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Natural Transmission of Avian-Lymphomatosis
Natural Transmission of Avian-Lymphomatosis NELSON F. WATERS Geneticist, Regional Poultry Research Laboratory, Agricultural Research Administration, United States Department of Agriculture, East Lansing, Michigan
MATERIAL AND METHODS
A
KEY to the control of lymphomatosis may be present in studies which involve natural transmission of the disease. Doyle (1927) was one of the first investigators to suggest that this disease was transmitted by way of the egg. Later numerous investigators, Warrack and Dalling (1932), Biely et at. (1932), Seager (1933), Blakemore and Glover (1935), Gibbs (1936), Tower (1937), McClary and Upp (1939), and Lee and Wilcke (1941), made suggestions and presented evidence which incriminated both the egg and infected birds as carriers of some form of lymphomatosis. Further evidence by Waters and Prickett (1944) has strengthened the conclusion that the egg and the infected bird serve as a means of transmission of the disease. The object of the present study, from data accumulated over a period of five years, is to add further factual evidence that the disease can be transmitted under natural conditions both through the egg and by contact. Many variables by the method of experimentation cannot be fully evaluated as yet, such as the most susceptible age in the life of the chicken, the natural resistance, the degree of exposure, the mode of transmission and the diet. Each of these factors when known will contribute to the knowledge of the etiological agent and contributing causes of this disease.
The foundation birds used in this study represented nine different flocks and were obtained as hatching eggs originating from widely separated geographic regions. No other chickens have been brought onto the Laboratory premises since the original introduction made in 1939. The first chickens obtained at this Laboratory were housed in newly constructed buildings. A rigid sanitary program, with quarantine regulations, was established with the beginning of the research program and remains in force. Each year, before brooding facilities are placed in operation, the houses and equipment are thoroughly cleaned and fumigated before a new population enters a unit. The brooder house is provided with a service room wherein personnel must change all outside clothing and shoes before entering the various pens. All young chicks are placed in a brooder house not used by adult chickens. Growing stock is transferred from the brooding house to a laying house at approximately four months of age. Sanitary precautions similar to those used in the brooder house are also required in all other poultry buildings. All the birds used in this experiment were kept in complete, confinement throughout their entire life. All the pens are provided with windows screened with 16mesh wire to prevent the entrance of in-
226
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(Received for publication, September 1,1944)
NATURAL TRANSMISSION OF AVIAN-LYMPHOMATOSIS
None of the birds reported on in this study were inoculated and they consisted of females only. Mortality- statistics prior to 30 days of age are not included in this report. Necropsies were performed on all birds over 30 days of age and diagnoses were based on gross necrospy findings only. EXPERIMENTAL RESULTS
Within a few months after the hatching of chickens on the Laboratory premises, numerous cases of lymphomatosis appeared among the chickens of the first, or 1939, population. Despite the fact that all chickens were introduced as eggs and reared in newly constructed houses under complete confinement with rigid sanitary and quarantine precautions, the disease made its first appearance 40 days after the first chicks were hatched. The total
lymphomatosis mortality during the first 300 days was 12.3 percent and at the end of 600 days it had increased to 21.9 percent as shown in Table 1. The appearance of lymphomatosis in this first population is especially sigificant in that none of the common infectious diseases of poultry and no parasites, except coccidia, have been observed among the Laboratory chickens. These first year data demonstrate that in this case lymphomatosis could not have been spread through direct contact with infected chickens. Thus, these results definitely incriminate the egg as a source of lymphomatosis infection. Table 1 provides additional evidence that lymphomatosis may be transmitted by way of the egg. Each of the four subsequent populations, 1940 to 1943, were placed in the brooder house at hatching and at no time up to four months of age was direct contact with older and infected birds possible. Nevertheless, each year lymphomatosis made its appearance in the brooder house during this early period. At approximately four months of age all chickens in the last four populations were transferred each year from the brooder house to a laying house in which birds from a previous population or populations were housed. However, direct bird to bird contact with adult chickens of previous populations was not possible in the laying house in that only birds hatched during the same year were placed in the same pen. The gradual and continuous 30-day increase in lymphomatosis up to 600 days of age in all five populations under study raises the question as to whether all the chickens which died from the disease received their initial infection from the egg. Evidence will be submitted later to show that a considerable portion of the observed incidence of lymphomatosis was transmitted by direct contact with birds
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sects. All birds were fed a similar ration, consistent with the availability of feedstuffs, throughout the experimental period. This report involves approximately 4,700 White Leghorn chickens which were divided into groups and subjected to several different environmental modifications in an attempt to measure the influence of such environment as it affected the incidence of lymphomatosis. Each year, for five years, 1939 to 1943 inclusive, a main population of chickens was incubated, hatched, and reared as a mixed group. In addition certain groups of chickens were incubated, hatched, and reared by families, in complete isolation. Still other groups originated as eggs from chickens held in complete isolation, but such eggs were incubated, hatched, and the birds reared in contact with a mixed population or group throughout life. One group which was hatched and reared in complete isolation was exposed to the mixed population after 300 days of age.
227
308 e h i e k e n s Lympho. m o r t a l i t y 3 0 0 d a y a - 8.1J. 600 days - 2 3 . 1 $
1074 e h i e k e n s Lympho. m o r t a l i t y 300 days - IS.7% 600 daya - 3 4 . ( 9
1059 ehiekana Lympho. m o r t a l i t y 300 days - 19.Tit 600 daya - 3 4 . 9
101.4 chickens Lympho. m o r t a l i t y 300 d a y s . - 21.2<
1940
1M1
1942
1943
1
494 e h i e k e n s Lympho* m o r t a l i t y 300 days • 1 2 . 3 * 600 days - 21.9%
1959
11
I
1
eggs
1 3 "progeny f r e e
^
100 progeny incubated and reared i n i s o l a t i o n * • f r e e from lympho. a t 300** days o f a g e .
1
eggs
4
4 dame from f r e e s t o c k
1 1 I 1 |
egge 91 progeny 1Incubated and reared i n i a p l a t l o n . Tree from lympho. a t 300 daya of a g e .
1
4 daa» from f r e e stock
1 ^—
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92 progeny incubated and reared i n i s o l a t i o n * Free from lympho* a t 300 days o f a g e .
I i
I n e l d a n e s o f lymphomatosis among ehlokens r e c e i v i n g v a r i o u s d e g r e s s o f i s o l a t i o n from or e o n t a o t w i t h , a mixed p o p u l a t i o n having a knom lneldenee of the d i s e a s e .
4
4
,
i
40 progeny incubated and reared with mixed flock. 2056 lympho. a t 300 daya of a g e .
51 progeny incubated and reared with nixed f l o c k . 285C lympho. a t 300 daya of a g e .
0
83 progeny incubated and reared with mixed f l o c k . « l £ lympho. a t 300 days of a g e .
dams
.Lymphomatosis. 28 b i r d s k i l l e d a n d a u t o p s i e d a t about 200 days of a g e .
1
Incubated and reared with mixed f l o c k . 3Cg& lympho. a t 300 daya o f a g e .
«125 progeny4
13 dame from f r e e atoc k • eggs
and reared w i t h mixed flock* 32jS lympho. a t 300 daya of a g e .
.71 progeny4 incubated
egga
i
6 dams from f r e e stock
and reared In i s o l a t i o n . 16* lympho. a t 300 daya of a g e .
»64 progeny4incubated
9 "progeny i n f e e t e d eggs
1
122 daiu were e e l e c t e d a t about 300 daya of a g e from t h e 1940 (mixed p o p u l a t i o n t o proyide egga f o r t r a n s m i s s i o n a t u d l e e . i
»
11.
B1TOHAL T8aBSMISSI0» Of IT IIS LYMPHOMATOSIS
S o l i d boxed l i n e s represent- contact' with mixed p o p u l a t i o n throughout l i f e . Broken boxed l i n e s r e p r e s e n t c o n t a c t with mixed p o p u l a t i o n a f t e r 300 days of a g e .
I . I n c i d e n c e of lymphomatosis f o r v a r i o u s y e a r s among e h i c k s n e hatched and reared a s a mixed p o p u l a t i o n .
TMr
CHaBT 1 .
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.
4
H progeny incubated and reared w i t h mixed f l o c k . 3 # lympho. a t 300 daya o f a g e .
dam*
^*i« ot age.
w
_ ^ _
J
1 ,, r i f t e r 300 daya o f age 2 0 * 1 f r e e stock c h i c k e n s from 1 F were p l a c e d i s c o n t a c t * w i t h t h e mixed, p o p u l a t i o n . ' Pr«e from lympho. a t 700 1
1
NATURAL TRANSMISSION OF AVIAN-LYMPHOMATOSIS
which presumably receive their infection through the egg. The data on all five populations, however, do provide evidence that egg transmission plays an important part in that none of the chickens in the different populations had any direct contact with infected birds up to four months of age. Indeed, the first population had no possible contact with other chickens up to at least one year of age.
229
exposed to a mixed population with a known incidence of lymphomatosis throughout their lives. The broken-boxed lines enclose data having contact with the mixed population after 300 days of age. The prevalence of lymphomatosis in the first population seriously handicapped the general Laboratory program and an attempt was made to eliminate or reduce the occurrence of this disease.
1942 (1,059 birds) No. Percent
1943 (1,014 birds) No. Percent
Age distribution in days
1939 (494 birds) No. Percent
3 1 - 60 6 1 - 90 91-120 ' 121-150 151-180 181-210 211-240 241-270 271-300 301-330 331-360 361-390 391-420 421-450 451-480 481-510 511-540 541-570 571-600
1 3 - 8 10 6 8 11 7 7 7 7 9 2 3 5 6 2 2 4
.2 .8 2.4 4.5 5.7 7.3 9.5 10.9 12.3 13.8 15.2 17.0 17.4 18.0 19.0 20.2 20.6 21.1 21.9
0 0 1 2 1 4 5 8 4 5 3 6 1 7 4 6 7 4 3
. .0 .0 .3 1.0 1.3 2.6 3.9 6.8 8.1 9.7 10.7 12.7 13.0 15.3 16.6 18.5 20.8 22.1 23.1
1 2 11 11 28 43 48 37, 31 24 31 14 18 11 11 11 9 15 9
.1 .3 1.3 2.3 4.9 8.9 13.4 16.9 19.7 22.0 24.9 26.2 27.8 28.9 30.0 30.9 31.8 33.1 34.0
2 10 8 15 23 43 32 40 36 25 18 25 16 16 12 14 11 8 8
.2 1,1 1.9 3.3 5.5 9.5 12.6 16.3 19.7 22.1 23.8 26.2 27.7 29.2 30.3 31.6 32.7 33.4 34.2
3 12 14 22 29 41 37 27 30
.3 1.5 2.9 5.0 7.9 11.9 15.7 18.2 21.2
Total
108
21.9
71
23.1
365
34.0
362
34.2
215
21.2*
%
•
1940 (308 birds) No. Percent
1941 (1,074 birds) No. Percent
* 300 days of age. OTHER EXPERIMENTS ON NATURAL TRANSMISSION
A summary of all experiments by years involving studies on natural transmission of lymphomatosis is presented in chart 1. For comparative purposes the five populations which were previously discussed are summarized in this chart also. Each phase of the transmission experiments is identified by a letter for easy reference. The mixed populations for the different years act as controls for other phases of the transmission studies. All solid-boxed lines enclose data on chickens which had been
It will be observed in Table 1 that the incidence of lymphomatosis in the 1940 population is considerably less during the early periods than in all other populations. This is due to a biased condition in that for the first 100 days of age all chickens in a family (progeny from one sire and dam) were discarded when the first case of lymphomatosis appeared. This procedure was adopted in an attempt to free this population from the disease. I t was discontinued when it became apparent that such a procedure would eventually eliminate the entire population.
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TABLE 1.—Accumulative percentage incidence of lymphomatosis mortality at intervals of 30 days for five different populations of chickens
230
NELSON F. WATERS
reared with the mixed flock showed 32 percent lymphomatosis. These experiments which provided both free and infected progeny under strict isolation are interpreted to mean that the progeny which showed no lymphomatosis at 300 days of age were free because their dams were free, whereas at least some of the progeny which showed 16 percent lymphomatosis were infected because the disease was transmitted from dam to progeny by way of the egg. Evidence for contact transmission is most convincing in that representative sibs, which were derived from each of the same dams providing chickens for the isolated pens, showed a relatively high incidence of lymphomatosis when exposed to the mixed flock. In an attempt to increase the number of free stock chickens during the year 1942 a total of 19 females and 1 male of the free chickens from B of 1941 were selected for breeding purposes and were allowed to remain in complete isolation after 300 days of age. It is important to point out that these 20 chickens were still free of lymphomatosis at 600 days of age. Eventually 4 of the 19 females were selected to produce progeny, representatives of which were to be hatched and reared both in isolation and in contact with the mixed flock. At the end of 300 days of age none of the progeny, F, which were hatched and reared in strict isolation, showed gross evidence of lymphomatosis. Sib progeny, G, of each family placed in strict isolation, were hatched and reared with the mixed flock. The incidence of lymphomatosis within these exposed progeny was 28 percent at 300 days of age. In 1943 (I and J) the procedures followed in 1942 were repeated and, with the exception of 28 sib's in I which were confined alone in one isolated pen since hatching and which were removed at 210 days
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Following this initial failure to eliminate lymphomatosis other attempts were made to obtain free stock by hatching and rearing chickens by families in strict isolation. The success of these subsequent attempts to obtain free stocks, together with evidence for egg and contact transmission of lymphomatosis, is described by Waters and Prickett (1944). From the 1940 mixed population 22 dams, A, were selected at about 300 days of age to provide eggs for transmission studies. At the time these birds were selected no lymphomatosis had appeared in the families from which they were chosen. During the time eggs were being obtained for subsequent studies all the 22 dams were maintained in contact with the mixed flock. Eggs from each of the 22 dams were transferred each day. to rigidly quarantined and isolated quarters. At the time of transfer the eggs were dipped and cleaned in a 70 percent solution of alcohol maintained at room temperature. The eggs from each of these hens were incubated and the progeny reared by families in strictly isolated pens. The behavior of these families of progeny with reference to lymphomatosis mortality is shown for the year 1941. In all, 13 dams now termed "progeny free" produced 92 progeny, B, which when incubated, hatched and reared in strict isolation were free of lymphomatosis at 300 days of age. Eggs from these same "progeny free" dams also produced 83 progeny, C, which when incubated, hatched, and reared with the mixed flock, showed 21 percent of lymphomatosis at 300 days of age. Nine of the 22 dams, now termed "progeny infected," produced 64 progeny, D, which when incubated, hatched, and reared in isolation showed 16 percent lymphomatosis at 300 days of age. Eggs from these "progeny infected" dams also produced 47 progeny, E, which when incubated, hatched and
NATURAL TRANSMISSION OF AVIAN-LYMPHOMATOSIS
Additional evidence for contact infection was provided in the years 1942 and 1943. Facilities were not available to test in isolation the progeny of all free stock dams. It was possible, however, from a few of these free dams to hatch and rear progeny in contact with the mixed infected flock. In chart 1, under H and K for the years 1942 and 1943 respectively, 19 such families representing 196 chickens were tested. The data for these two years show about 31 percent of lymphomatosis at 300 days of age. In all the contact experiments, which include the C, E, G, H, J, and K phases, the incidence of lymphomatosis at 300 or more days of age is, for the most part, in excess of that found in the mixed populations at the same age. This fact provides evidence that the related free stock groups B, F, and I, were free because the disease was not present and not because they were resistant to the disease. During 1943 evidence was obtained which indicates that there may be some association between the age at exposure and the subsequent incidence of lymphomatosis. At the end of a 300-day period 20 chickens, L in chart 1, representing 4 free families which had been isolated under strict quarantine during 1942, were trans-
ferred to quarters where a high incidence of the disease was present. After 400 days of this type of exposure no losses from lymphomatosis have occurred. A total of 51 sibs, G, of the 20 chickens which were continually exposed to the mixed flock from hatching to 300 days of age showed 28 percent of lymphomatosis, and at 600 days the incidence had increased to 47 percent. DISCUSSION AND CONCLUSIONS
Evidence obtained at this Laboratory that lymphomatosis is transmitted through the egg and spread by direct contact with infected chickens has been presented. The first chickens introduced as hatching eggs were placed in new buildings and serviced with new equipment. A most rigid sanitary and quarantine program was adopted and put into operation. Despite these precautions lymphomatosis made its appearance within 40 days after the first chickens were hatched and placed in the brooder house. Subsequent populations of chickens" -were placed in the brooder house immediately after hatching. All chickens within each year were confined to the brooder house until they were about four months of age. The data show that in every population some chickens had lymphomatosis while they were still in the brooder house. The occurrence of this disease among young chickens during the first four months of their lives and before they had had any contact with infected birds, raises ., the question from whence did i t arise. Control of the enviroment did prevent, for a period of five years, the entrance of all common infectious diseases and all parasites, except coccidia, but it did not prevent the entrance of lymphomatosis. Data have been presented which indicate that the incidence of lymphomatosis among the five different populations of
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of age, the results were essentially the same. At about 200 days of age, 2 of the 28 sibs mentioned above showed certain clinical symptoms of lymphomatosis. As a possible safeguard to the birds in other pens, the 28 sibs were immediately killed and autopsied. Gross examination on all of these birds showed no evidence of lymphomatosis. Microscopic examination, however, did show pathology, commonly interpreted as lymphomatosis, in 57 percent of the birds. It should be stressed that the parental chickens of the progeny in I and J for 1943, were still free of lymphomatosis at 700 days of age.
231
232
NELSON F. WATERS
It would seem clear from the data presented that lymphomatosis is transmitted from parent to offspring by way of the egg and that no method which includes isolation, confinement, sanitation, or quarantine during the early period of the
chicken's life will eliminate or control the disease unless the eggs are also free. The evidence for contact transmission of lymphomatosis is most conclusive, although the precise period or the exact manner of greatest exposure is not known. Three separate trials extending over a three-year period show that chickens which were hatched and reared with infected birds showed a high incidence of lymphomatosis, whereas their sibs hatched and reared in isolation remained free. The data, though in part meagre, indicate that there is an association between the age of a bird at the time it is exposed to lymphomatosis and its ability to resist infection. The evidence is clear that when representatives of four different families, which were hatched and reared in strict isolation for 300 days, are exposed to a mixed flock with a known incidence of lymphomatosis, for an additional period of 400 days, no deaths from the disease result. In contrast, when the sibs of these same four families were exposed to an infected mixed flock, from hatching to 600 days of age, a very high lymphomatosis mortality results. The presence of microscopic lesions showing pathology commonly interpreted as lymphomatosis in certain of the free stocks during 1943 may have important implications which cannot be discounted. It should be remembered that none of the birds showing microscopic lesions could be classed as grossly, positive for lymphomatosis at necropsy. It is not the intent at this time to pass on the merits of present methods for diagnosing lymphomatosis microscopically. It may be pertinent to ask, however, whether the tissues of a chicken will show abnormal extravascular accumulations of mononuclear cells only when stimulated by the
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chickens reported on could not be attributed, at least during the first four months of the brooding period, to direct contact with previously infected fowl. Indeed, the first population of chickens in 1939 had no contact, up to at least one year of age with any other chickens whatsoever. The criticism may be advanced that lymphomatosis may have been introduced to the Laboratory chickens by the same channels as were the sporoblasts of coccidia. Such a method of introduction cannot be ruled out, but if this were true it would be pertinent to ask why, with the above exceptions, none of the other infectious diseases or parasites common to poultry have made an entrance during a five-year period? Additional and more exacting evidence on egg transmission is provided in experiments which involved the incubation and hatching of families of chickens in individually isolated and strictly quarantined pens. Some of these families showed cases of lymphomatosis while other families remained entirely free up to at least 700 days of age. These results are interpreted to mean that in some instances lymphomatosis was transmitted from parent to offspring by way of the egg. The criticism may arise that these free families were free because they were resistant to the disease. That such a criticism is not valid is demonstrated by the fact that sibs of these free families, when hatched and reared with a known infected flock, show a relatively high incidence of lymphomatosis.
NATURAL TRANSMISSION OF AVIAN-LYMPHOMATOSIS
233
6. The hatching and rearing of chickens in strict isolation, even though placed in an environment where the best known sanitary practices or quarantine measures exist, will not prevent per se the occurrence of lymphomatosis unless the parents of such birds are free of the disease.
disease, lymphomatosis. If lymphomatosis were present in these free chickens in excess of all others involved in actual contact experiments at 200 days of age, then some indirect means of transmission must be held accountable. SUMMARY x
REFERENCES
Biely, Jacob, Elvira Palmer and V. S. Asmundson, 1932. Inheritance of resistance to fowl paralysis (neurolymphomatosis gallinarum). Can. Jour. Res. 6:374-380. Blakemore, F., and R. E. Glover, 1935. Fowl paralysis. I I . Experiments on transmission 4th rpt. director Camb. Univ., Inst. An. Path. 1934-35:5164. Doyle, L. P., 1927. Neuritis or paralysis in chickens. Proc. 64th Annual Meeting A.V.M.A. Gibbs, Charles S., 1936. Observations and experiments with neurolymphomatosis and the leukotic diseases. Mass. Agri. Exp. Sta. Bui. 337:1-31. Led, C. D., and H. L. Wilcke, 1941. Transmission experiments with iritis of fowls. Am. Jour. Vet. Res. 2:292-294. McClary, C. F., and Chas. W. Upp, 1939. Is paralysis of fowls, as manifested by iritis, transmitted through the egg? Poultry Sci. 18:210-219. Seager, E. A., 1933. The pathology of fowl paralysis-, with some aspects of its cause and control. Vet. Jour. 89:454^473. Tower, Benjamin A., 1937. Breeding as a factor in the control of blindness and paralysis in fowls. La. State Univ. Thesis: 1-71. Warrack, G. H., and T. Dalling, 1932. So-called "fowl paralysis." Vet. Jour. 88:28-43. Waters, Nelson F., and C. O. Prickett, 1944. The development of families of chickens free of lymphomatosis. Poultry Sci. 23:321-333.
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1. This report involves approximately 4,700 White Leghorn chickens which were divided into groups and subjected to several different environmental modifications in an attempt to measure the influence of such environment as it affected the natural transmission of lymphomatosis. 2. All chickens were reared throughout life in complete confinement and were maintained in an environment of strict sanitation and quarantine. 3. Lymphomatosis was present in chickens under four months of age despite the fact that direct contact with previously infected birds did not exist. 4. Evidence is presented to show that lymphomatosis is transmitted both by way of the egg and by contact with infected birds. 5. Careful selection and testing of certain families hatched and reared in isolation and maintained under rigid quarantine have prevented the occurrence of lymphomatosis to at least 700 days of age.
MATERIAL AND METHODS
A
KEY to the control of lymphomatosis may be present in studies which involve natural transmission of the disease. Doyle (1927) was one of the first investigators to suggest that this disease was transmitted by way of the egg. Later numerous investigators, Warrack and Dalling (1932), Biely et at. (1932), Seager (1933), Blakemore and Glover (1935), Gibbs (1936), Tower (1937), McClary and Upp (1939), and Lee and Wilcke (1941), made suggestions and presented evidence which incriminated both the egg and infected birds as carriers of some form of lymphomatosis. Further evidence by Waters and Prickett (1944) has strengthened the conclusion that the egg and the infected bird serve as a means of transmission of the disease. The object of the present study, from data accumulated over a period of five years, is to add further factual evidence that the disease can be transmitted under natural conditions both through the egg and by contact. Many variables by the method of experimentation cannot be fully evaluated as yet, such as the most susceptible age in the life of the chicken, the natural resistance, the degree of exposure, the mode of transmission and the diet. Each of these factors when known will contribute to the knowledge of the etiological agent and contributing causes of this disease.
The foundation birds used in this study represented nine different flocks and were obtained as hatching eggs originating from widely separated geographic regions. No other chickens have been brought onto the Laboratory premises since the original introduction made in 1939. The first chickens obtained at this Laboratory were housed in newly constructed buildings. A rigid sanitary program, with quarantine regulations, was established with the beginning of the research program and remains in force. Each year, before brooding facilities are placed in operation, the houses and equipment are thoroughly cleaned and fumigated before a new population enters a unit. The brooder house is provided with a service room wherein personnel must change all outside clothing and shoes before entering the various pens. All young chicks are placed in a brooder house not used by adult chickens. Growing stock is transferred from the brooding house to a laying house at approximately four months of age. Sanitary precautions similar to those used in the brooder house are also required in all other poultry buildings. All the birds used in this experiment were kept in complete, confinement throughout their entire life. All the pens are provided with windows screened with 16mesh wire to prevent the entrance of in-
226
Downloaded from http://ps.oxfordjournals.org/ at FU BerlinFB Humanmedizin on May 4, 2015
(Received for publication, September 1,1944)
NATURAL TRANSMISSION OF AVIAN-LYMPHOMATOSIS
None of the birds reported on in this study were inoculated and they consisted of females only. Mortality- statistics prior to 30 days of age are not included in this report. Necropsies were performed on all birds over 30 days of age and diagnoses were based on gross necrospy findings only. EXPERIMENTAL RESULTS
Within a few months after the hatching of chickens on the Laboratory premises, numerous cases of lymphomatosis appeared among the chickens of the first, or 1939, population. Despite the fact that all chickens were introduced as eggs and reared in newly constructed houses under complete confinement with rigid sanitary and quarantine precautions, the disease made its first appearance 40 days after the first chicks were hatched. The total
lymphomatosis mortality during the first 300 days was 12.3 percent and at the end of 600 days it had increased to 21.9 percent as shown in Table 1. The appearance of lymphomatosis in this first population is especially sigificant in that none of the common infectious diseases of poultry and no parasites, except coccidia, have been observed among the Laboratory chickens. These first year data demonstrate that in this case lymphomatosis could not have been spread through direct contact with infected chickens. Thus, these results definitely incriminate the egg as a source of lymphomatosis infection. Table 1 provides additional evidence that lymphomatosis may be transmitted by way of the egg. Each of the four subsequent populations, 1940 to 1943, were placed in the brooder house at hatching and at no time up to four months of age was direct contact with older and infected birds possible. Nevertheless, each year lymphomatosis made its appearance in the brooder house during this early period. At approximately four months of age all chickens in the last four populations were transferred each year from the brooder house to a laying house in which birds from a previous population or populations were housed. However, direct bird to bird contact with adult chickens of previous populations was not possible in the laying house in that only birds hatched during the same year were placed in the same pen. The gradual and continuous 30-day increase in lymphomatosis up to 600 days of age in all five populations under study raises the question as to whether all the chickens which died from the disease received their initial infection from the egg. Evidence will be submitted later to show that a considerable portion of the observed incidence of lymphomatosis was transmitted by direct contact with birds
Downloaded from http://ps.oxfordjournals.org/ at FU BerlinFB Humanmedizin on May 4, 2015
sects. All birds were fed a similar ration, consistent with the availability of feedstuffs, throughout the experimental period. This report involves approximately 4,700 White Leghorn chickens which were divided into groups and subjected to several different environmental modifications in an attempt to measure the influence of such environment as it affected the incidence of lymphomatosis. Each year, for five years, 1939 to 1943 inclusive, a main population of chickens was incubated, hatched, and reared as a mixed group. In addition certain groups of chickens were incubated, hatched, and reared by families, in complete isolation. Still other groups originated as eggs from chickens held in complete isolation, but such eggs were incubated, hatched, and the birds reared in contact with a mixed population or group throughout life. One group which was hatched and reared in complete isolation was exposed to the mixed population after 300 days of age.
227
308 e h i e k e n s Lympho. m o r t a l i t y 3 0 0 d a y a - 8.1J. 600 days - 2 3 . 1 $
1074 e h i e k e n s Lympho. m o r t a l i t y 300 days - IS.7% 600 daya - 3 4 . ( 9
1059 ehiekana Lympho. m o r t a l i t y 300 days - 19.Tit 600 daya - 3 4 . 9
101.4 chickens Lympho. m o r t a l i t y 300 d a y s . - 21.2<
1940
1M1
1942
1943
1
494 e h i e k e n s Lympho* m o r t a l i t y 300 days • 1 2 . 3 * 600 days - 21.9%
1959
11
I
1
eggs
1 3 "progeny f r e e
^
100 progeny incubated and reared i n i s o l a t i o n * • f r e e from lympho. a t 300** days o f a g e .
1
eggs
4
4 dame from f r e e s t o c k
1 1 I 1 |
egge 91 progeny 1Incubated and reared i n i a p l a t l o n . Tree from lympho. a t 300 daya of a g e .
1
4 daa» from f r e e stock
1 ^—
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92 progeny incubated and reared i n i s o l a t i o n * Free from lympho* a t 300 days o f a g e .
I i
I n e l d a n e s o f lymphomatosis among ehlokens r e c e i v i n g v a r i o u s d e g r e s s o f i s o l a t i o n from or e o n t a o t w i t h , a mixed p o p u l a t i o n having a knom lneldenee of the d i s e a s e .
4
4
,
i
40 progeny incubated and reared with mixed flock. 2056 lympho. a t 300 daya of a g e .
51 progeny incubated and reared with nixed f l o c k . 285C lympho. a t 300 daya of a g e .
0
83 progeny incubated and reared with mixed f l o c k . « l £ lympho. a t 300 days of a g e .
dams
.Lymphomatosis. 28 b i r d s k i l l e d a n d a u t o p s i e d a t about 200 days of a g e .
1
Incubated and reared with mixed f l o c k . 3Cg& lympho. a t 300 daya o f a g e .
«125 progeny4
13 dame from f r e e atoc k • eggs
and reared w i t h mixed flock* 32jS lympho. a t 300 daya of a g e .
.71 progeny4 incubated
egga
i
6 dams from f r e e stock
and reared In i s o l a t i o n . 16* lympho. a t 300 daya of a g e .
»64 progeny4incubated
9 "progeny i n f e e t e d eggs
1
122 daiu were e e l e c t e d a t about 300 daya of a g e from t h e 1940 (mixed p o p u l a t i o n t o proyide egga f o r t r a n s m i s s i o n a t u d l e e . i
»
11.
B1TOHAL T8aBSMISSI0» Of IT IIS LYMPHOMATOSIS
S o l i d boxed l i n e s represent- contact' with mixed p o p u l a t i o n throughout l i f e . Broken boxed l i n e s r e p r e s e n t c o n t a c t with mixed p o p u l a t i o n a f t e r 300 days of a g e .
I . I n c i d e n c e of lymphomatosis f o r v a r i o u s y e a r s among e h i c k s n e hatched and reared a s a mixed p o p u l a t i o n .
TMr
CHaBT 1 .
m http://ps.oxfordjournals.org/ at FU BerlinFB Humanmedizin on May 4, 2015
.
4
H progeny incubated and reared w i t h mixed f l o c k . 3 # lympho. a t 300 daya o f a g e .
dam*
^*i« ot age.
w
_ ^ _
J
1 ,, r i f t e r 300 daya o f age 2 0 * 1 f r e e stock c h i c k e n s from 1 F were p l a c e d i s c o n t a c t * w i t h t h e mixed, p o p u l a t i o n . ' Pr«e from lympho. a t 700 1
1
NATURAL TRANSMISSION OF AVIAN-LYMPHOMATOSIS
which presumably receive their infection through the egg. The data on all five populations, however, do provide evidence that egg transmission plays an important part in that none of the chickens in the different populations had any direct contact with infected birds up to four months of age. Indeed, the first population had no possible contact with other chickens up to at least one year of age.
229
exposed to a mixed population with a known incidence of lymphomatosis throughout their lives. The broken-boxed lines enclose data having contact with the mixed population after 300 days of age. The prevalence of lymphomatosis in the first population seriously handicapped the general Laboratory program and an attempt was made to eliminate or reduce the occurrence of this disease.
1942 (1,059 birds) No. Percent
1943 (1,014 birds) No. Percent
Age distribution in days
1939 (494 birds) No. Percent
3 1 - 60 6 1 - 90 91-120 ' 121-150 151-180 181-210 211-240 241-270 271-300 301-330 331-360 361-390 391-420 421-450 451-480 481-510 511-540 541-570 571-600
1 3 - 8 10 6 8 11 7 7 7 7 9 2 3 5 6 2 2 4
.2 .8 2.4 4.5 5.7 7.3 9.5 10.9 12.3 13.8 15.2 17.0 17.4 18.0 19.0 20.2 20.6 21.1 21.9
0 0 1 2 1 4 5 8 4 5 3 6 1 7 4 6 7 4 3
. .0 .0 .3 1.0 1.3 2.6 3.9 6.8 8.1 9.7 10.7 12.7 13.0 15.3 16.6 18.5 20.8 22.1 23.1
1 2 11 11 28 43 48 37, 31 24 31 14 18 11 11 11 9 15 9
.1 .3 1.3 2.3 4.9 8.9 13.4 16.9 19.7 22.0 24.9 26.2 27.8 28.9 30.0 30.9 31.8 33.1 34.0
2 10 8 15 23 43 32 40 36 25 18 25 16 16 12 14 11 8 8
.2 1,1 1.9 3.3 5.5 9.5 12.6 16.3 19.7 22.1 23.8 26.2 27.7 29.2 30.3 31.6 32.7 33.4 34.2
3 12 14 22 29 41 37 27 30
.3 1.5 2.9 5.0 7.9 11.9 15.7 18.2 21.2
Total
108
21.9
71
23.1
365
34.0
362
34.2
215
21.2*
%
•
1940 (308 birds) No. Percent
1941 (1,074 birds) No. Percent
* 300 days of age. OTHER EXPERIMENTS ON NATURAL TRANSMISSION
A summary of all experiments by years involving studies on natural transmission of lymphomatosis is presented in chart 1. For comparative purposes the five populations which were previously discussed are summarized in this chart also. Each phase of the transmission experiments is identified by a letter for easy reference. The mixed populations for the different years act as controls for other phases of the transmission studies. All solid-boxed lines enclose data on chickens which had been
It will be observed in Table 1 that the incidence of lymphomatosis in the 1940 population is considerably less during the early periods than in all other populations. This is due to a biased condition in that for the first 100 days of age all chickens in a family (progeny from one sire and dam) were discarded when the first case of lymphomatosis appeared. This procedure was adopted in an attempt to free this population from the disease. I t was discontinued when it became apparent that such a procedure would eventually eliminate the entire population.
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TABLE 1.—Accumulative percentage incidence of lymphomatosis mortality at intervals of 30 days for five different populations of chickens
230
NELSON F. WATERS
reared with the mixed flock showed 32 percent lymphomatosis. These experiments which provided both free and infected progeny under strict isolation are interpreted to mean that the progeny which showed no lymphomatosis at 300 days of age were free because their dams were free, whereas at least some of the progeny which showed 16 percent lymphomatosis were infected because the disease was transmitted from dam to progeny by way of the egg. Evidence for contact transmission is most convincing in that representative sibs, which were derived from each of the same dams providing chickens for the isolated pens, showed a relatively high incidence of lymphomatosis when exposed to the mixed flock. In an attempt to increase the number of free stock chickens during the year 1942 a total of 19 females and 1 male of the free chickens from B of 1941 were selected for breeding purposes and were allowed to remain in complete isolation after 300 days of age. It is important to point out that these 20 chickens were still free of lymphomatosis at 600 days of age. Eventually 4 of the 19 females were selected to produce progeny, representatives of which were to be hatched and reared both in isolation and in contact with the mixed flock. At the end of 300 days of age none of the progeny, F, which were hatched and reared in strict isolation, showed gross evidence of lymphomatosis. Sib progeny, G, of each family placed in strict isolation, were hatched and reared with the mixed flock. The incidence of lymphomatosis within these exposed progeny was 28 percent at 300 days of age. In 1943 (I and J) the procedures followed in 1942 were repeated and, with the exception of 28 sib's in I which were confined alone in one isolated pen since hatching and which were removed at 210 days
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Following this initial failure to eliminate lymphomatosis other attempts were made to obtain free stock by hatching and rearing chickens by families in strict isolation. The success of these subsequent attempts to obtain free stocks, together with evidence for egg and contact transmission of lymphomatosis, is described by Waters and Prickett (1944). From the 1940 mixed population 22 dams, A, were selected at about 300 days of age to provide eggs for transmission studies. At the time these birds were selected no lymphomatosis had appeared in the families from which they were chosen. During the time eggs were being obtained for subsequent studies all the 22 dams were maintained in contact with the mixed flock. Eggs from each of the 22 dams were transferred each day. to rigidly quarantined and isolated quarters. At the time of transfer the eggs were dipped and cleaned in a 70 percent solution of alcohol maintained at room temperature. The eggs from each of these hens were incubated and the progeny reared by families in strictly isolated pens. The behavior of these families of progeny with reference to lymphomatosis mortality is shown for the year 1941. In all, 13 dams now termed "progeny free" produced 92 progeny, B, which when incubated, hatched and reared in strict isolation were free of lymphomatosis at 300 days of age. Eggs from these same "progeny free" dams also produced 83 progeny, C, which when incubated, hatched, and reared with the mixed flock, showed 21 percent of lymphomatosis at 300 days of age. Nine of the 22 dams, now termed "progeny infected," produced 64 progeny, D, which when incubated, hatched, and reared in isolation showed 16 percent lymphomatosis at 300 days of age. Eggs from these "progeny infected" dams also produced 47 progeny, E, which when incubated, hatched and
NATURAL TRANSMISSION OF AVIAN-LYMPHOMATOSIS
Additional evidence for contact infection was provided in the years 1942 and 1943. Facilities were not available to test in isolation the progeny of all free stock dams. It was possible, however, from a few of these free dams to hatch and rear progeny in contact with the mixed infected flock. In chart 1, under H and K for the years 1942 and 1943 respectively, 19 such families representing 196 chickens were tested. The data for these two years show about 31 percent of lymphomatosis at 300 days of age. In all the contact experiments, which include the C, E, G, H, J, and K phases, the incidence of lymphomatosis at 300 or more days of age is, for the most part, in excess of that found in the mixed populations at the same age. This fact provides evidence that the related free stock groups B, F, and I, were free because the disease was not present and not because they were resistant to the disease. During 1943 evidence was obtained which indicates that there may be some association between the age at exposure and the subsequent incidence of lymphomatosis. At the end of a 300-day period 20 chickens, L in chart 1, representing 4 free families which had been isolated under strict quarantine during 1942, were trans-
ferred to quarters where a high incidence of the disease was present. After 400 days of this type of exposure no losses from lymphomatosis have occurred. A total of 51 sibs, G, of the 20 chickens which were continually exposed to the mixed flock from hatching to 300 days of age showed 28 percent of lymphomatosis, and at 600 days the incidence had increased to 47 percent. DISCUSSION AND CONCLUSIONS
Evidence obtained at this Laboratory that lymphomatosis is transmitted through the egg and spread by direct contact with infected chickens has been presented. The first chickens introduced as hatching eggs were placed in new buildings and serviced with new equipment. A most rigid sanitary and quarantine program was adopted and put into operation. Despite these precautions lymphomatosis made its appearance within 40 days after the first chickens were hatched and placed in the brooder house. Subsequent populations of chickens" -were placed in the brooder house immediately after hatching. All chickens within each year were confined to the brooder house until they were about four months of age. The data show that in every population some chickens had lymphomatosis while they were still in the brooder house. The occurrence of this disease among young chickens during the first four months of their lives and before they had had any contact with infected birds, raises ., the question from whence did i t arise. Control of the enviroment did prevent, for a period of five years, the entrance of all common infectious diseases and all parasites, except coccidia, but it did not prevent the entrance of lymphomatosis. Data have been presented which indicate that the incidence of lymphomatosis among the five different populations of
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of age, the results were essentially the same. At about 200 days of age, 2 of the 28 sibs mentioned above showed certain clinical symptoms of lymphomatosis. As a possible safeguard to the birds in other pens, the 28 sibs were immediately killed and autopsied. Gross examination on all of these birds showed no evidence of lymphomatosis. Microscopic examination, however, did show pathology, commonly interpreted as lymphomatosis, in 57 percent of the birds. It should be stressed that the parental chickens of the progeny in I and J for 1943, were still free of lymphomatosis at 700 days of age.
231
232
NELSON F. WATERS
It would seem clear from the data presented that lymphomatosis is transmitted from parent to offspring by way of the egg and that no method which includes isolation, confinement, sanitation, or quarantine during the early period of the
chicken's life will eliminate or control the disease unless the eggs are also free. The evidence for contact transmission of lymphomatosis is most conclusive, although the precise period or the exact manner of greatest exposure is not known. Three separate trials extending over a three-year period show that chickens which were hatched and reared with infected birds showed a high incidence of lymphomatosis, whereas their sibs hatched and reared in isolation remained free. The data, though in part meagre, indicate that there is an association between the age of a bird at the time it is exposed to lymphomatosis and its ability to resist infection. The evidence is clear that when representatives of four different families, which were hatched and reared in strict isolation for 300 days, are exposed to a mixed flock with a known incidence of lymphomatosis, for an additional period of 400 days, no deaths from the disease result. In contrast, when the sibs of these same four families were exposed to an infected mixed flock, from hatching to 600 days of age, a very high lymphomatosis mortality results. The presence of microscopic lesions showing pathology commonly interpreted as lymphomatosis in certain of the free stocks during 1943 may have important implications which cannot be discounted. It should be remembered that none of the birds showing microscopic lesions could be classed as grossly, positive for lymphomatosis at necropsy. It is not the intent at this time to pass on the merits of present methods for diagnosing lymphomatosis microscopically. It may be pertinent to ask, however, whether the tissues of a chicken will show abnormal extravascular accumulations of mononuclear cells only when stimulated by the
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chickens reported on could not be attributed, at least during the first four months of the brooding period, to direct contact with previously infected fowl. Indeed, the first population of chickens in 1939 had no contact, up to at least one year of age with any other chickens whatsoever. The criticism may be advanced that lymphomatosis may have been introduced to the Laboratory chickens by the same channels as were the sporoblasts of coccidia. Such a method of introduction cannot be ruled out, but if this were true it would be pertinent to ask why, with the above exceptions, none of the other infectious diseases or parasites common to poultry have made an entrance during a five-year period? Additional and more exacting evidence on egg transmission is provided in experiments which involved the incubation and hatching of families of chickens in individually isolated and strictly quarantined pens. Some of these families showed cases of lymphomatosis while other families remained entirely free up to at least 700 days of age. These results are interpreted to mean that in some instances lymphomatosis was transmitted from parent to offspring by way of the egg. The criticism may arise that these free families were free because they were resistant to the disease. That such a criticism is not valid is demonstrated by the fact that sibs of these free families, when hatched and reared with a known infected flock, show a relatively high incidence of lymphomatosis.
NATURAL TRANSMISSION OF AVIAN-LYMPHOMATOSIS
233
6. The hatching and rearing of chickens in strict isolation, even though placed in an environment where the best known sanitary practices or quarantine measures exist, will not prevent per se the occurrence of lymphomatosis unless the parents of such birds are free of the disease.
disease, lymphomatosis. If lymphomatosis were present in these free chickens in excess of all others involved in actual contact experiments at 200 days of age, then some indirect means of transmission must be held accountable. SUMMARY x
REFERENCES
Biely, Jacob, Elvira Palmer and V. S. Asmundson, 1932. Inheritance of resistance to fowl paralysis (neurolymphomatosis gallinarum). Can. Jour. Res. 6:374-380. Blakemore, F., and R. E. Glover, 1935. Fowl paralysis. I I . Experiments on transmission 4th rpt. director Camb. Univ., Inst. An. Path. 1934-35:5164. Doyle, L. P., 1927. Neuritis or paralysis in chickens. Proc. 64th Annual Meeting A.V.M.A. Gibbs, Charles S., 1936. Observations and experiments with neurolymphomatosis and the leukotic diseases. Mass. Agri. Exp. Sta. Bui. 337:1-31. Led, C. D., and H. L. Wilcke, 1941. Transmission experiments with iritis of fowls. Am. Jour. Vet. Res. 2:292-294. McClary, C. F., and Chas. W. Upp, 1939. Is paralysis of fowls, as manifested by iritis, transmitted through the egg? Poultry Sci. 18:210-219. Seager, E. A., 1933. The pathology of fowl paralysis-, with some aspects of its cause and control. Vet. Jour. 89:454^473. Tower, Benjamin A., 1937. Breeding as a factor in the control of blindness and paralysis in fowls. La. State Univ. Thesis: 1-71. Warrack, G. H., and T. Dalling, 1932. So-called "fowl paralysis." Vet. Jour. 88:28-43. Waters, Nelson F., and C. O. Prickett, 1944. The development of families of chickens free of lymphomatosis. Poultry Sci. 23:321-333.
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1. This report involves approximately 4,700 White Leghorn chickens which were divided into groups and subjected to several different environmental modifications in an attempt to measure the influence of such environment as it affected the natural transmission of lymphomatosis. 2. All chickens were reared throughout life in complete confinement and were maintained in an environment of strict sanitation and quarantine. 3. Lymphomatosis was present in chickens under four months of age despite the fact that direct contact with previously infected birds did not exist. 4. Evidence is presented to show that lymphomatosis is transmitted both by way of the egg and by contact with infected birds. 5. Careful selection and testing of certain families hatched and reared in isolation and maintained under rigid quarantine have prevented the occurrence of lymphomatosis to at least 700 days of age.