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Response of Chicken Embryos to Tissues of Chickens Affected with the Avian Leukosis Complex and to Tissues of Normal Birds
Response of Chicken Embryos to Tissues of Chickens Affected with the Avian Leukosis Complex and to Tissues of Normal Birds C. A. BRANDLY,1 FRANK THORP, JR., 2 AND C. O. PRICKETT 3 {From the U. S. Regional Poultry Research Laboratory. East Lansing, Michigan) (Received for publication November 17, 1948)
/ ~ \ N L Y limited work has been done ^ ^ with the cultivation of tumors in avian eggs since the original studies with the chicken sarcoma, CT1 by Rous and Murphy (1911), and Murphy and Rous (1912). With the recent extensive use of embryonated eggs in the study of disease agents and processes, emphasis has been placed largely on classical viruses, bacteria and higher forms of pathogenic agents. The methods and procedures which have evolved from the latter work would, however, appear to have certain limitations in investigations of fowl tumors. The object of the studies here described was to determine whether the several expressions of the avian leukosis complex (Second Collaborators' Conference, 1941) . could be induced by injection of embryonated chicken eggs, and if this were possible, to attempt serial passage of the disease or diseases in incubating eggs. Early in the study it was observed that when chicken eggs of 10 to 14 day's incubation were inoculated with fresh blood from advanced cases of erythro-
Eggs and chickens from the pedigreed White Leghorn flock of the Regional Laboratory were used for this study. Donors of blood and other tissues were represented by chickens of both sexes inoculated with and showing manifestations typical of the following serial passage strains of the avian leukosis complex: Strain 1 (lymphoma tosis-Brandly, 1941); Strain 3 (lymphomatosis-osteopetrosisBrandly et ah, 1942) and Strain 4 (erythrogranuloblastosis, Srain A of Hall, Bean and Pollard, 1941). These donors ranged in age from 12 to 548 days.
Resigned, present address, 1. University of Wisconsin, Madison, 2. Michigan State College, East Lansing and, 3. Graselli Chemicals Department, E. I. Dupont de Nemours & Company, Wilmington, Delaware.
* The term erythrogranuloblastosis3 signifies the condition associated with generalized malignancy of the erythroblastic and granuloblastic elements. The term is synonymous with the designations, granuloblastic leukosis and in the restricted sense, leukosis.
granuloblastosis* of chickens, death of the embryos often followed within 5 to 10 days. At necropsy, marked visceral and skeletal changes were observed which at first were thought to be the specific lesions of erythrogranuloblastosis in the embryo. However, normal blood as well as blood from aleukemic lymphomatsis donors induced grosslyand microscopically similar lymphomatoid and leukemoid changes. MATERIALS AND METHODS
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TISSUE TRANSPLANTS AND AVIAN LEUKOSIS COMPLEX
The number and age range of donors of apparently normal tissue were as follows: 18 chickens, 2-1296 days old; 5 turkeys, 64-713 days old; 3 domestic ducks, 44-170 days old. The ages of mature birds of other species (2 wild ducks, 1 goose, 1 pheasant, 1 partridge, 2 sparrows, 1 pigeon) were not known. With the exception of the wild species and 3 chickens still living at approximately 1300 days of age, all donors were subjected to necropsy examination. Some birds served as donors on several occasions at intervals up to a month or more. Donors were classed as normal in the absence of symptoms or gross lesions of lymphomatosis. In several instances, apparently normal donors later developed evidence of the disease and the results from the embryos involved were excluded from the data. Only a minor proportion of donors have been examined microscopically. Eggs with embryos of 10 to 14 days' development were inoculated by several routes: chorio-allantoic instillation (Burnet, 1936), intravenous and amnionic chamber (Brandly, 1941), and allantoic chamber and yolk chamber injection. The quantity of inoculum varied somewhat according to the route—a maximum of 0.2 cc. for the chorio-allantoic membrane, yolk and allantoic chamber; 0.05 cc. for the amnionic chamber and intravenous routes. Decimal fractions of the latter quantities were made to the original volume in 0.85 percent sodium cholride solution for amnionic and intravenous titration. Control and inoculated embryos were examined at various intervals, postinoculation to hatching. All embryos which died within 72 hours after injection were excluded from consideration in order to minimize the influence of trauma. The organs and tissues of a representative
487
number of embryos and chicks hatched from injected eggs were studied from impression preparations, paraffin sections and blood smears. With minor exceptions, blood examinations of the chicks were made at weekly or semi-weekly periods from hatching or inoculation until death or termination of the 8-12 weeks' observation period. Blood smears were stained by Wright's method or Wright's-Giemsa combination. EXPERIMENTAL
Necropsy Findings. Various degrees of impaired or delayed development of the' embryo were apparent within 3 to 10 days after injection. At hatching time control embryos frequently weighed 35 to 40 percent more than affected embryos. Splenomegaly was observed within 3 days after injection and increased progressively with resultant weights as great as 5 times the normal (250 mgm. vs. 50 mgm. after 8-9 days). Color changes varied from a uniform deep red or a yellow-gray to a distinct red-gray with mottling. Minute focal areas as well as 2-3 mm. nodules were commonly seen. At the outset the nodules were white in Color and uniform in structure but later became somewhat yellow with central necrosis. The nodules were frequently raised above the surface to give a bosselated effect. Bone alterations consisting of changes in color and enlargement of the shafts of the long bones often accompanied or followed early spleen changes. The color varied from a reddish gray to a pale yellow, the diameter of the shaft was often increased three times, the walls were thickened and the lumen frequently narrowed almost to obliteration. On stress there was a tendency toward transverse fracture of the thickened shaft. The livers showed slight to extreme alterations in color and size. A light olive
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C. A. BRANDLY, F R A N K T H O R P , J R . , AND C. 0 . P R I C K E T T
color, sometimes seen in exsanguinated embryos not injected or injected with other agents, was encountered early after injection or in slightly affected individuals. In the advanced stages a deep green to yellow red color, together with weight increases of two or more times normal, was found. Very infrequently yellowish
With advanced visceral involvement, the heart was frequently enlarged and quite atonic. Occasionally single small nodules were found in the walls of the auricles. T h e chorio-allantoic membrane commonly showed diffuse degenerative changes and, occasionally, definite focal or tumor-
FIG. 1.—Spleen (16478)—Phloxine—Methylene Blue—X200 Small necrotic focus having for center a clump of disintegrated red blood cells surrounded by a few giant cells and a thin capsule of fibrotic tissue.
white nodules, pinpoint to 2 mm. in diameter, were present in the liver. T h e kidneys occasionally showed definite enlargement. I n the advanced stages, impaired kidney function associated with accumulation of urates in the tubules and ureters was also observed. Nodules were only rarely seen in these organs or in the peritoneum, subcutaneous tissue or skin. These nodules showed less tendency toward central necrosis than those which developed in the spleen.
like masses. Several series of membranes which had been inoculated by instilling leukotic tissue upon the ectodermal layer, revealed an almost hemispherical b u t slightly flattened tumor protruding into the allantoic chamber from the adjacent entodermal surface. These tumors varied in size up to 10 mm. in diameter. Tumor-like nodules were, however, not seen following introduction of normal and lymphomatous blood. Microscopic
Findings.
Microscopic ex-
T I S S U E TRANSPLANTS AND AVIAN L E U K O S I S COMPLEX
amination of the spleen revealed areas of degeneration, necrosis and hemorrhage corresponding to the nodules observed a t necropsy. I n later stages these lesions were surrounded by macrophages a n d / o r giant cells. With normal and lymphomatotic blood the necrotic process predominated, while hemoblastic cells were seen in a few spleens (Figure 1). Leukotic blood produced a general leukemic state
constant following the injection of normal, lymphomatotic and leukotic bolod. Necrotic foci were sometimes observed in the marrow cavity a t either end of the shaft as well as in the interstices of the newly formed cancellous bone. These foci varied from those t h a t showed only recent death of cells, without peripheral reaction, to those t h a t were surrounded by macrophages a n d / o r giant cells (Figure 4).
FIG. 2.—Tibia (16478)—Phloxine—Methylene Blue—X200 Thickening of the periosteum.
in most spleens with a minimal number of necrotic lesions. Microscopically the bone enlargement was characterized by an active proliferation of the periosteum (Figure 2). Osteoclastic and osteoblastic activity was quite pronounced. The new bone tended toward oblique and transverse arrangement. Bone formation was not compact, for between the spicules much cellular tissue was found (Figure 3). At times the marrow cavity was completely obliterated and the new bone formed a mosaic throughout. These lesions were quite
A study of the bone marrow from sections and impressions revealed some striking differences. Marrow from uninoculated embryos showed all stages of blood cell development with a normal cellular and vascular bed. In the embryos inoculated with normal, lymphomatotic and leukotic blood the bone marrow was usually packed with blastic cells in different phases of development. There was an apparent lack of normal vascularity. The injection of normal and lymphomatotic blood caused an increase of erythroid and myeloid elements with a
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jection of leukotic blood resembled those seen in chickens after successful inoculation and there was usually a generalized leukemic state with many cells in the sinusoids. The perivascular infiltrations observed following the injection of the other tissues employed were characterized by the presence of blastic cells. These cells differ from the above in t h a t t h e y have a more vesicular, chromatin-poor nucleus with scant, faintly pink staining cytoplasm. The bile duct proliferations were observed following injection with normal and lymphomatotic b u t not with erythrogranuloblastic blood. T h e hyperplastic bile ducts varied in size and number, were perivenous in location and replaced the adjacent parenchyma. Some of the ducts contained what appeared to be cellular debris.
FIG. 3.—Femur (16504)— Phloxine—Methylene Blue—X200 Proliferating periosteum; considerable numbers of osteoclasts are observed; fibrosis of the bone marrow.
preponderance of promyelocytes and myelocytes (Figure 5). With leukotic blood the reaction induced was basically the same except in place of the excess meylocytes there was a large number of what appeared to be cells of the erythroblastic series. T h e liver showed various alterations, focal necrosis, perivascular infiltration, bile duct hyperplasia and enlargement of the sinusoids with leukemic cells. T h e necrotic areas of the liver were similar to those seen in the bone marrow and spleen except t h a t they were occasionally surrounded b y some blastic cells. This lesion was observed only once following the injection of leukotic blood and was so infrequent as to be considered a rarity. Perivascular infiltration consisted largely of very young cells. The cells observed in embryos following the in-
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FIG. 4.—Tibia (16478)—Phloxine—Methylene Blue—X200 Round focus of necrosis, showing some surrounding giant cell reaction.
T I S S U E TRANSPLANTS AND AVIAN L E U K O S I S COMPLEX
Necrosis of the kidney was not observed in any of the sections, b u t infiltrations with blastic cells were occasionally seen after the embryos were inoculated with normal, leukotic and lymphomatotic blood. Necrotic foci were not observed in any of the heart sections. After inoculation with leukotic blood, mild infiltrations with blastic cells were commonly observed. The incidence and extent of immature cell infiltrations following injection of normal and aleukemic lymphomatotic blood were definitely decreased.
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Incidence and Hatchability. Comparisons of the response of the embryo to intravenous injection of leukotic (leukemic) blood as well as the blood of lymphomatotic (aleukemic) and normal birds m a y be made from the data in Table 1. The incidence of embryo-induced mor-
Sections of the chorio-allantoic tumors showed keratinization, vacuolization, and "piling u p " of the ectodermal cells. There was a general thickening of the mesoderm in which the developing cells resembled the granulocytic series. I n some of the tumors, necrotic foci were seen. Epithelial pearls were frequently observed in the mesenchyma. Leukotic cells when present were usually in small nests, perivascular in position. The presence of polychrome and erythroblastic elements in embryonic blood renders the detection of early and mild leukemoid and erythroblastic changes somewhat difficult. Toward the latter p a r t of the incubation period, however, there were occasionally noticed, with Strain 4 inoculum, earlier stages in erythroblastic development than were encountered following injection of blood from normal and lymphomatotic donors. Although the erythrogranuloblastosis Strain A of Hall and co-workers (1941) was employed in these studies there were not encountered in embryos the marked leukotic blood changes pictured by them. T h e embryonic changes suggest t h a t the leukotic state accompanies or is superimposed upon the disturbances induced b y injection with normal and non-leukemic tissue components.
FIG. 5.—Tibia (16478)—Phloxine—Methylene Blue—X200 Hyperplastic bone marrow showing increased cellularity.
bidity and percentage of hatchability were employed as criteria. With lymphomatotic blood, a somewhat, but perhaps not a significantly, lower incidence of embryo lesions was induced t h a n with leukotic blood and with normal blood. The percentage of hatch was, however, considerably below t h a t in the latter two groups. Blood from turkeys, which showed no evidence of the avain leukosis complex, produced the embryo disease. These changes were absent in embryos injected with blood
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C. A. BRANDLY, FRANK THORP, J R . , AND C. 0 . PRICKETT TABLE 1.—Incidence of lesions and halchability
Inoculum Strain 4 blood Strain 4 plasma Strain 1 and 3 blood Normal chicken blood Normal chicken plasma Normal turkey blood Other species blood Saline or broth Uninoculated
No. embryos inoculated
Gross lesions
Chicks hatched
—
413 236 167 100 44 78' 136 94 292
of eight other species of birds (goose, pheasant, partridge, pigeon, sparrow and 3 ducks) and several samples of human blood. The reason why the blood of the pheasant, which species is more closely related to the chicken than is the turkey, did not induce a reaction similar to that obtained with turkey blood, warrants further consideration. However, the induction of osteal changes with human myeloid leukemia tissue (Thiersch, 1944) may suggest implication of non-specific factors. The tissues from male and female donors appeared to be equally effective in inducing leukemoid changes in embryos of both sexes. '•Furthermore, the blood of the sire and dam of embryos of several matings studied appeared to produce the disturbance as readily as the blood of other individuals of unrelated families. Only 3 percent of the 236 embryos which received injections of erythrogranulo-
No.
Percent
No.
Percent
206 7 59 46 2 16 0 1 1
49.9 3.0 35.3 46.0 4.5 20.5 0.0 0.94 .03
127 143 40 34 24 37 73 50 193
30.8 60.6 23.9 34.0 54.5 47.4 53.7 53.2 66.1
blastotic plasma and 4.5 percent of 844 embryos which received normal plasma showed gross lesions. The suggested greater concentration of the embryodisease-inducing factor in blood than in plasma would seem to be confirmed by the results of titrations. Quantities of plasma less than 0.05 cc. in no instance gave positive results whereas lesions of the embryo were provoked by quantities of blood as small as 0.00005 cc. When the whole blood was subjected to drying, laking with distilled water or rapid freezing to — 76°C, the embryo-aberrative effect appeared to be largely or entirely destroyed. Only one embryo in each of the saline and broth control groups showed gross visceral changes which resembled those provoked by the blood. The gross findings in these instances were not confirmed microscopically.
TABLE 2.—Production and serial passage of embryo aberration by normal blood
Inoculum
Normal chicken blood Normal chick blood* Normal embryo blood 1st and 2nd embryo passage of chicken blood 3rd and 4th embryo passage of chicken blood
Positive gross changes
No. embryos inoculated
No.
Chicks hatched
Percent
No.
Percent
100 27 46
46 1 0
46.0 3.7 0.0
34 20 27
34.0 74.1 58.7
105
22
21.0
32
30.5
28
0
0.0
18
64.3
* Includes chickens less than twenty-nine days of age.
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TISSUE TRANSPLANTS AND AVIAN LEUKOSIS COMPLEX
Passage of Embryo-induced Disease. The results of attempts to perpetuate the embryo disease by serial passage are summarized in Table 2. Blood collected from 18 to 19 day embryos which had been injected at the 11th and 12th days of incubation was used for the subsequent series of inoculations. Sufficient data have not been obtained to determine the approximate age of the chick when its blood will cause the homoioplastic reaction in the embryo. It would appear that the normal-tissue-induced disease may not be perpetuated beyond the second passage. Blood from uninjected control
chamber injection was next in order (11.1). Inoculation into the allantoic chamber and into the yolk appeared to be virtually without effect in inducing the embryo disease. The absence of the latter condition probably accounts for the increase in percentage of chicks hatched. Incidence of A vian Leukosis Complex in Chicks Hatched from Injected Eggs. None of 58 chicks which hatched from normal blood- or plasma-injected eggs developed erythrogranuloblastosis within the 90-day observation period post hatching. In contrast, approximately 20 percent of the 25 chicks hatched from eggs inoculated
TABLE 3.—Influence of route of injection of chicken blood upon incidence of embryo disease and hatchability
Route
No. embryos inoculated
Intravenous Chorio-allantoic Amnionic sac Allantoic sac Yolk sac
100 44 324 51 42
Positive gross changes
Chicks hatched
No.
Percent
No.
Percent
46 14 36 1 0
46.0 31.8 11.1 2.0 0.0
34 13 181 27 29
34.0 29.5 55.9 53.0 69.0
embryos and from chicks 12 days or less in age did not, with the one exception noted, produce any gross changes. Thus, the factor which induces the embryonic disturbance would not appear to be present in the tissues during embryonic life or for several weeks after hatching. Furthermore, inability to perpetuate this disease after the 2nd passage in the embryo indicates that it is not capable of producing malignancy nor of continuous propagation under the circumstances which existed. Effect of Route of Injection. A summary of the effects of the route of injection of chicken blood upon the incidence of embryo disease and hatchability is given in Table 3. The intravenous route induced the highest percentage of gross lesions, (46) the chorio-allantoic route a lower incidence (31.8). The intra-amnionic
with Strain 4 blood and 33 percent of the 57 chicks which received Strain 4 plasma developed erythroganuloblastosis during the 90-days period post hatching (Table 4). Quantities of blood and plasma as small as 0.000005 cc. caused erythrogranuloblastosis in these chicks. Attention is drawn to the fact that quantities of chicken blood larger than 0.005 cc. usually prevented hatching; whereas this effect was not marked with quantities of plasma as large as 0.05 cc. The inoculation of chicks, 1 to 26 days of age, brought forth marked differences in response to blood and to plasma from leukotic donors (Table 4). The incidence of erythrogranuloblastosis was approximately 70 and 7 percent in the Strain 4 blood and plasma inoculated groups, respectively. It is seen that the average
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C. A. BRANDLY, FRANK THORP, JR., AND C. 0.
incubation period (the time in days from inoculation until the development of an erythrogranuloblastotic blood picture)was much longer after plasma than after blood inoculation. These results indicated a much greater concentration and/or activity of Strain 4 agent in the blood cells and unfiltered plasma than in the plasma filtrate. On the basis of incubation period and incidence of the disease, it appears that
PRICKETT
also inoculated into a second series of embryos but blood from the latter failed to induce erythrogranuloblastosis in any of the 8 chicks inoculated with it. The blood of embryos inoculated with Berkefeld filtrates and leukotic chicken plasma failed to induce erythrogranuloblastosis in 10 chicks into which it was injected. Since the concentration of the oncogenic agent may be considered invariably to be greater in whole blood than
TABLE 4.—Incidence and mcubatton period of erythrogranuloblastosis in chicks hatched from inoculated eggs and chicks inoculated after hatching
Inoculum
Strain 4 erythrogranuloblastosis blood plasma Strain 4 erythrogranuloblastosis blood plasma
Quantum inoculum intravenously
No. inoculum
Age at inoculum
Erythrogranuloblastosis positive No.
Percentage
Average incubation period (days)
' 0.000005 to 0.05 cc. ea.
127
Embryos, 11—14 days development
25
19.7
43.8
0.000005 to 0.05 cc. ea.
143
Embryos, 11—14 days development
57
32.9
47.0
0.2 cc. per 100 gm. body wt.
191
134
70.2
24.8
0.5 cc. per 100 gm.
42
3
6.6
56.6
Chicks 1-26 days of age Chicks 1-26 days of age
the embryo was no more susceptible than the young chick, and possibly less so, to the inoculation of Strain 4 agent. Quantitative differences in the inocula received by the embryos and chicks would not, on the basis of experience with this and other strains, account for the degrees of difference observed. Incidence of the Avian Leukosis Complex in Chicks Inoculated with Embryo Tissue. Blood taken from 19-day embryos, 7 days after inoculation with Strain 4 blood, was inoculated intravenously into a limited number of chicks ranging in age from 2 to 8 days. Five of 8 chicks so inoculated developed erythrogranuloblastosis. The first embryo passage material was
in plasma or plasma filtrate, evidence for or against its multiplication in embryos cannot be deduced from these findings. Nevertheless, it would appear that under the circumstances serial passage in embryos did not provide optimal conditions for propagation of the strain in question. When Strain 3 lymphomatosisosteopetrosis blood was used for embryo passage and subsequent chick injection, the incidence of combined lymphomatosis and osteopetrosis in the injected chicks was as follows: 1st passage, 30 of 45 positive (66.6 percent); 2nd passage, 6 of 16 positive (37.5 percent); and 3rd passage, 8 of 10 positive (80.0 percent). Twenty, four and five birds of these
TISSUE TRANSPLANTS AND AVIAN LEUKOSIS COMPLEX
respective groups showed osteopetrosis either alone or in combination with visceral or neural lymphomatosis. Six of 20 uninoculated control chickens developed lymphomatosis but none showed osteopetrosis. The prevalence of lymphomatosis in the controls (30 percent) raises doubt as to the significance of lymphomatosis in the inoculated group even though the incidence was twice as great in the latter. The assumption that an osteopetrotic factor was transmitted may be open to question in view of Gohs (1934) observation that injection of embryonic marrow may induce osteopetrotic or similar changes in chickens. Likewise, Theirsch (1944) has recently recorded the induction of an osteopetrosis-like condition in 3 of 9 chickens hatched from embryos injected intra-amnionically with blood from two human cases of chronic myeloid leukemia. DISCUSSION AND SUMMARY
The injection of apparently normal chicken blood and tissues may produce pathological changes of the chicken embryo which are grossly, and to a certain extent, microscopically similar to those induced by leukotic material. This emphasizes the need for caution in interpretation of results. The disease of the embryo and extra-embryonic membranes here described is apparently identical with t h a t ' found by Murphy (1916) and Dantschakoff (1920) to be associated with chorioallantoic grafts of certain organs of older chickens. The normal organ implants were, however, made only in younger embryos, i.e., those of 7 days' incubation. In addition to the changes seen by Murphy and Dantschakoff our work has revealed significant bone and liver lesions. Furthermore, the embryonic disease resulted when chicken and turkey bloods were introduced by several channels—
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the intravenous, supra-chorio-allantoicand intra-amnionic, but only in a negligible number, or not at all, by the allantoic or yolk chamber routes. The tissues of both male and female chicken and turkey donors were found to produce the leukemoid disease in embryos of both sexes. The additional observation that the blood of both sire and dam of certain matings studied was as effective as that of unrelated normal individuals, suggests that the response of the embryo to normal homologous tissue is a function of age and individual differentials. With Strain 4, for example, a quantity of inoculum so small as not to induce leukemoid embryo disease yet capable of producing erythrogranuloblastosis, required an incubation period greatly in excess of the remaining period of intra-shell life. The possibility does exist, however, that this or other strains of the agent may be adapted to a shorter incubation period in the embryo and thus to serial propagation within the egg. Erythrogranuloblastosis developed in some of the chicks hatched from eggs inoculated with Strain 4 material by each of the routes employed. None of the uninoculated controls or those injected with blood from apparently normal birds or lymphomatotic donors developed erythrogranuloblastosis. However, lymphomatosis occurred in all groups although in a higher percentage in the recipients of leukosis inoculum (15 percent) than in the controls (8 percent). A number of other workers have found that chicks hatched from eggs inoculated by several routes may develop leukosis. Jarmai and co-workers (1932, 1933) obtained positive results following injection into the albumen after the 9th day of incubation. Storti and Mezzadra (1938) concluded that leukosis virus could survive in tissues instilled upon the chorio-
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C. A. BRANDLY, FRANK THORP, J R . , AND C. 0 . PRICKETT
allantoic membrane at the 4th day of development but that it did not multiply. Van den Berghe and d'Ursel (1939) reported serial passage of fowl leukosis on the chorio-allantoic membrane of chicken embryos and 5 of 6 chicks hatched from eggs of the 1st to 4th serial passage developed the disease 8-19 days after hatching. Using Furth's Strain 1, Kirschbaum, Stern and Hooker (1940) found that leukosis developed in chicks hatched from eggs inoculated via the albumen on the 12th day but not in those inoculated on the 7th day of incubation. Two of four chickens hatched after intra-amnionic sac injection of plasma on the 7th day developed leukosis when 46 and 58 days of age. Hall, Bean and Pollard (1941) reported leukotic changes in the embryo following intravenous injection of leukotic blood but not after chorio-allantoic and yolk injection. They failed to induce "leukotic" changes with filtrates of leukotic tissue or plasma and did not report the fate of chicks which hatched from injected eggs. Pierce (1942) failed to observe leukosis in chicks hatched from eggs treated after the 8th day of incubation by instilling filtrates of leukotic tissue and plasma upon the chorio-allantois. However, positive results were obtained with unfiltered tissue suspensions and plasma. Our observations suggest that inoculation of the embryo, particularly by the intravenous route, with relatively large quantities of leukotic blood may result in a successful transplantation or graft similar to that described by Crank and Furth (1932) after massive inoculation of susceptible chickens. The host apparently may be overwhelmed by such a graft. Upon injection of considerably smaller quantities of leukotic cells, the graft may apparently fail to become established although as with the introduction of tissue
and plasma freed of leukotic cells, erythrogranuloblastosis may be initiated by the oncogenic agent present in the leukotic fluids. The requisite incubation period in the latter instances would seerri to preclude the direct transfer of most strains of erythrogranuloblastosis from chickens to serial passage in embryos. Aside from serving the function of a culture medium for autonomous multiplication of implanted leukotic cells, the tissues of the embryo may obviously respond to cell-free leukosis-producing agent. Initiation of the specific disease processes is followed by an appropriate incubation period and the postnatal appearance of the disease. The early leukemoid and lymphomatoid changes brought about by normal and non-leukemic tissue components obviously tend to complicate these phenomena and their interpretation. In earlier observations Storti and Mezzadra (1938), and Van den Berghe and d'Ursel (1939) found that the leukosis agent placed on the chorio-allantoic membrane may remain alive for a considerable period without growth. Pierce also found no evidence that the infectivity of leukotic filtrates was increased by incubation in the chorio-allantoic membrane. In view of these findings together with the observation of Pierce (1942) that transplanted cells may migrate from the chorioallantois into the embryo, it is not surprising that Hall et al. (1941), and Pollard and Hall (1942) demonstrated the agent to be present in the embryo after one or several "passages" of leukotic chicken blood by the intravenous route. Demonstration of the agent under these circumstances may not be taken as proof that multiplication has occurred nor indisputable evidence that the "leukemic" and leukemoid changes were provoked exclusively by the leukosis virus. Negative results were obtained by Hall
TISSUE TRANSPLANTS AND AVIAN LEUKOSIS COMPLEX
and co-workers (loc. cit.) from injection of embryos with filtrates of leukosis-inoculated embryos or the serum of leukotic chicks, whereas these materials when inoculated into chicks produced leukosis after an appropriate incubation period. Pierce (1942) obtained similar results with plasma filtrates. These experiences would further suggest that the gross lesions in the embryos were associated at least in part with homoioplastic tissue reactions. CONCLUSIONS
1. The tissues of apparently normal chickens as well as those of aleukemic lymphomatosis and of erythrogranuloblastosis affected birds may induce pronounced and extensive leukemoid and related processes in chicken embryos injected with them. 2. Specific erythrogranuloblastotic processes may accompany or follow these phenomena in embryos injected with relatively large quantities of blood representing a single strain of erythrogranulobastosis. 3. The injection of relatively small quantities of erythrogranuloblastotic blood or of cell-free plasma may result in development of the uncomplicated specific disease, after a suitable incubation period, in chickens hatched from inoculated eggs. 4. Attempts to propagate two strains of lymphomatosis and one of erythrogranuloblastosis by continuous serial passage in chicken embryos were unsuccessful. The developmental period of the uncomplicated disease after injection, exceeded the remaining period of embryonal life. 5. Recognition of the role of nonspecific influences or factors in the response of the embryo to these and other neoplasms is essential to proper interpretation of the several phenomena involved. The authors are indebted to Dr. S. B.
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Wolbach for assistance in the evaluation of the histologic changes and to Dr. Paulo Dacorso for preparation of the photographs. REFERENCES
Brandly, C. A., 1941. Progress report on several phases of pathology research. Second Collaborators' Conference, U. S. Bureau of Animal Industry, Regional Poultry Research Laboratory, East Lansing, Michigan, pp. 23-38. Brandly, C. A„ N. M. Nelson and G. E. Cottral, 1942. Serial passage of lymphomatosis-osteopetrosis in chickens. Am. J. Vet. Res. 3: 289-295. Burnet, F. M., 1936. The use of the developing egg in virus research. Medical Research Council, Special Report Series No. 220. His Majesty's Stationery Office, London. Crank, F. P. and J. Furth, 1932. Transfusion experiments with the blood of leukemic chickens. Arch. Path. 14: 660-670. Dantschakoff, Vera, 1920. Myeloid metaplasia of the embryonic mesenchyme in relation to cell potentialities and differential factors. Carnegie Institution of Washington, Contributions to Embryology No. 49: 3-32. Gohs, W., 1934. Uber die Wirkung arteigener Knochen- und Knochenmarkzerfallstoffe aud die Knochen- und Blutbildung der Huhner. (Experimentele bei Huhner erzeugte Osteo dystrophia fibrosa, Myelosiche Leukose, Erythramia und schwere Anamie). Frankfurt Ztschr. f. Path. 46: 453-476. Hall, W. J., C. W. Bean and M. Pollard, 1941. Transmission of fowl leucosis through chick embryos and young chicks. Am. J. Vet. Res. 2: 272279. Jarmai, Karl, Tibor Stenszky and Ladislaus Farkas, 1932. Neuere Beitrage zur Kenntnis der iibertragbaren HUhnerleukose. Arch. f. Wlsensch. u. prakt. Tierh. 65:46-71. Jarmai, Karl, 1933. Infektionsversuche bebriiteter Eier mit dem "virus" der Huhnererythroleukose. Deut. tierarztl. Wchnschr. 79: 418-420. Kirschbaum, Arthur, K. G. Stern and C. W. Hooker, 1940. Studies on fowl leukosis. Transfer with fractions obtained by ultracentrifugation of leukemic plasma and bone marrow extracts. Yale J. Biol. & Med. 13: 1-14. Murphy, J. B. and P. Rous, 1912. The behavior of chicken sarcoma implanted in the developing embryo. Am. Med. Assoc. J. 56: 119-132. Murphy, J. B., 1916. The effect of adult and chicken organ grafts on the chick embryo. J. Exper. Med. 24: 1-5.
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BOOK REVIEW
Pierce, Mila, 1942. Studies on fowl leukosis. Transfer to the chick embryo. Am. J. Path. 18:1127-1136. Pollard, Morris, and W. J. Hall, 1942. Interspecies transmission of avian leucosis in embryos. Am. J. Vet. Res. 3:247-252. Rous, Peyton, and J. B. Murphy, 1911. Tumor implantations in the developing embryos. Experiments with a transmissable sarcoma of the fowl. Am. Med. Assoc. J. 56: 741-742. Second Collaborators' Conference, 1941. Recommendations of the pathology committee. TJ. S. Bureau of Animal Industry, Regional Poultry Research Laboratory, East Lansing, Michigan, p. 119.
Storti, E., and G. Mezzadra, 1938. Tentatives de culture du virus de la leuceme des poules dans la membrane chorio-allantoide (Note preliminaire). Sang. 12:533-539. Thiersch, J. B., 1944. Attempts to transmit leucaemia of man and of mice to the chick embryo and to the young chick by the amnionic and intravenous routes. Austr. J. Exp. Biol, and Med. Sci. 22: 57-61. Van den Berghe, L., and F. d'Ursel, 1939. Culture entissus du virus de l'erythroblastose de la poule (Souche O. G.) Compt. rend. Soc. de Biol. 131: 1301-1302.
Book Review H. C. Fryer Fortune. Cosmos Publishing Company, Hollywood, California. 1949. 115 pages plus 41-page supplement. Price $7.50.
SHAW,
The first 115 pages of this booklet are printed in excessively large type with about 7 words to a line and only on one side of each page. There are a number of full page diagrams and illustrations. The entire booklet is printed on a heavy weight paper and bound by 12 rigid metallic rings. The subject matter of the main part of the booklet is devoted almost exclusively to one type of battery raising of fryers which was used by the author in the relatively mild winter area of the California
coast. It is written in the first person and describes the author's experiences and conclusions. Thirty one pages of the supplement, in normal size type, are occupied by "Brooding and Rearing Chickens" credited to California Extension Bulletin 127. The floor method of growing fryers is said to be used extensively in the eastern section of the United States. Is it not used extensively in all sections of the country? Brooding in batteries continues to have some sort of glamorous attraction. Even with this glamor for the novice, it is difficult to understand how the first edition of 1000 copies was sold by the author at $12.50 each.
/ ~ \ N L Y limited work has been done ^ ^ with the cultivation of tumors in avian eggs since the original studies with the chicken sarcoma, CT1 by Rous and Murphy (1911), and Murphy and Rous (1912). With the recent extensive use of embryonated eggs in the study of disease agents and processes, emphasis has been placed largely on classical viruses, bacteria and higher forms of pathogenic agents. The methods and procedures which have evolved from the latter work would, however, appear to have certain limitations in investigations of fowl tumors. The object of the studies here described was to determine whether the several expressions of the avian leukosis complex (Second Collaborators' Conference, 1941) . could be induced by injection of embryonated chicken eggs, and if this were possible, to attempt serial passage of the disease or diseases in incubating eggs. Early in the study it was observed that when chicken eggs of 10 to 14 day's incubation were inoculated with fresh blood from advanced cases of erythro-
Eggs and chickens from the pedigreed White Leghorn flock of the Regional Laboratory were used for this study. Donors of blood and other tissues were represented by chickens of both sexes inoculated with and showing manifestations typical of the following serial passage strains of the avian leukosis complex: Strain 1 (lymphoma tosis-Brandly, 1941); Strain 3 (lymphomatosis-osteopetrosisBrandly et ah, 1942) and Strain 4 (erythrogranuloblastosis, Srain A of Hall, Bean and Pollard, 1941). These donors ranged in age from 12 to 548 days.
Resigned, present address, 1. University of Wisconsin, Madison, 2. Michigan State College, East Lansing and, 3. Graselli Chemicals Department, E. I. Dupont de Nemours & Company, Wilmington, Delaware.
* The term erythrogranuloblastosis3 signifies the condition associated with generalized malignancy of the erythroblastic and granuloblastic elements. The term is synonymous with the designations, granuloblastic leukosis and in the restricted sense, leukosis.
granuloblastosis* of chickens, death of the embryos often followed within 5 to 10 days. At necropsy, marked visceral and skeletal changes were observed which at first were thought to be the specific lesions of erythrogranuloblastosis in the embryo. However, normal blood as well as blood from aleukemic lymphomatsis donors induced grosslyand microscopically similar lymphomatoid and leukemoid changes. MATERIALS AND METHODS
486
TISSUE TRANSPLANTS AND AVIAN LEUKOSIS COMPLEX
The number and age range of donors of apparently normal tissue were as follows: 18 chickens, 2-1296 days old; 5 turkeys, 64-713 days old; 3 domestic ducks, 44-170 days old. The ages of mature birds of other species (2 wild ducks, 1 goose, 1 pheasant, 1 partridge, 2 sparrows, 1 pigeon) were not known. With the exception of the wild species and 3 chickens still living at approximately 1300 days of age, all donors were subjected to necropsy examination. Some birds served as donors on several occasions at intervals up to a month or more. Donors were classed as normal in the absence of symptoms or gross lesions of lymphomatosis. In several instances, apparently normal donors later developed evidence of the disease and the results from the embryos involved were excluded from the data. Only a minor proportion of donors have been examined microscopically. Eggs with embryos of 10 to 14 days' development were inoculated by several routes: chorio-allantoic instillation (Burnet, 1936), intravenous and amnionic chamber (Brandly, 1941), and allantoic chamber and yolk chamber injection. The quantity of inoculum varied somewhat according to the route—a maximum of 0.2 cc. for the chorio-allantoic membrane, yolk and allantoic chamber; 0.05 cc. for the amnionic chamber and intravenous routes. Decimal fractions of the latter quantities were made to the original volume in 0.85 percent sodium cholride solution for amnionic and intravenous titration. Control and inoculated embryos were examined at various intervals, postinoculation to hatching. All embryos which died within 72 hours after injection were excluded from consideration in order to minimize the influence of trauma. The organs and tissues of a representative
487
number of embryos and chicks hatched from injected eggs were studied from impression preparations, paraffin sections and blood smears. With minor exceptions, blood examinations of the chicks were made at weekly or semi-weekly periods from hatching or inoculation until death or termination of the 8-12 weeks' observation period. Blood smears were stained by Wright's method or Wright's-Giemsa combination. EXPERIMENTAL
Necropsy Findings. Various degrees of impaired or delayed development of the' embryo were apparent within 3 to 10 days after injection. At hatching time control embryos frequently weighed 35 to 40 percent more than affected embryos. Splenomegaly was observed within 3 days after injection and increased progressively with resultant weights as great as 5 times the normal (250 mgm. vs. 50 mgm. after 8-9 days). Color changes varied from a uniform deep red or a yellow-gray to a distinct red-gray with mottling. Minute focal areas as well as 2-3 mm. nodules were commonly seen. At the outset the nodules were white in Color and uniform in structure but later became somewhat yellow with central necrosis. The nodules were frequently raised above the surface to give a bosselated effect. Bone alterations consisting of changes in color and enlargement of the shafts of the long bones often accompanied or followed early spleen changes. The color varied from a reddish gray to a pale yellow, the diameter of the shaft was often increased three times, the walls were thickened and the lumen frequently narrowed almost to obliteration. On stress there was a tendency toward transverse fracture of the thickened shaft. The livers showed slight to extreme alterations in color and size. A light olive
488
C. A. BRANDLY, F R A N K T H O R P , J R . , AND C. 0 . P R I C K E T T
color, sometimes seen in exsanguinated embryos not injected or injected with other agents, was encountered early after injection or in slightly affected individuals. In the advanced stages a deep green to yellow red color, together with weight increases of two or more times normal, was found. Very infrequently yellowish
With advanced visceral involvement, the heart was frequently enlarged and quite atonic. Occasionally single small nodules were found in the walls of the auricles. T h e chorio-allantoic membrane commonly showed diffuse degenerative changes and, occasionally, definite focal or tumor-
FIG. 1.—Spleen (16478)—Phloxine—Methylene Blue—X200 Small necrotic focus having for center a clump of disintegrated red blood cells surrounded by a few giant cells and a thin capsule of fibrotic tissue.
white nodules, pinpoint to 2 mm. in diameter, were present in the liver. T h e kidneys occasionally showed definite enlargement. I n the advanced stages, impaired kidney function associated with accumulation of urates in the tubules and ureters was also observed. Nodules were only rarely seen in these organs or in the peritoneum, subcutaneous tissue or skin. These nodules showed less tendency toward central necrosis than those which developed in the spleen.
like masses. Several series of membranes which had been inoculated by instilling leukotic tissue upon the ectodermal layer, revealed an almost hemispherical b u t slightly flattened tumor protruding into the allantoic chamber from the adjacent entodermal surface. These tumors varied in size up to 10 mm. in diameter. Tumor-like nodules were, however, not seen following introduction of normal and lymphomatous blood. Microscopic
Findings.
Microscopic ex-
T I S S U E TRANSPLANTS AND AVIAN L E U K O S I S COMPLEX
amination of the spleen revealed areas of degeneration, necrosis and hemorrhage corresponding to the nodules observed a t necropsy. I n later stages these lesions were surrounded by macrophages a n d / o r giant cells. With normal and lymphomatotic blood the necrotic process predominated, while hemoblastic cells were seen in a few spleens (Figure 1). Leukotic blood produced a general leukemic state
constant following the injection of normal, lymphomatotic and leukotic bolod. Necrotic foci were sometimes observed in the marrow cavity a t either end of the shaft as well as in the interstices of the newly formed cancellous bone. These foci varied from those t h a t showed only recent death of cells, without peripheral reaction, to those t h a t were surrounded by macrophages a n d / o r giant cells (Figure 4).
FIG. 2.—Tibia (16478)—Phloxine—Methylene Blue—X200 Thickening of the periosteum.
in most spleens with a minimal number of necrotic lesions. Microscopically the bone enlargement was characterized by an active proliferation of the periosteum (Figure 2). Osteoclastic and osteoblastic activity was quite pronounced. The new bone tended toward oblique and transverse arrangement. Bone formation was not compact, for between the spicules much cellular tissue was found (Figure 3). At times the marrow cavity was completely obliterated and the new bone formed a mosaic throughout. These lesions were quite
A study of the bone marrow from sections and impressions revealed some striking differences. Marrow from uninoculated embryos showed all stages of blood cell development with a normal cellular and vascular bed. In the embryos inoculated with normal, lymphomatotic and leukotic blood the bone marrow was usually packed with blastic cells in different phases of development. There was an apparent lack of normal vascularity. The injection of normal and lymphomatotic blood caused an increase of erythroid and myeloid elements with a
490
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jection of leukotic blood resembled those seen in chickens after successful inoculation and there was usually a generalized leukemic state with many cells in the sinusoids. The perivascular infiltrations observed following the injection of the other tissues employed were characterized by the presence of blastic cells. These cells differ from the above in t h a t t h e y have a more vesicular, chromatin-poor nucleus with scant, faintly pink staining cytoplasm. The bile duct proliferations were observed following injection with normal and lymphomatotic b u t not with erythrogranuloblastic blood. T h e hyperplastic bile ducts varied in size and number, were perivenous in location and replaced the adjacent parenchyma. Some of the ducts contained what appeared to be cellular debris.
FIG. 3.—Femur (16504)— Phloxine—Methylene Blue—X200 Proliferating periosteum; considerable numbers of osteoclasts are observed; fibrosis of the bone marrow.
preponderance of promyelocytes and myelocytes (Figure 5). With leukotic blood the reaction induced was basically the same except in place of the excess meylocytes there was a large number of what appeared to be cells of the erythroblastic series. T h e liver showed various alterations, focal necrosis, perivascular infiltration, bile duct hyperplasia and enlargement of the sinusoids with leukemic cells. T h e necrotic areas of the liver were similar to those seen in the bone marrow and spleen except t h a t they were occasionally surrounded b y some blastic cells. This lesion was observed only once following the injection of leukotic blood and was so infrequent as to be considered a rarity. Perivascular infiltration consisted largely of very young cells. The cells observed in embryos following the in-
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FIG. 4.—Tibia (16478)—Phloxine—Methylene Blue—X200 Round focus of necrosis, showing some surrounding giant cell reaction.
T I S S U E TRANSPLANTS AND AVIAN L E U K O S I S COMPLEX
Necrosis of the kidney was not observed in any of the sections, b u t infiltrations with blastic cells were occasionally seen after the embryos were inoculated with normal, leukotic and lymphomatotic blood. Necrotic foci were not observed in any of the heart sections. After inoculation with leukotic blood, mild infiltrations with blastic cells were commonly observed. The incidence and extent of immature cell infiltrations following injection of normal and aleukemic lymphomatotic blood were definitely decreased.
491
Incidence and Hatchability. Comparisons of the response of the embryo to intravenous injection of leukotic (leukemic) blood as well as the blood of lymphomatotic (aleukemic) and normal birds m a y be made from the data in Table 1. The incidence of embryo-induced mor-
Sections of the chorio-allantoic tumors showed keratinization, vacuolization, and "piling u p " of the ectodermal cells. There was a general thickening of the mesoderm in which the developing cells resembled the granulocytic series. I n some of the tumors, necrotic foci were seen. Epithelial pearls were frequently observed in the mesenchyma. Leukotic cells when present were usually in small nests, perivascular in position. The presence of polychrome and erythroblastic elements in embryonic blood renders the detection of early and mild leukemoid and erythroblastic changes somewhat difficult. Toward the latter p a r t of the incubation period, however, there were occasionally noticed, with Strain 4 inoculum, earlier stages in erythroblastic development than were encountered following injection of blood from normal and lymphomatotic donors. Although the erythrogranuloblastosis Strain A of Hall and co-workers (1941) was employed in these studies there were not encountered in embryos the marked leukotic blood changes pictured by them. T h e embryonic changes suggest t h a t the leukotic state accompanies or is superimposed upon the disturbances induced b y injection with normal and non-leukemic tissue components.
FIG. 5.—Tibia (16478)—Phloxine—Methylene Blue—X200 Hyperplastic bone marrow showing increased cellularity.
bidity and percentage of hatchability were employed as criteria. With lymphomatotic blood, a somewhat, but perhaps not a significantly, lower incidence of embryo lesions was induced t h a n with leukotic blood and with normal blood. The percentage of hatch was, however, considerably below t h a t in the latter two groups. Blood from turkeys, which showed no evidence of the avain leukosis complex, produced the embryo disease. These changes were absent in embryos injected with blood
492
C. A. BRANDLY, FRANK THORP, J R . , AND C. 0 . PRICKETT TABLE 1.—Incidence of lesions and halchability
Inoculum Strain 4 blood Strain 4 plasma Strain 1 and 3 blood Normal chicken blood Normal chicken plasma Normal turkey blood Other species blood Saline or broth Uninoculated
No. embryos inoculated
Gross lesions
Chicks hatched
—
413 236 167 100 44 78' 136 94 292
of eight other species of birds (goose, pheasant, partridge, pigeon, sparrow and 3 ducks) and several samples of human blood. The reason why the blood of the pheasant, which species is more closely related to the chicken than is the turkey, did not induce a reaction similar to that obtained with turkey blood, warrants further consideration. However, the induction of osteal changes with human myeloid leukemia tissue (Thiersch, 1944) may suggest implication of non-specific factors. The tissues from male and female donors appeared to be equally effective in inducing leukemoid changes in embryos of both sexes. '•Furthermore, the blood of the sire and dam of embryos of several matings studied appeared to produce the disturbance as readily as the blood of other individuals of unrelated families. Only 3 percent of the 236 embryos which received injections of erythrogranulo-
No.
Percent
No.
Percent
206 7 59 46 2 16 0 1 1
49.9 3.0 35.3 46.0 4.5 20.5 0.0 0.94 .03
127 143 40 34 24 37 73 50 193
30.8 60.6 23.9 34.0 54.5 47.4 53.7 53.2 66.1
blastotic plasma and 4.5 percent of 844 embryos which received normal plasma showed gross lesions. The suggested greater concentration of the embryodisease-inducing factor in blood than in plasma would seem to be confirmed by the results of titrations. Quantities of plasma less than 0.05 cc. in no instance gave positive results whereas lesions of the embryo were provoked by quantities of blood as small as 0.00005 cc. When the whole blood was subjected to drying, laking with distilled water or rapid freezing to — 76°C, the embryo-aberrative effect appeared to be largely or entirely destroyed. Only one embryo in each of the saline and broth control groups showed gross visceral changes which resembled those provoked by the blood. The gross findings in these instances were not confirmed microscopically.
TABLE 2.—Production and serial passage of embryo aberration by normal blood
Inoculum
Normal chicken blood Normal chick blood* Normal embryo blood 1st and 2nd embryo passage of chicken blood 3rd and 4th embryo passage of chicken blood
Positive gross changes
No. embryos inoculated
No.
Chicks hatched
Percent
No.
Percent
100 27 46
46 1 0
46.0 3.7 0.0
34 20 27
34.0 74.1 58.7
105
22
21.0
32
30.5
28
0
0.0
18
64.3
* Includes chickens less than twenty-nine days of age.
493
TISSUE TRANSPLANTS AND AVIAN LEUKOSIS COMPLEX
Passage of Embryo-induced Disease. The results of attempts to perpetuate the embryo disease by serial passage are summarized in Table 2. Blood collected from 18 to 19 day embryos which had been injected at the 11th and 12th days of incubation was used for the subsequent series of inoculations. Sufficient data have not been obtained to determine the approximate age of the chick when its blood will cause the homoioplastic reaction in the embryo. It would appear that the normal-tissue-induced disease may not be perpetuated beyond the second passage. Blood from uninjected control
chamber injection was next in order (11.1). Inoculation into the allantoic chamber and into the yolk appeared to be virtually without effect in inducing the embryo disease. The absence of the latter condition probably accounts for the increase in percentage of chicks hatched. Incidence of A vian Leukosis Complex in Chicks Hatched from Injected Eggs. None of 58 chicks which hatched from normal blood- or plasma-injected eggs developed erythrogranuloblastosis within the 90-day observation period post hatching. In contrast, approximately 20 percent of the 25 chicks hatched from eggs inoculated
TABLE 3.—Influence of route of injection of chicken blood upon incidence of embryo disease and hatchability
Route
No. embryos inoculated
Intravenous Chorio-allantoic Amnionic sac Allantoic sac Yolk sac
100 44 324 51 42
Positive gross changes
Chicks hatched
No.
Percent
No.
Percent
46 14 36 1 0
46.0 31.8 11.1 2.0 0.0
34 13 181 27 29
34.0 29.5 55.9 53.0 69.0
embryos and from chicks 12 days or less in age did not, with the one exception noted, produce any gross changes. Thus, the factor which induces the embryonic disturbance would not appear to be present in the tissues during embryonic life or for several weeks after hatching. Furthermore, inability to perpetuate this disease after the 2nd passage in the embryo indicates that it is not capable of producing malignancy nor of continuous propagation under the circumstances which existed. Effect of Route of Injection. A summary of the effects of the route of injection of chicken blood upon the incidence of embryo disease and hatchability is given in Table 3. The intravenous route induced the highest percentage of gross lesions, (46) the chorio-allantoic route a lower incidence (31.8). The intra-amnionic
with Strain 4 blood and 33 percent of the 57 chicks which received Strain 4 plasma developed erythroganuloblastosis during the 90-days period post hatching (Table 4). Quantities of blood and plasma as small as 0.000005 cc. caused erythrogranuloblastosis in these chicks. Attention is drawn to the fact that quantities of chicken blood larger than 0.005 cc. usually prevented hatching; whereas this effect was not marked with quantities of plasma as large as 0.05 cc. The inoculation of chicks, 1 to 26 days of age, brought forth marked differences in response to blood and to plasma from leukotic donors (Table 4). The incidence of erythrogranuloblastosis was approximately 70 and 7 percent in the Strain 4 blood and plasma inoculated groups, respectively. It is seen that the average
494
C. A. BRANDLY, FRANK THORP, JR., AND C. 0.
incubation period (the time in days from inoculation until the development of an erythrogranuloblastotic blood picture)was much longer after plasma than after blood inoculation. These results indicated a much greater concentration and/or activity of Strain 4 agent in the blood cells and unfiltered plasma than in the plasma filtrate. On the basis of incubation period and incidence of the disease, it appears that
PRICKETT
also inoculated into a second series of embryos but blood from the latter failed to induce erythrogranuloblastosis in any of the 8 chicks inoculated with it. The blood of embryos inoculated with Berkefeld filtrates and leukotic chicken plasma failed to induce erythrogranuloblastosis in 10 chicks into which it was injected. Since the concentration of the oncogenic agent may be considered invariably to be greater in whole blood than
TABLE 4.—Incidence and mcubatton period of erythrogranuloblastosis in chicks hatched from inoculated eggs and chicks inoculated after hatching
Inoculum
Strain 4 erythrogranuloblastosis blood plasma Strain 4 erythrogranuloblastosis blood plasma
Quantum inoculum intravenously
No. inoculum
Age at inoculum
Erythrogranuloblastosis positive No.
Percentage
Average incubation period (days)
' 0.000005 to 0.05 cc. ea.
127
Embryos, 11—14 days development
25
19.7
43.8
0.000005 to 0.05 cc. ea.
143
Embryos, 11—14 days development
57
32.9
47.0
0.2 cc. per 100 gm. body wt.
191
134
70.2
24.8
0.5 cc. per 100 gm.
42
3
6.6
56.6
Chicks 1-26 days of age Chicks 1-26 days of age
the embryo was no more susceptible than the young chick, and possibly less so, to the inoculation of Strain 4 agent. Quantitative differences in the inocula received by the embryos and chicks would not, on the basis of experience with this and other strains, account for the degrees of difference observed. Incidence of the Avian Leukosis Complex in Chicks Inoculated with Embryo Tissue. Blood taken from 19-day embryos, 7 days after inoculation with Strain 4 blood, was inoculated intravenously into a limited number of chicks ranging in age from 2 to 8 days. Five of 8 chicks so inoculated developed erythrogranuloblastosis. The first embryo passage material was
in plasma or plasma filtrate, evidence for or against its multiplication in embryos cannot be deduced from these findings. Nevertheless, it would appear that under the circumstances serial passage in embryos did not provide optimal conditions for propagation of the strain in question. When Strain 3 lymphomatosisosteopetrosis blood was used for embryo passage and subsequent chick injection, the incidence of combined lymphomatosis and osteopetrosis in the injected chicks was as follows: 1st passage, 30 of 45 positive (66.6 percent); 2nd passage, 6 of 16 positive (37.5 percent); and 3rd passage, 8 of 10 positive (80.0 percent). Twenty, four and five birds of these
TISSUE TRANSPLANTS AND AVIAN LEUKOSIS COMPLEX
respective groups showed osteopetrosis either alone or in combination with visceral or neural lymphomatosis. Six of 20 uninoculated control chickens developed lymphomatosis but none showed osteopetrosis. The prevalence of lymphomatosis in the controls (30 percent) raises doubt as to the significance of lymphomatosis in the inoculated group even though the incidence was twice as great in the latter. The assumption that an osteopetrotic factor was transmitted may be open to question in view of Gohs (1934) observation that injection of embryonic marrow may induce osteopetrotic or similar changes in chickens. Likewise, Theirsch (1944) has recently recorded the induction of an osteopetrosis-like condition in 3 of 9 chickens hatched from embryos injected intra-amnionically with blood from two human cases of chronic myeloid leukemia. DISCUSSION AND SUMMARY
The injection of apparently normal chicken blood and tissues may produce pathological changes of the chicken embryo which are grossly, and to a certain extent, microscopically similar to those induced by leukotic material. This emphasizes the need for caution in interpretation of results. The disease of the embryo and extra-embryonic membranes here described is apparently identical with t h a t ' found by Murphy (1916) and Dantschakoff (1920) to be associated with chorioallantoic grafts of certain organs of older chickens. The normal organ implants were, however, made only in younger embryos, i.e., those of 7 days' incubation. In addition to the changes seen by Murphy and Dantschakoff our work has revealed significant bone and liver lesions. Furthermore, the embryonic disease resulted when chicken and turkey bloods were introduced by several channels—
495
the intravenous, supra-chorio-allantoicand intra-amnionic, but only in a negligible number, or not at all, by the allantoic or yolk chamber routes. The tissues of both male and female chicken and turkey donors were found to produce the leukemoid disease in embryos of both sexes. The additional observation that the blood of both sire and dam of certain matings studied was as effective as that of unrelated normal individuals, suggests that the response of the embryo to normal homologous tissue is a function of age and individual differentials. With Strain 4, for example, a quantity of inoculum so small as not to induce leukemoid embryo disease yet capable of producing erythrogranuloblastosis, required an incubation period greatly in excess of the remaining period of intra-shell life. The possibility does exist, however, that this or other strains of the agent may be adapted to a shorter incubation period in the embryo and thus to serial propagation within the egg. Erythrogranuloblastosis developed in some of the chicks hatched from eggs inoculated with Strain 4 material by each of the routes employed. None of the uninoculated controls or those injected with blood from apparently normal birds or lymphomatotic donors developed erythrogranuloblastosis. However, lymphomatosis occurred in all groups although in a higher percentage in the recipients of leukosis inoculum (15 percent) than in the controls (8 percent). A number of other workers have found that chicks hatched from eggs inoculated by several routes may develop leukosis. Jarmai and co-workers (1932, 1933) obtained positive results following injection into the albumen after the 9th day of incubation. Storti and Mezzadra (1938) concluded that leukosis virus could survive in tissues instilled upon the chorio-
496
C. A. BRANDLY, FRANK THORP, J R . , AND C. 0 . PRICKETT
allantoic membrane at the 4th day of development but that it did not multiply. Van den Berghe and d'Ursel (1939) reported serial passage of fowl leukosis on the chorio-allantoic membrane of chicken embryos and 5 of 6 chicks hatched from eggs of the 1st to 4th serial passage developed the disease 8-19 days after hatching. Using Furth's Strain 1, Kirschbaum, Stern and Hooker (1940) found that leukosis developed in chicks hatched from eggs inoculated via the albumen on the 12th day but not in those inoculated on the 7th day of incubation. Two of four chickens hatched after intra-amnionic sac injection of plasma on the 7th day developed leukosis when 46 and 58 days of age. Hall, Bean and Pollard (1941) reported leukotic changes in the embryo following intravenous injection of leukotic blood but not after chorio-allantoic and yolk injection. They failed to induce "leukotic" changes with filtrates of leukotic tissue or plasma and did not report the fate of chicks which hatched from injected eggs. Pierce (1942) failed to observe leukosis in chicks hatched from eggs treated after the 8th day of incubation by instilling filtrates of leukotic tissue and plasma upon the chorio-allantois. However, positive results were obtained with unfiltered tissue suspensions and plasma. Our observations suggest that inoculation of the embryo, particularly by the intravenous route, with relatively large quantities of leukotic blood may result in a successful transplantation or graft similar to that described by Crank and Furth (1932) after massive inoculation of susceptible chickens. The host apparently may be overwhelmed by such a graft. Upon injection of considerably smaller quantities of leukotic cells, the graft may apparently fail to become established although as with the introduction of tissue
and plasma freed of leukotic cells, erythrogranuloblastosis may be initiated by the oncogenic agent present in the leukotic fluids. The requisite incubation period in the latter instances would seerri to preclude the direct transfer of most strains of erythrogranuloblastosis from chickens to serial passage in embryos. Aside from serving the function of a culture medium for autonomous multiplication of implanted leukotic cells, the tissues of the embryo may obviously respond to cell-free leukosis-producing agent. Initiation of the specific disease processes is followed by an appropriate incubation period and the postnatal appearance of the disease. The early leukemoid and lymphomatoid changes brought about by normal and non-leukemic tissue components obviously tend to complicate these phenomena and their interpretation. In earlier observations Storti and Mezzadra (1938), and Van den Berghe and d'Ursel (1939) found that the leukosis agent placed on the chorio-allantoic membrane may remain alive for a considerable period without growth. Pierce also found no evidence that the infectivity of leukotic filtrates was increased by incubation in the chorio-allantoic membrane. In view of these findings together with the observation of Pierce (1942) that transplanted cells may migrate from the chorioallantois into the embryo, it is not surprising that Hall et al. (1941), and Pollard and Hall (1942) demonstrated the agent to be present in the embryo after one or several "passages" of leukotic chicken blood by the intravenous route. Demonstration of the agent under these circumstances may not be taken as proof that multiplication has occurred nor indisputable evidence that the "leukemic" and leukemoid changes were provoked exclusively by the leukosis virus. Negative results were obtained by Hall
TISSUE TRANSPLANTS AND AVIAN LEUKOSIS COMPLEX
and co-workers (loc. cit.) from injection of embryos with filtrates of leukosis-inoculated embryos or the serum of leukotic chicks, whereas these materials when inoculated into chicks produced leukosis after an appropriate incubation period. Pierce (1942) obtained similar results with plasma filtrates. These experiences would further suggest that the gross lesions in the embryos were associated at least in part with homoioplastic tissue reactions. CONCLUSIONS
1. The tissues of apparently normal chickens as well as those of aleukemic lymphomatosis and of erythrogranuloblastosis affected birds may induce pronounced and extensive leukemoid and related processes in chicken embryos injected with them. 2. Specific erythrogranuloblastotic processes may accompany or follow these phenomena in embryos injected with relatively large quantities of blood representing a single strain of erythrogranulobastosis. 3. The injection of relatively small quantities of erythrogranuloblastotic blood or of cell-free plasma may result in development of the uncomplicated specific disease, after a suitable incubation period, in chickens hatched from inoculated eggs. 4. Attempts to propagate two strains of lymphomatosis and one of erythrogranuloblastosis by continuous serial passage in chicken embryos were unsuccessful. The developmental period of the uncomplicated disease after injection, exceeded the remaining period of embryonal life. 5. Recognition of the role of nonspecific influences or factors in the response of the embryo to these and other neoplasms is essential to proper interpretation of the several phenomena involved. The authors are indebted to Dr. S. B.
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Wolbach for assistance in the evaluation of the histologic changes and to Dr. Paulo Dacorso for preparation of the photographs. REFERENCES
Brandly, C. A., 1941. Progress report on several phases of pathology research. Second Collaborators' Conference, U. S. Bureau of Animal Industry, Regional Poultry Research Laboratory, East Lansing, Michigan, pp. 23-38. Brandly, C. A„ N. M. Nelson and G. E. Cottral, 1942. Serial passage of lymphomatosis-osteopetrosis in chickens. Am. J. Vet. Res. 3: 289-295. Burnet, F. M., 1936. The use of the developing egg in virus research. Medical Research Council, Special Report Series No. 220. His Majesty's Stationery Office, London. Crank, F. P. and J. Furth, 1932. Transfusion experiments with the blood of leukemic chickens. Arch. Path. 14: 660-670. Dantschakoff, Vera, 1920. Myeloid metaplasia of the embryonic mesenchyme in relation to cell potentialities and differential factors. Carnegie Institution of Washington, Contributions to Embryology No. 49: 3-32. Gohs, W., 1934. Uber die Wirkung arteigener Knochen- und Knochenmarkzerfallstoffe aud die Knochen- und Blutbildung der Huhner. (Experimentele bei Huhner erzeugte Osteo dystrophia fibrosa, Myelosiche Leukose, Erythramia und schwere Anamie). Frankfurt Ztschr. f. Path. 46: 453-476. Hall, W. J., C. W. Bean and M. Pollard, 1941. Transmission of fowl leucosis through chick embryos and young chicks. Am. J. Vet. Res. 2: 272279. Jarmai, Karl, Tibor Stenszky and Ladislaus Farkas, 1932. Neuere Beitrage zur Kenntnis der iibertragbaren HUhnerleukose. Arch. f. Wlsensch. u. prakt. Tierh. 65:46-71. Jarmai, Karl, 1933. Infektionsversuche bebriiteter Eier mit dem "virus" der Huhnererythroleukose. Deut. tierarztl. Wchnschr. 79: 418-420. Kirschbaum, Arthur, K. G. Stern and C. W. Hooker, 1940. Studies on fowl leukosis. Transfer with fractions obtained by ultracentrifugation of leukemic plasma and bone marrow extracts. Yale J. Biol. & Med. 13: 1-14. Murphy, J. B. and P. Rous, 1912. The behavior of chicken sarcoma implanted in the developing embryo. Am. Med. Assoc. J. 56: 119-132. Murphy, J. B., 1916. The effect of adult and chicken organ grafts on the chick embryo. J. Exper. Med. 24: 1-5.
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Pierce, Mila, 1942. Studies on fowl leukosis. Transfer to the chick embryo. Am. J. Path. 18:1127-1136. Pollard, Morris, and W. J. Hall, 1942. Interspecies transmission of avian leucosis in embryos. Am. J. Vet. Res. 3:247-252. Rous, Peyton, and J. B. Murphy, 1911. Tumor implantations in the developing embryos. Experiments with a transmissable sarcoma of the fowl. Am. Med. Assoc. J. 56: 741-742. Second Collaborators' Conference, 1941. Recommendations of the pathology committee. TJ. S. Bureau of Animal Industry, Regional Poultry Research Laboratory, East Lansing, Michigan, p. 119.
Storti, E., and G. Mezzadra, 1938. Tentatives de culture du virus de la leuceme des poules dans la membrane chorio-allantoide (Note preliminaire). Sang. 12:533-539. Thiersch, J. B., 1944. Attempts to transmit leucaemia of man and of mice to the chick embryo and to the young chick by the amnionic and intravenous routes. Austr. J. Exp. Biol, and Med. Sci. 22: 57-61. Van den Berghe, L., and F. d'Ursel, 1939. Culture entissus du virus de l'erythroblastose de la poule (Souche O. G.) Compt. rend. Soc. de Biol. 131: 1301-1302.
Book Review H. C. Fryer Fortune. Cosmos Publishing Company, Hollywood, California. 1949. 115 pages plus 41-page supplement. Price $7.50.
SHAW,
The first 115 pages of this booklet are printed in excessively large type with about 7 words to a line and only on one side of each page. There are a number of full page diagrams and illustrations. The entire booklet is printed on a heavy weight paper and bound by 12 rigid metallic rings. The subject matter of the main part of the booklet is devoted almost exclusively to one type of battery raising of fryers which was used by the author in the relatively mild winter area of the California
coast. It is written in the first person and describes the author's experiences and conclusions. Thirty one pages of the supplement, in normal size type, are occupied by "Brooding and Rearing Chickens" credited to California Extension Bulletin 127. The floor method of growing fryers is said to be used extensively in the eastern section of the United States. Is it not used extensively in all sections of the country? Brooding in batteries continues to have some sort of glamorous attraction. Even with this glamor for the novice, it is difficult to understand how the first edition of 1000 copies was sold by the author at $12.50 each.