an average of only 1.71 percent visceral lymphomatosis and 0.73 percent neural lymphomatosis occurred in populations hatched during 1951 to 1954, inclusive. In view of the constancy of test chicks with respect to breeding, management practices, and amount of natural infection, it * Unpublished data.
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show a distinct drop in general level of virus shed; however, with this group the drop occurred between the first and second tests, which were on eggs laid at about 9 and IS months of age. Thus, shedding of the virus appeared to decrease with the age of the hens. In the first series it occurred after 18 months of age, and in the progeny series after only 9 months of age. This is a rather important variation inasmuch as the first occurred after the first laying year and before the second; whereas, in the progeny series, the decrease occurred shortly after the birds were one year of age. Further examination of the data shows that the first generally low transmission results, in both the dam and the progeny series, occurred with eggs laid at about the same period, i.e., March 10 to April IS, 1954. It may be suggested, therefore, that the general, apparent decrease, in shedding observed, may not be so much related to age of the hens laying the eggs as to other factors generally affecting latent infections with the virus. The dams were maintained in one pen of a mating house beginning with the laying of eggs for the second test. The progeny were in several other pens of the same mating house, during the periods of the first and second tests, and were placed in the pen with the dams shortly thereafter. Thus, during the period of apparent reduction in shedding, the two groups of hens were in the same house, but in different pens. Therefore, the general level of infection in the mating house may have decreased during the same period, which may account for the change in the amount of virus shed.
943
944
B. R. BURMESTER AND N. F. WATERS
SUMMARY
The deposition of the virus of visceral lymphomatosis in the eggs of 17 normal appearing hens was determined at specific intervals over a period of 2 years, and similar determinations were made on 13 progeny over a 1J4 year period. Detection of the virus was based on reproducing the disease in susceptible chickens inoculated with a preparation of a pool of livers from four 15-day embryos of each hen to be tested. All of 9 daughters from 5 inbred line 9 hens tested, shed as much or more virus in their eggs when 9 months of age as did their mothers at 12 months of age. However, only 1 of 4 daughters of two line-15 hens gave indication of significant shedding of virus into their eggs, even though both of the dams had shed the virus. Eggs laid by the hens when 12, 15, and
18 months of age gave transmission indicative of a fairly consistent, high level of shedding of the virus. Eggs laid by the same hens when 2 and 3 years of age, gave transmissions indicative of a generally low level of deposition of virus into the eggs. The progeny showed a high level of shedding at 9 months of age, but tests on eggs laid at 15 months and 2 years of age indicated a low level of shedding. Results obtained indicated a reduction in the shedding of the virus of visceral lymphomatosis into eggs as hens became older. However, such reduction may not occur at a uniform age, and may be influenced by other, yet unknown, factors. REFERENCES Burmester, B. R., and R. F. Gentry, 1954a. The presence of the virus causing visceral lymphomatosis in the secretions and excretions of chickens. Poultry Sci. 3 3 : 836-842. Burmester, B. R., and R. F. Gentry, 1954b. A study of possible avenues of infection with the virus of avian visceral lymphomatosis. Proc. 91st Annual Meeting Am. Vet. Med. Assoc: 311-316. Burmester, B. R., R. F. Gentry and N. F. Waters, 1955. The presence of the virus of visceral lymphomatosis in embryonated eggs of normal appearing hens. Poultry Sci. 34: 609-617. Burmester, B. R., and N. F. Waters, 1955. The role of the infected egg in the transmission of visceral lymphomatosis. Poultry Sci. 34: 14151429. Carr, J. G., 1952. A survey of the leucosis complex. Modern Poultry Keeping, Oct. 8, 1952: 360-361. Cottral, G. E., B. R. Burmester and N. F. Waters, 1954. Egg transmission of avian lymphomatosis. Poultry Sci. 33 : 1174-1184. Waters, N. F., and C. O. Prickett, 1944. The development of families of chickens free of lymphomatosis. Poultry Sci. 23: 321-333. Waters, N. F., 1954a. Etiological relationship of visceral and neural lymphomatosis. Poultry Sci. 33:365-373. Waters, N. F., 1954b. Avian lymphomatosis mortality among inbred line crosses. Proc. 10th World's Poultry Congress, Article 61.
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seems unlikely that the test chicks were a source of significant variation during the period in question. With few exceptions, the 15 dams and 10 progeny which were tested more than once, and which were involved in a total of 73 infectivity tests, showed a general consistency in the shedding of the virus of visceral lymphomatosis into their eggs. In each generation there appeared to be a generally high level of shedding which was followed by a much lower level. Age may have been a factor in this marked decrease; however, it does not appear to be of overriding importance, since the decrease occurred at distinctly different ages in the two generations tested. Moreover, the decrease appeared to have taken place in both generations at about the same time, indicating that a possible common environmental factor or factors were involved. Possible identity of the latter was not detected.