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The Presence of the Virus Causing Visceral Lymphomatosis in the Secretions and Excretions of Chickens
836
B . R . BURMESTER AND R . F . GENTRY REFERENCES
The Presence of the Virus Causing Visceral Lymphomatosis in the Secretions and Excretions of Chickens B. R. BURMESTER AND R. F. GENTRY Regional Poultry Research Laboratory, Agricultural Research Service, United Stales Department of Agriculture, East Lansing, Michigan (Received for publication November 23, 1953)
M
UCH has been written on the natural transmission of lymphomatosis although little is actually known concerning the specific methods and avenues by which the causative agent is transferred from an infected host to a noninfected susceptible animal. It has long been suspected that lymphomatosis is transmitted from parent to offspring by way of the hatching egg but it is only recently that an adequate basis for this mode of transmission has been established Experiments reported by Cottral, Burmester and Waters (1949) showed that significant quantities of the infectious agent of visceral lymphomatosis were present in embryonated eggs from some but not all normal appearing chickens of a population in which the disease was clinically evident.
Other investigations pertaining to the method of shedding the causative agent or agents of lymphomatosis have been confined primarily to the excreta of the intestinal and urinary tracts. Since these are excreted together in all birds, the term "feces," as used in this paper and in other published reports, includes both the intestinal and urinary excretions, and in this sense may be regarded as synonymous with droppings or manure. In considering the literature on the infectivity of feces one must recognize that there is increasing evidence (Davis and Doyle, 1947; Burmester, 1947; Burmester, and Gentry, 1954; Waters, 1954) indicating that the various forms of lymphomatosis; i.e., neural, ocular, and visceral; are caused by distinctly different viral agents. These agents may well have different
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
Almquist, H. J., 1937. The anti-hemorrhagic vitamin. Poultry Sci. 16:166-172. Almquist, H. J., and E. L. R. Stokstad, 1936. Factors influencing the incidence of dietary hemorrhagic disease in chicks. J. Nutrition, 12:329-335. Anonymous, 1953. Hemorrhagic disease in chickens. J. Am. Vet. Med. Assn. 123: 37. Ansbacher, S., 1938. Vitamin K deficiency of the chick. Science, 88:221. Balloun, S. L., and E. L. Johnson, 1952. Underheated soybean oil meal increases blood clotting time of chicks. Poultry Sci. 31: 905-906. Bird, H. R., 0 . L. Kline, C. A. Elvehjem, E. B. Hart and J. G. Halpin, 1936. The distribution and properties of the anti-gizzard-erosion factor required by chicks. J. Nutrition, 12:571-582. Eveleth, D. F., and A. I. Goldsby, 1953. Toxicosis of chickens caused by trichloroethylene-ex-
tracted soybean meal. J. Am. Vet. Med. Assn. 123:38-39. Griminger, P., H. M. Scott and L. E. Card, 1953a. Hemorrhaging in broilers. Flour and Feed, 54 (2): 18. Griminger, P., H. Fisher, W. D. Morrison, J. M. Snyder and H. M. Scott, 1953b. The influence of different levels of alfalfa meal added to a cornsoybean meal type of ration on blood clotting time. Poultry Sci. 32:902. Griminger, P., H. Fisher, W. D. Morrison, J. M. Snyder and H. M. Scott, 1953c. Factors influencing blood clotting time in the chick. Science, 118: 379-380. Hare, J. H., G. C. Anderson, C. E. Weakley, Jr. and J. K. Bletner, 1953. Factors contributing to a hemorrhagic condition in experimental chicks fed simplified rations. Poultry Sci. 32: 904. Jukes, T. H., 1937. Recent studies of vitamins required by chicks. J. Nutrition, 13:359-385.
A VIRUS CAUSING LYMPHOMATOSIS
i.e., intraperitoneal, was used. This paper is not concerned with the question of whether these materials under test would be effective by a natural portal of entry. MATERIALS AND METHODS
General.—Oral washings and feces were collected from clinically normal birds, from cases of visceral lymphomatosis, and from inoculated birds. Inoculums were prepared from the collections and their infectivity was tested by the intraperitoneal inoculation of baby chicks. The results are in terms of the relative incidence of visceral lymphomatosis obtained in the various inoculated and control groups duran experimental period of 270 days. Recipients.—The chicks used were of a# inbred line of S. C. White Leghorns selected §t this Laboratory (line 15) for susceptibility to lymphomatosis and which had been propagated in isolation during the past twelve years for the purpose of reducing natural infection with lymphomatosis (Waters and Prickett, 1944). The chicks were banded and inoculated the day after hatching and immediately after inoculation each group was placed in a cubicle where they were maintained for the first 75 days of life. The cubicles were 3X6 feet in size with walls of J inch plywood 4 feet high and open on top. The chicks were maintained on wire mesh floor which was about 12 inches above the concrete floor of the pen. There were 25 such cubicles in the one pen having common ventilation and source of heat. Each cubicle was equipped with an electric heat lamp, a feed hopper, and a continuously running, self-cleaning, watering device. A cubicle with the front wall removed is shown in Figure 1. Before the chicks were placed in the cubicle, all surfaces of the pen, cubicle, and equipment were thoroughly cleaned and sanitized with Roccal disinfectant. During the
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transmission characteristics. Another consideration of importance is that different investigators used different methods of testing the infectivity of the excreta. Recent results (unpublished) indicate that certain routes of administration are much less effective than others. Successful transmission of neural lymphomatosis (fowl paralysis) by the feeding of feces and by the parenteral injection of aqueous extracts was reported by Seager (1933) and Fritzsche (1938). Jungherr (1937) obtained equivocal result. Feces from cases of neural lymphomatosis were fed to chickens in three tests. In the first, 2 of 5 chickens became positive, one developed erythroblastosis and the other visceral lymphomatosis. In the next two tests, involving a total of 6 chickens, none developed the disease; however, in a fourth test in which 3 birds were fed feces of a case of erythroblastosis, one developed neural and one visceral lymphomatosis. Thorp and Graham (1936) using 221 birds in four tests obtained no evidence of transmission when feces from cases of ocular lymphomatosis and erythroblastosis were fed. Similar results were obtained by Barber (1948) who fed fresh manure, collected from cases of neural lymphomatosis, to 133 chicks. The incidence of all forms of lymphomatosis in this lot was no different than in control lots fed manure from normal turkeys. Barber also obtained negative results with saline nasal washings given by the intranasal and ocular routes. The purpose of this paper is to report an experiment designed to determine whether the virus of visceral lymphomatosis was present in fresh feces and in secretions of the oral mucosa. Since the purpose of these tests was to answer only the question of whether the virus was present in infectious levels, a standard route of administration of known efficacy,
837
838
B . R . BURMESTER AND R . F . GENTRY
FIG. 1. Cubicles used in the brooding of inoculated chicks to 75 days of age.
75-day period in the cubicles, all servicing, such as removal of dead birds and \ inch wire mesh screen used during the first three weeks, was done with a metal grapple which was disinfected just prior to use. It has been shown (Burmester and Gentry, 1954) that by the use of such equipment and procedures, cross infection between inoculated groups and with noninoculated controls was effectively reduced to an insignificant level. At 75 days of age the birds were moved to regular pens with wire floors, 9 X 1 2 ft. in size, and then at about 150 days of age they were moved to laying house pens where they remained until the termination of the experiment. All birds t h a t died were given a complete necropsy examination and the diagnosis was based on gross findings with
Three collections of oral washings were tested. Inoculum R l was prepared from the washings of 12 normal appearing pullets selected at random from a flock which developed a moderate incidence of lymphomatosis. Collections were made when the birds were about 220 days of age. During the succeeding 280 days, 5 of the 12 birds died, but only 2 of these died of lymphomatosis, and these at 18 and 30 days after the collections were made. Inoculum R2 was prepared from pooled oral washings from 5 advanced cases of naturally occurring visceral lymphomatosis. The donors were identified by the clinical symptoms, the collections were made and then the birds were killed and necropsy examination performed to confirm or reject the tentative clinical diagnosis. T h e y were all from the same L a b o r a t o r y flock which supplied chickens for the R l inoculum and were all about 250 days of age. Inoculum R3 was prepared from pooled
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confirmation by microscopic study of the livers and in questionable cases other suspected tissues. Inoculums.—Oral washings were obtained by using a rubber tipped 10 ml. syringe and irrigating with 10 ml. of Simms' salt solution (Simms and Sanders, 1942) the mucosa of the oral cavity, the pharynx and to a limited extent the nasal cavity, the nasolacrimal duct and the anterior nares. The washings were collected in a beaker and the irrigation was repeated two times using the same solution. The washings from several birds of the same treatment or similar pathology were pooled and 10 percent by weight of celite 512 was added. The mixture was then passed through a sterile grade F Pyrex frilled glass filter. The filtrate was transferred to serum tubes, sealed, frozen and stored in a CO2 ice box until used for inoculation.
839
A VIRUS CAUSING LYMPHOMATOSIS
TABLE 1.—Response of chickens inoculated intraperitoneally with fecal extracts and oral washings of normal and inoculated chickens and of cases of visceral lymphomatosis. {Experimental period of 270 days)
Inoculum
Other deaths (percent)
Ave. age at death; cases of V. Lym.
0 0 4.6
1.8 0 0
9.0 3.2 7.7
237 222
9.0 66.2
3.0 0
0 8.1
9.0 9.8
231 178
69 66 67
65.2 10.6 73.2
2.8 0 1.5
11.8 0 4.5
7.2 7.6 3.0
187 220 186
62 67
64.5 7.5
0 1.5
0 0
3.2 6.0
170 252
Total number chickens
Fl F2 F6
56 62 65
0 6.5 13.8
Rl R2
67 62
F3 F4 F5 R3 None
Visceral Neural (percent)
* Inoculated at one day of age.
oral washings of 4 advanced cases of induced viscera] lymphomatosis. These donors were identified by the same method used for donors in the preparation of inoculum R2, but were from an experiment in which a large number of birds were inoculated intraperitoneally with a cell-free filtrate preparation of strain RPL 12 with the result that 53 percent of the birds inoculated developed visceral lymphomatosis (Burmester and Gentry, 1954). Feces used for infectivity test were collected on new waterproof paper placed under the clean wire mesh floor of individual hen batteries. The collection period was 2 to 6 hours. The droppings were either processed immediately or were transferred to pyrex test tubes, sealed, frozen, and stored in a CO2 ice box until other samples had been collected. The fecal extracts were prepared by mixing the pooled collections in a Waring blendor with 3 parts by weight of Simms' salt solution. The mixture was centrifuged for 40 minutes at an R.F.C. of 2,000 times gravity. Ten grams of celite 512 were added
to each 100 ml. of supernatant and then filtered through a celite pad formed on a No. 1 Whatman filter paper. The filtrate was then passed through a Selas 02 porcelain filter candle, transferred to serum vials, sealed, and stored in a CO2 ice box until the time of inoculation. Fecal extract Fl was prepared from feces of the same birds used for the pool of oral washings R l . Preparation F2 was made from the same feces and in the same manner as Fl, except that a preparation of strain RPL 12 virus in the form of a lymphomatous liver filtrate of known potency was added to a part of the pooled feces and the fecal extract prepared in the usual manner. Extract F3 was prepared from the feces of 9 clinically normal birds, 154 days of age, which had been inoculated at one day of age with strain 12 virus preparation. Two of the 9 birds developed visceral lymphomatosis at 213 and 302 days of age. Inoculum F4 was obtained from the feces of 5 birds about 400 days of age which had received 25 injections of a strain RPL 12 virus preparation of known
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Natural exposure Extract of feces from: Normal hens Normal hens-|-RPL 12 virus Cases of vise, lympho. Oral washings from: Normal hens Cases of vise, lympho. Inoculated with RPL 12 virus Extract of feces from: Normal hens* Normal hens inoc. as adults Cases of vise, lympho.* Oral washings from: Cases of vise, lympho.* Noninoculated controls
Osteopetrosis (percent)
Lymphomatosis
Inoculum No.
840
B . R . BURMESTER AND R . F . GENTRY
potency during a period of 6 weeks just preceding the collection of the feces used. All birds were normal at the time of collection and for a period of 322 days thereafter. Inoculum F5 was from feces collected from the same cases of visceral lymphomatosis induced by inoculation as were used for the oral washing preparation R3, and fecal extract F6 came from feces of the same cases of naturally occurring visceral lymphomatosis used for inoculum R2. Results of the inoculations were measured in terms of the incidences of the various forms of lymphomatosis and osteopetrosis in birds that died during an experimental period of 270 days, and of the average age of death of chickens that died of visceral lymphomatosis. A summary of these results is presented in Table 1 and Figure 1. The results show that the extract of feces from clinically normal birds from a naturally infected population (Fl) was not infectious. Oral washings from the same birds (Rl) were similarly negative. Thus both tests were negative despite the fact that two of the twelve donors used died of visceral lymphomatosis within 30 days after the collections were made. Since the feces from several birds were pooled it was important to determine whether or not normal feces itself was detrimental to the virus under test. For this reason, an amount of RPL 12 lymphomatous liver nitrate preparation was added to the feces of clinically normal birds which, when an extract was prepared in the usual manner, contained an equivalent of 3.5X10- 1 gm. tumor (F2). The potency of this same liver filtrate preparation was tested in a titration experiment at about the same time as the fecal infectivity experiments were conducted. It
The extract of a pool of feces from several cases of visceral lymphomatosis (F6) caused by natural means of infection was without significant effect in recipient chicks. An incidence of 13.8 percent obtained in this group was not significantly different from 7.5 percent obtained in the controls. Oral washings from clinically normal birds (Rl) were noninfectious but those collected from several cases of visceral lymphomatosis (R2) produced a highly significant incidence (66.2 percent) of the disease in recipient chickens. Extracts of feces of clinically normal birds which had been inoculated at 1 day of age with the virus of visceral lymphomatosis (F3), as well as fecal extracts of birds of the same group which had developed large lymphomatous livers (F5), were highly infectious as indicated by the incidence of tumors induced in recipient chickens. These results are in contrast with the negative result obtained with the fecal extract of naturally infected birds. This difference may be related to the particular strain of inoculum employed or due to the fact that the feces from individual
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RESULTS AND DISCUSSION
was found that the highest dilution used, which was a dosage equivalent to 1X 10~7 gm. of tumor, produced an incidence of 70 percent visceral lymphomatosis in 55 chickens inoculated. Thus, the liver filtrate preparation added was of known high activity. However, the extract of the feces-liver filtrate mixture (F2) did not produce a significant incidence of tumors in 62 chicks inoculated. It would appear that the pooled feces from noninoculated normal birds, neutralized or inactivated the causative virus in the liver preparation. However, all feces do not appear to act in this manner since results to be presented show that active virus was present in feces of certain groups of inoculated chickens.
A VIRUS CAUSING LYMPHOMATOSIS
Oral washings from cases of visceral lymphomatosis induced by inoculation (R3) were found to be highly infectious, producing an incidence of 64.5 percent in 62 chickens. This rate is almost identical to that produced by oral washings of naturally occurring cases. The average ages at death due to visceral lymphomatosis in the 4 groups which had a significant incidence, ranged from 170 to 187 days. The averages for the
other groups were much higher (220-252 days). Cumulative lymphomatosis mortality by 30-day periods for groups inoculated with R2 and R3 oral washings and fecal extracts F3 and F4, and for the noninoculated control groups are presented graphically in Fig. 2. The regression lines for each type of inoculum also are shown. The graph shows a remarkable similarity of rate of mortality for the 4 inoculated groups. During the experimental period of 270 days the rate was quite constant after the initial incubation period.
oxmwumms Xs s natiBm/KTj Ts
„ so •
% 1 few
A
comxoi x
I
10
60
SO Ml
120 at
ISO
ISO
140
U0
DEATH-days
FIG. 2. Cumulative percentage of visceral lymphomatosis occurring in susceptible groups of chickens inoculated with filtered preparations of oral washings and fecal extracts.
The percentages of neural lymphomatosis in any of the inoculated groups were not significantly different from those in the controls, nor was there any indication of a correlation in the incidence of the two forms of lymphomatosis. A few cases of osteopetrosis occurred in 4 of the 9 inoculated groups. Three of these 4 groups had a high incidence of visceral lymphomatosis. The results presented show that chickens that were inoculated when young with the virus of visceral lymphomatosis shed this virus in the feces as adults
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birds were pooled. In the inoculated flock all birds were infected; whereas, in the naturally infected flock it is likely that part of the birds were not infected and the feces from these birds may have inactivated the feces of infected birds in the same manner that inactivation of the liver preparation of known potency was obtained in inoculum F2. Results obtained with inoculum F4 are of particular interest in view of the positive reaction elicited by inoculum F3. Hens which supplied feces for F4 had received 18.75 ml. of liver filtrate preparation of known potency in 25 intraperitoneal injections over a period of 6 weeks just prior to the collection of the feces. However, in spite of the large amount of virus injected, insufficient quantities appeared in the feces to be detected by the methods employed; whereas, F3 inoculum came from feces of chickens, that had received a single intraperitoneal injection of only 0.2 ml. In addition to the dosage difference between the two groups of birds the other primary difference was that of age at inoculation. Chickens of the F3 inoculum were inoculated at 1 day of age; whereas, those of F4 were over 400 days of age when inoculated. It would appear that a latent infection had been established in the chickens inoculated at one day of age but such was not the case in birds injected when about 400 days of age.
841
842
B. R. BURMESTER AND R. F. GENTRY
SUMMARY AND CONCLUSIONS
The presence of the causative agent of visceral lymphomatosis in secretions of the oral and nasal cavities and in excretions of the intestinal and urinary tracts was determined by the inoculation of filtered preparations into groups of dayold chicks. Highly significant incidences of visceral lymphomatosis (65-73 percent), during an experimental period of 270 days, were obtained in groups of chicks inoculated with oral-nasal washings of cases of naturally occurring and cases of induced visceral lymphomatosis. Fecal extracts from the latter cases, as well as from clinically normal birds, produced a high incidence of tumors; however, extracts of feces from chickens inoculated as adults were noninfectious.
The tumor producing activity of a lymphomatous liver filtrate preparation of known high potency was lost when it was mixed with fresh feces of normal chickens. REFERENCES Barber, C. W., 1948. Transmission experiments on the avian leucosis complex. Cornell Vet. 38: 130-134. Burmester, B. R., 1947. Studies on the transmission of avian visceral lymphomatosis. II. Propagation of lymphomatosis with cellular and cell-free preparations. Cancer Res. 7: 786-797. Burmester, B. R., and R. F. Gentry, 1954. The transmission of avian visceral lymphomatosis by contact. Cancer Res. 14: 34-42. Cottral, G. E., B. R. Burmester and N. F. Waters, 1949. The transmission of visceral lymphomatosis with tissue from embryonated eggs and chicks from "normal" parents. Poultry Sci. 28: 761. Davis, O. S., and L. P. Doyle, 1947. Studies in avian leucosis. I. The transmissibility of visceral lymphomatosis. Am. J. Vet. Res. 8:103-112. Fritzsche, K., 1938. Versuche zur Erforschung und Bekampfung der Marekschen Huhnerlahme. I. Versuche iiber die Ausscheidung des Hiihnerlahmevirus mit dem Kot und iiber die naturliche Infektionsweise. Zeitschr. Infekt-Krankh. der Haustiere 52: 51-69. Jungherr, E., 1937. Studies on fowl paralysis. 2. Transmission experiments. Storrs Agr. Exp. Sta. Bull. 218. Seager, E. A., 1933. Pathology of fowl paralysis with some aspects of its cause and control. Vet. J. 89: 454-573. Simms, H. S., and M. Sanders, 1942. Use of serum ultrafiltrate in tissue cultures for studying deposition of fat and for propagation of viruses. Arch. Path. 33:619-635. Thorp, F., and R. Graham, 1936. Transmission studies in leucemia. Vet. Med. 31: 82. Waters, N. F., 1954. Etiological relationship of visceral and neural lymphomatosis. Poultry Sci. 33: 365-373. Waters, N. F., and C. O. Prickett, 1944. The development of families of chickens free of lymphomatosis. Poultry Sci. 23: 321-333.
DEDICATION OF JULL HALL AND POULTRY INDUSTRY HALL OF FAME, UNIVERSITY OF MARYLAND, SEPTEMBER 15
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whether they had tumors or were clinically normal. This virus also was present in the secretion of the oral mucosa of induced and naturally occurring cases. Thus, cases of visceral lymphomatosis resulting from natural infection shed the virus in their saliva and in this manner presumably may have contributed to an infected environment. Whether the virus is shed also in the intestinal and urinary excretions remains to be determined. Results of the one test reported were negative; however, as indicated earlier, inactivation of the virus of some of the cases may have taken place when mixed with feces of other cases in preparation of the pooled extract. The nature and significance of the virus inactivating principle or condition in the feces of normal noninoculated birds also remains to be determined.
B . R . BURMESTER AND R . F . GENTRY REFERENCES
The Presence of the Virus Causing Visceral Lymphomatosis in the Secretions and Excretions of Chickens B. R. BURMESTER AND R. F. GENTRY Regional Poultry Research Laboratory, Agricultural Research Service, United Stales Department of Agriculture, East Lansing, Michigan (Received for publication November 23, 1953)
M
UCH has been written on the natural transmission of lymphomatosis although little is actually known concerning the specific methods and avenues by which the causative agent is transferred from an infected host to a noninfected susceptible animal. It has long been suspected that lymphomatosis is transmitted from parent to offspring by way of the hatching egg but it is only recently that an adequate basis for this mode of transmission has been established Experiments reported by Cottral, Burmester and Waters (1949) showed that significant quantities of the infectious agent of visceral lymphomatosis were present in embryonated eggs from some but not all normal appearing chickens of a population in which the disease was clinically evident.
Other investigations pertaining to the method of shedding the causative agent or agents of lymphomatosis have been confined primarily to the excreta of the intestinal and urinary tracts. Since these are excreted together in all birds, the term "feces," as used in this paper and in other published reports, includes both the intestinal and urinary excretions, and in this sense may be regarded as synonymous with droppings or manure. In considering the literature on the infectivity of feces one must recognize that there is increasing evidence (Davis and Doyle, 1947; Burmester, 1947; Burmester, and Gentry, 1954; Waters, 1954) indicating that the various forms of lymphomatosis; i.e., neural, ocular, and visceral; are caused by distinctly different viral agents. These agents may well have different
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
Almquist, H. J., 1937. The anti-hemorrhagic vitamin. Poultry Sci. 16:166-172. Almquist, H. J., and E. L. R. Stokstad, 1936. Factors influencing the incidence of dietary hemorrhagic disease in chicks. J. Nutrition, 12:329-335. Anonymous, 1953. Hemorrhagic disease in chickens. J. Am. Vet. Med. Assn. 123: 37. Ansbacher, S., 1938. Vitamin K deficiency of the chick. Science, 88:221. Balloun, S. L., and E. L. Johnson, 1952. Underheated soybean oil meal increases blood clotting time of chicks. Poultry Sci. 31: 905-906. Bird, H. R., 0 . L. Kline, C. A. Elvehjem, E. B. Hart and J. G. Halpin, 1936. The distribution and properties of the anti-gizzard-erosion factor required by chicks. J. Nutrition, 12:571-582. Eveleth, D. F., and A. I. Goldsby, 1953. Toxicosis of chickens caused by trichloroethylene-ex-
tracted soybean meal. J. Am. Vet. Med. Assn. 123:38-39. Griminger, P., H. M. Scott and L. E. Card, 1953a. Hemorrhaging in broilers. Flour and Feed, 54 (2): 18. Griminger, P., H. Fisher, W. D. Morrison, J. M. Snyder and H. M. Scott, 1953b. The influence of different levels of alfalfa meal added to a cornsoybean meal type of ration on blood clotting time. Poultry Sci. 32:902. Griminger, P., H. Fisher, W. D. Morrison, J. M. Snyder and H. M. Scott, 1953c. Factors influencing blood clotting time in the chick. Science, 118: 379-380. Hare, J. H., G. C. Anderson, C. E. Weakley, Jr. and J. K. Bletner, 1953. Factors contributing to a hemorrhagic condition in experimental chicks fed simplified rations. Poultry Sci. 32: 904. Jukes, T. H., 1937. Recent studies of vitamins required by chicks. J. Nutrition, 13:359-385.
A VIRUS CAUSING LYMPHOMATOSIS
i.e., intraperitoneal, was used. This paper is not concerned with the question of whether these materials under test would be effective by a natural portal of entry. MATERIALS AND METHODS
General.—Oral washings and feces were collected from clinically normal birds, from cases of visceral lymphomatosis, and from inoculated birds. Inoculums were prepared from the collections and their infectivity was tested by the intraperitoneal inoculation of baby chicks. The results are in terms of the relative incidence of visceral lymphomatosis obtained in the various inoculated and control groups duran experimental period of 270 days. Recipients.—The chicks used were of a# inbred line of S. C. White Leghorns selected §t this Laboratory (line 15) for susceptibility to lymphomatosis and which had been propagated in isolation during the past twelve years for the purpose of reducing natural infection with lymphomatosis (Waters and Prickett, 1944). The chicks were banded and inoculated the day after hatching and immediately after inoculation each group was placed in a cubicle where they were maintained for the first 75 days of life. The cubicles were 3X6 feet in size with walls of J inch plywood 4 feet high and open on top. The chicks were maintained on wire mesh floor which was about 12 inches above the concrete floor of the pen. There were 25 such cubicles in the one pen having common ventilation and source of heat. Each cubicle was equipped with an electric heat lamp, a feed hopper, and a continuously running, self-cleaning, watering device. A cubicle with the front wall removed is shown in Figure 1. Before the chicks were placed in the cubicle, all surfaces of the pen, cubicle, and equipment were thoroughly cleaned and sanitized with Roccal disinfectant. During the
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
transmission characteristics. Another consideration of importance is that different investigators used different methods of testing the infectivity of the excreta. Recent results (unpublished) indicate that certain routes of administration are much less effective than others. Successful transmission of neural lymphomatosis (fowl paralysis) by the feeding of feces and by the parenteral injection of aqueous extracts was reported by Seager (1933) and Fritzsche (1938). Jungherr (1937) obtained equivocal result. Feces from cases of neural lymphomatosis were fed to chickens in three tests. In the first, 2 of 5 chickens became positive, one developed erythroblastosis and the other visceral lymphomatosis. In the next two tests, involving a total of 6 chickens, none developed the disease; however, in a fourth test in which 3 birds were fed feces of a case of erythroblastosis, one developed neural and one visceral lymphomatosis. Thorp and Graham (1936) using 221 birds in four tests obtained no evidence of transmission when feces from cases of ocular lymphomatosis and erythroblastosis were fed. Similar results were obtained by Barber (1948) who fed fresh manure, collected from cases of neural lymphomatosis, to 133 chicks. The incidence of all forms of lymphomatosis in this lot was no different than in control lots fed manure from normal turkeys. Barber also obtained negative results with saline nasal washings given by the intranasal and ocular routes. The purpose of this paper is to report an experiment designed to determine whether the virus of visceral lymphomatosis was present in fresh feces and in secretions of the oral mucosa. Since the purpose of these tests was to answer only the question of whether the virus was present in infectious levels, a standard route of administration of known efficacy,
837
838
B . R . BURMESTER AND R . F . GENTRY
FIG. 1. Cubicles used in the brooding of inoculated chicks to 75 days of age.
75-day period in the cubicles, all servicing, such as removal of dead birds and \ inch wire mesh screen used during the first three weeks, was done with a metal grapple which was disinfected just prior to use. It has been shown (Burmester and Gentry, 1954) that by the use of such equipment and procedures, cross infection between inoculated groups and with noninoculated controls was effectively reduced to an insignificant level. At 75 days of age the birds were moved to regular pens with wire floors, 9 X 1 2 ft. in size, and then at about 150 days of age they were moved to laying house pens where they remained until the termination of the experiment. All birds t h a t died were given a complete necropsy examination and the diagnosis was based on gross findings with
Three collections of oral washings were tested. Inoculum R l was prepared from the washings of 12 normal appearing pullets selected at random from a flock which developed a moderate incidence of lymphomatosis. Collections were made when the birds were about 220 days of age. During the succeeding 280 days, 5 of the 12 birds died, but only 2 of these died of lymphomatosis, and these at 18 and 30 days after the collections were made. Inoculum R2 was prepared from pooled oral washings from 5 advanced cases of naturally occurring visceral lymphomatosis. The donors were identified by the clinical symptoms, the collections were made and then the birds were killed and necropsy examination performed to confirm or reject the tentative clinical diagnosis. T h e y were all from the same L a b o r a t o r y flock which supplied chickens for the R l inoculum and were all about 250 days of age. Inoculum R3 was prepared from pooled
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
confirmation by microscopic study of the livers and in questionable cases other suspected tissues. Inoculums.—Oral washings were obtained by using a rubber tipped 10 ml. syringe and irrigating with 10 ml. of Simms' salt solution (Simms and Sanders, 1942) the mucosa of the oral cavity, the pharynx and to a limited extent the nasal cavity, the nasolacrimal duct and the anterior nares. The washings were collected in a beaker and the irrigation was repeated two times using the same solution. The washings from several birds of the same treatment or similar pathology were pooled and 10 percent by weight of celite 512 was added. The mixture was then passed through a sterile grade F Pyrex frilled glass filter. The filtrate was transferred to serum tubes, sealed, frozen and stored in a CO2 ice box until used for inoculation.
839
A VIRUS CAUSING LYMPHOMATOSIS
TABLE 1.—Response of chickens inoculated intraperitoneally with fecal extracts and oral washings of normal and inoculated chickens and of cases of visceral lymphomatosis. {Experimental period of 270 days)
Inoculum
Other deaths (percent)
Ave. age at death; cases of V. Lym.
0 0 4.6
1.8 0 0
9.0 3.2 7.7
237 222
9.0 66.2
3.0 0
0 8.1
9.0 9.8
231 178
69 66 67
65.2 10.6 73.2
2.8 0 1.5
11.8 0 4.5
7.2 7.6 3.0
187 220 186
62 67
64.5 7.5
0 1.5
0 0
3.2 6.0
170 252
Total number chickens
Fl F2 F6
56 62 65
0 6.5 13.8
Rl R2
67 62
F3 F4 F5 R3 None
Visceral Neural (percent)
* Inoculated at one day of age.
oral washings of 4 advanced cases of induced viscera] lymphomatosis. These donors were identified by the same method used for donors in the preparation of inoculum R2, but were from an experiment in which a large number of birds were inoculated intraperitoneally with a cell-free filtrate preparation of strain RPL 12 with the result that 53 percent of the birds inoculated developed visceral lymphomatosis (Burmester and Gentry, 1954). Feces used for infectivity test were collected on new waterproof paper placed under the clean wire mesh floor of individual hen batteries. The collection period was 2 to 6 hours. The droppings were either processed immediately or were transferred to pyrex test tubes, sealed, frozen, and stored in a CO2 ice box until other samples had been collected. The fecal extracts were prepared by mixing the pooled collections in a Waring blendor with 3 parts by weight of Simms' salt solution. The mixture was centrifuged for 40 minutes at an R.F.C. of 2,000 times gravity. Ten grams of celite 512 were added
to each 100 ml. of supernatant and then filtered through a celite pad formed on a No. 1 Whatman filter paper. The filtrate was then passed through a Selas 02 porcelain filter candle, transferred to serum vials, sealed, and stored in a CO2 ice box until the time of inoculation. Fecal extract Fl was prepared from feces of the same birds used for the pool of oral washings R l . Preparation F2 was made from the same feces and in the same manner as Fl, except that a preparation of strain RPL 12 virus in the form of a lymphomatous liver filtrate of known potency was added to a part of the pooled feces and the fecal extract prepared in the usual manner. Extract F3 was prepared from the feces of 9 clinically normal birds, 154 days of age, which had been inoculated at one day of age with strain 12 virus preparation. Two of the 9 birds developed visceral lymphomatosis at 213 and 302 days of age. Inoculum F4 was obtained from the feces of 5 birds about 400 days of age which had received 25 injections of a strain RPL 12 virus preparation of known
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Natural exposure Extract of feces from: Normal hens Normal hens-|-RPL 12 virus Cases of vise, lympho. Oral washings from: Normal hens Cases of vise, lympho. Inoculated with RPL 12 virus Extract of feces from: Normal hens* Normal hens inoc. as adults Cases of vise, lympho.* Oral washings from: Cases of vise, lympho.* Noninoculated controls
Osteopetrosis (percent)
Lymphomatosis
Inoculum No.
840
B . R . BURMESTER AND R . F . GENTRY
potency during a period of 6 weeks just preceding the collection of the feces used. All birds were normal at the time of collection and for a period of 322 days thereafter. Inoculum F5 was from feces collected from the same cases of visceral lymphomatosis induced by inoculation as were used for the oral washing preparation R3, and fecal extract F6 came from feces of the same cases of naturally occurring visceral lymphomatosis used for inoculum R2. Results of the inoculations were measured in terms of the incidences of the various forms of lymphomatosis and osteopetrosis in birds that died during an experimental period of 270 days, and of the average age of death of chickens that died of visceral lymphomatosis. A summary of these results is presented in Table 1 and Figure 1. The results show that the extract of feces from clinically normal birds from a naturally infected population (Fl) was not infectious. Oral washings from the same birds (Rl) were similarly negative. Thus both tests were negative despite the fact that two of the twelve donors used died of visceral lymphomatosis within 30 days after the collections were made. Since the feces from several birds were pooled it was important to determine whether or not normal feces itself was detrimental to the virus under test. For this reason, an amount of RPL 12 lymphomatous liver nitrate preparation was added to the feces of clinically normal birds which, when an extract was prepared in the usual manner, contained an equivalent of 3.5X10- 1 gm. tumor (F2). The potency of this same liver filtrate preparation was tested in a titration experiment at about the same time as the fecal infectivity experiments were conducted. It
The extract of a pool of feces from several cases of visceral lymphomatosis (F6) caused by natural means of infection was without significant effect in recipient chicks. An incidence of 13.8 percent obtained in this group was not significantly different from 7.5 percent obtained in the controls. Oral washings from clinically normal birds (Rl) were noninfectious but those collected from several cases of visceral lymphomatosis (R2) produced a highly significant incidence (66.2 percent) of the disease in recipient chickens. Extracts of feces of clinically normal birds which had been inoculated at 1 day of age with the virus of visceral lymphomatosis (F3), as well as fecal extracts of birds of the same group which had developed large lymphomatous livers (F5), were highly infectious as indicated by the incidence of tumors induced in recipient chickens. These results are in contrast with the negative result obtained with the fecal extract of naturally infected birds. This difference may be related to the particular strain of inoculum employed or due to the fact that the feces from individual
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RESULTS AND DISCUSSION
was found that the highest dilution used, which was a dosage equivalent to 1X 10~7 gm. of tumor, produced an incidence of 70 percent visceral lymphomatosis in 55 chickens inoculated. Thus, the liver filtrate preparation added was of known high activity. However, the extract of the feces-liver filtrate mixture (F2) did not produce a significant incidence of tumors in 62 chicks inoculated. It would appear that the pooled feces from noninoculated normal birds, neutralized or inactivated the causative virus in the liver preparation. However, all feces do not appear to act in this manner since results to be presented show that active virus was present in feces of certain groups of inoculated chickens.
A VIRUS CAUSING LYMPHOMATOSIS
Oral washings from cases of visceral lymphomatosis induced by inoculation (R3) were found to be highly infectious, producing an incidence of 64.5 percent in 62 chickens. This rate is almost identical to that produced by oral washings of naturally occurring cases. The average ages at death due to visceral lymphomatosis in the 4 groups which had a significant incidence, ranged from 170 to 187 days. The averages for the
other groups were much higher (220-252 days). Cumulative lymphomatosis mortality by 30-day periods for groups inoculated with R2 and R3 oral washings and fecal extracts F3 and F4, and for the noninoculated control groups are presented graphically in Fig. 2. The regression lines for each type of inoculum also are shown. The graph shows a remarkable similarity of rate of mortality for the 4 inoculated groups. During the experimental period of 270 days the rate was quite constant after the initial incubation period.
oxmwumms Xs s natiBm/KTj Ts
„ so •
% 1 few
A
comxoi x
I
10
60
SO Ml
120 at
ISO
ISO
140
U0
DEATH-days
FIG. 2. Cumulative percentage of visceral lymphomatosis occurring in susceptible groups of chickens inoculated with filtered preparations of oral washings and fecal extracts.
The percentages of neural lymphomatosis in any of the inoculated groups were not significantly different from those in the controls, nor was there any indication of a correlation in the incidence of the two forms of lymphomatosis. A few cases of osteopetrosis occurred in 4 of the 9 inoculated groups. Three of these 4 groups had a high incidence of visceral lymphomatosis. The results presented show that chickens that were inoculated when young with the virus of visceral lymphomatosis shed this virus in the feces as adults
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birds were pooled. In the inoculated flock all birds were infected; whereas, in the naturally infected flock it is likely that part of the birds were not infected and the feces from these birds may have inactivated the feces of infected birds in the same manner that inactivation of the liver preparation of known potency was obtained in inoculum F2. Results obtained with inoculum F4 are of particular interest in view of the positive reaction elicited by inoculum F3. Hens which supplied feces for F4 had received 18.75 ml. of liver filtrate preparation of known potency in 25 intraperitoneal injections over a period of 6 weeks just prior to the collection of the feces. However, in spite of the large amount of virus injected, insufficient quantities appeared in the feces to be detected by the methods employed; whereas, F3 inoculum came from feces of chickens, that had received a single intraperitoneal injection of only 0.2 ml. In addition to the dosage difference between the two groups of birds the other primary difference was that of age at inoculation. Chickens of the F3 inoculum were inoculated at 1 day of age; whereas, those of F4 were over 400 days of age when inoculated. It would appear that a latent infection had been established in the chickens inoculated at one day of age but such was not the case in birds injected when about 400 days of age.
841
842
B. R. BURMESTER AND R. F. GENTRY
SUMMARY AND CONCLUSIONS
The presence of the causative agent of visceral lymphomatosis in secretions of the oral and nasal cavities and in excretions of the intestinal and urinary tracts was determined by the inoculation of filtered preparations into groups of dayold chicks. Highly significant incidences of visceral lymphomatosis (65-73 percent), during an experimental period of 270 days, were obtained in groups of chicks inoculated with oral-nasal washings of cases of naturally occurring and cases of induced visceral lymphomatosis. Fecal extracts from the latter cases, as well as from clinically normal birds, produced a high incidence of tumors; however, extracts of feces from chickens inoculated as adults were noninfectious.
The tumor producing activity of a lymphomatous liver filtrate preparation of known high potency was lost when it was mixed with fresh feces of normal chickens. REFERENCES Barber, C. W., 1948. Transmission experiments on the avian leucosis complex. Cornell Vet. 38: 130-134. Burmester, B. R., 1947. Studies on the transmission of avian visceral lymphomatosis. II. Propagation of lymphomatosis with cellular and cell-free preparations. Cancer Res. 7: 786-797. Burmester, B. R., and R. F. Gentry, 1954. The transmission of avian visceral lymphomatosis by contact. Cancer Res. 14: 34-42. Cottral, G. E., B. R. Burmester and N. F. Waters, 1949. The transmission of visceral lymphomatosis with tissue from embryonated eggs and chicks from "normal" parents. Poultry Sci. 28: 761. Davis, O. S., and L. P. Doyle, 1947. Studies in avian leucosis. I. The transmissibility of visceral lymphomatosis. Am. J. Vet. Res. 8:103-112. Fritzsche, K., 1938. Versuche zur Erforschung und Bekampfung der Marekschen Huhnerlahme. I. Versuche iiber die Ausscheidung des Hiihnerlahmevirus mit dem Kot und iiber die naturliche Infektionsweise. Zeitschr. Infekt-Krankh. der Haustiere 52: 51-69. Jungherr, E., 1937. Studies on fowl paralysis. 2. Transmission experiments. Storrs Agr. Exp. Sta. Bull. 218. Seager, E. A., 1933. Pathology of fowl paralysis with some aspects of its cause and control. Vet. J. 89: 454-573. Simms, H. S., and M. Sanders, 1942. Use of serum ultrafiltrate in tissue cultures for studying deposition of fat and for propagation of viruses. Arch. Path. 33:619-635. Thorp, F., and R. Graham, 1936. Transmission studies in leucemia. Vet. Med. 31: 82. Waters, N. F., 1954. Etiological relationship of visceral and neural lymphomatosis. Poultry Sci. 33: 365-373. Waters, N. F., and C. O. Prickett, 1944. The development of families of chickens free of lymphomatosis. Poultry Sci. 23: 321-333.
DEDICATION OF JULL HALL AND POULTRY INDUSTRY HALL OF FAME, UNIVERSITY OF MARYLAND, SEPTEMBER 15
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whether they had tumors or were clinically normal. This virus also was present in the secretion of the oral mucosa of induced and naturally occurring cases. Thus, cases of visceral lymphomatosis resulting from natural infection shed the virus in their saliva and in this manner presumably may have contributed to an infected environment. Whether the virus is shed also in the intestinal and urinary excretions remains to be determined. Results of the one test reported were negative; however, as indicated earlier, inactivation of the virus of some of the cases may have taken place when mixed with feces of other cases in preparation of the pooled extract. The nature and significance of the virus inactivating principle or condition in the feces of normal noninoculated birds also remains to be determined.