Canine Oral Papillomatosis. of the Experimental Infection*

Transcription

1 Canine Oral Papillomatosis IO Virus Assay and Observations on the Various Stages of the Experimental Infection* VELMA C. CHAMBERS AND CHARLES A. EVANS (Department of Microbiology, University of Washington, Seattle, Wash.) The infectious nature of canine oral papillomatosis was described by Penberthy (5) and by M'Fadyean and Hobday (4) in A virus was shown to be the causative agent of the disease by :Findlay (~) in Later, DeMonbreun and Goodpasture (1) and Goodlow (8) inoculated young dogs with tumor extracts and filtrates and studied the progression and the regression of the papillomas. The present study was undertaken to gain more comprehensive information about the virus and the infection it causes and to investigate new technics that might facilitate further work with this virus. MATERIALS AND METHODS The strain of canine oral papilloma virus used in most of this work originated from papillomas removed from a dog in Seattle, Washington, in April, 1955, and is known as strain N-211. The papillomas were collected in 50 per cent buffered glycerin in Locke's solution and were stored at 4 ~ C. Stock virus was prepared from the original material and from later passages in the following manner: Glycerinated papillomas were washed 2 times in Locke's solution, minced with scissors, and ground in a sterile mortar; sufficient Locke's solution was added to make a 10 per cent suspension. The suspension was centrifuged at 2000 r.p.m, for 10 minutes. The supernatant fluid was collected and stored at -25 ~ C. to -40 ~ C. for use as stock virus. Stock virus preparations derived from papillomas of the first five passages were used in the experiments to be described. Assay of N-211 stock virus is described in a later section of this report, and the results appear in Table 1. The ID.~0 was approximately 10 -~.4. Titration of stock virus N-211-1, prepared from tumors of the first passage, and stock virus N- 211-~, prepared from tumors of the second passage, gave ]Ds0 values of approximately 10 -a'5 and 10-4"5, respectively. Further attempts to titrate these virus preparations were complicated by sickness and death of inoculated dogs. Titrations were not * Initiation of the research reported in this paper was aided by a grant from the State of Washington Initiative 171 Fund for Research in Biology and Medicine. Since the early stages of the study, the principal financial support has been received from the National Cancer Institute, United States Public Health Service. Received for publication May 25, carried out for a second N virus preparation or for stock virus N Stock virus N-212 was used to a limited extent. It consisted of the supernatant fluid from a 10 per cent extract of papilloma tissue from four dogs. The papillomas had been preserved in 50 per cent buffered glycerin for 5 years. Canine oral papilloma infection was produced by applying the stock virus to the scarified oral mucosa or by injecting it into the mucosa of young dogs. After inoculation the dogs were routinely examined at intervals of 1 week. The dogs that were used for most of the experiments were mongrels approximately 2-6 months of age. They had received distemper vaccine and hepatitis vaccine from 1 to several weeks before they were given inoculations of papilloma virus. In spite of these and other precautions, the experiments were seriously hampered by a variety of intercurrent diseases including distemper, hepatitis, and severe mange. Tissue cultures were prepared from oral mucosa, bladder mucosa, conjunctiva, and kidney cortex from newborn or young pups. Finely minced tissues or trypsinized tissues were planted on the sides of tubes with 0.5 ml. of 0.5 per cent lactalbumin medium with 10 per cent horse serum, or with 0.5 ml. of medium 199 with either 2 per cent or 40 per cent horse serum. All media contained 100 units of penicillin and 100 mg of streptomycin/ml; most media also contained 25 units of Mycostatin/ml. Cultures that were to be used for virus studies were inoculated with the N-~ll strain of papilloma virus at the time the cultures were initiated, or 8-7 days later, at which time cell proliferation had begun, or after a longer period of maintenance as stock cultures. The medium of both inoculated an uninoculated cultures was usually changed every 8 or 4 days. RESULTS Virus assay methods.--two methods of assaying papilloma virus were compared. In both methods dogs were anesthetized by the intraperitoneal administration of 60 mg. of sodium pentobarbital per 5 pounds of body weight. The oral sites to be inoculated were wiped with dry cotton. No chemical disinfectant was used before or after inoculation. The simultaneous-injection method consisted of injection of ml. of each of several serial dilutions of stock virus into different sites in the oral mucosa. A bleb was produced at the site. Any visible leakage that occurred after the needle was withdrawn was absorbed with dry cotton. To de-

2 CHAMBERS AND EvAns--Canine Oral Papillomatosis 1189 termine the amount of virus that might spread from one inoculation site to another, three control sites in the oral mucosa of each dog were injected with diluent. :Papillomas developed at the sites of virus injections 31- days after inoculation. :Figure 1 shows the discrete lesions produced by this method of inoculation. It was feasible to inject twelve different sites in the mouth of each dog. The staggered-scarification method consisted of inoculation of serial dilutions of stock virus into small scarified areas of oral mucosa at intervals of ~ or 3 days. The purpose of this interval was to allow time for the virus from one inoculum to disappear from the secretions in the mouth before at one time, thus obviating the use of separate frozen samples of virus for each dilution. This method also eliminated the possibility that serum antibody, produced in response to the first inoculation by scarification, might interfere with later inoculations. Inoculation by scarification was useful for obtaining large amounts of papilloma tissue as a source of virus or antigen. Tissue-culture methods were investigated in an attempt to find a virus assay technic that could be accomplished without giving inoculations to dogs. We attempted unsuccessfully to find tissue that would support the growth of the virus and would show some recognizable change as the result of viral multiplication. The results of tissue-culture TABLE 1 ASSAY OF VIRUS BY INOCULATION OF MULTIPLE ORAL SITES IN EACH DOG DILUTION OF VIRUS METHOD Simultaneous injection* Staggered scarification:~ /3? 3/3w 10-2 lo-a /6 5/6 5/8 3/3 3/3 2/3, /6 1/ /6 1/s 10-7 O/3 0/3 Diluent only 1/l~ 1/9 * Each dog received three injections of each of three virus dilutions and three injections of diluent. s were made in close succession beginning with the diluent and proceeding from the more dilute to the less dilute virus suspensions. t Numerator = total number of injection sites at which papillomas developed. Denominator = total number of sites inoculated with the virus dilution or diluent as indicated. :~ Each dog received one inoculation of each virus dilution 10-1 through 10-7 and three inoculations of diluent without virus in separate scarified areas. Inoculations were staggered over a period of 21 days. w Numerator = total number of inoculated scarified sites at which papillomas developed. Denominator = total number of scarified sites that wele inoculated. the scarification was carried out for the next inoculation. :Figure ~ shows tumors that developed in response to this method of inoculation. The small single tumor resulting from the l0-5 inoculum, the few scattered tumors at the site of the 10-4 inoculation, and the confluent tumor mass at the site of the 10-3 inoculation are typical of the responses to graded doses of virus. The results of both titrations are presented in Table 1. A papilloma developed at one of the twelve sites that were injected with diluent and in one of nine scarified sites that received diluent. These results are typical of our experience and show that, although virus may spread from one inoculation site to another, it does so infrequently. Virus concentrations 10 or more times higher than the IDs0 level produced lesions in per cent of tests in susceptible dogs. The simultaneous-injection method was preferred, because all dilutions could be inoculated experiments involving primary viral inoculation of pup tissues are presented in Table ~. Tests for virus in tissue cultures consisted of injection of tissue-culture fluids, with or without cells, into the oral mucosa of dogs shortly after tissue components had been harvested or after a period of storage in the frozen state. The few test sites at which tumors developed suggested that virus probably persisted in small concentration for periods of several weeks in some of the cultures. There was no indication of extensive multiplication of virus in any of the cultures. The cellular degeneration, which was mild in some inoculated cultures and extensive in others, usually paralleled the degeneration in uninoculated controls and was not attributed to virus. Attempts to subculture the virus through two or three blind passages showed no degeneration which could be related to virus multiplication. The capacity of papilloma cells to grow in tissue

3 1190 Cancer Research Vol. 19, December, 1959 culture and to support the multiplication of the papilloma virus was investigated. Tissue cultures were prepared from oral papillomas of four dogs between 1 and days after the tumors first appeared. In 71 cultures that were observed for periods of 6 or 7 weeks, proliferation of cells occurred around approximately 15 per cent of the 850 explants. Fluids from several cultures were inoculated into the oral mucosa of dogs to determine whether the virus had multiplied. Unfortunately, intercurrent infections in our dogs at this time prevented satisfactory results. layer cultures of chick embryo cells gave irregular results. In another experiment canine oral papilloma virus failed to interfere with the cytopathogenic effect of the Rustigian monkey kidney virus (6) in cultures of strain L cells. The incubation period.--the incubation period, representing the interval between inoculation of virus and the first appearance of papillomas as tiny white papules, was usually between 4 and 8 weeks. Chart 1 shows the range of incubation periods in a group of 90 dogs. In 69 per cent of the dogs this interval was 4-5 weeks. The data in TABLE 2 CANINE ORAL PAPILLOMA VIRUS IN CULTURES OF PUP TISSUES TISSUE USED FOR CULTURES AGE OF DONOR PUP CULTURE PERIOD BEFORE VIRUS INOCULATION PERIOD OF OBSERVATION OF INOCULATED CULTURES (WEEKS) CELL PROLIFERATION TESTS FOR VIRUS In Around Weeks after eul- ex- inoc. of Results tures plants cultures Oral mucosa Urinary bladder mucosa 2-3 mo /5* 47/66t 2/~ Unknown 1/8 1/4s 5/5 52/64 2/2 Unknown 3w i/5 s 0/5 3 1/8 3w 1/5 Conjunctiva Kidney cortex 2-3 mo , 2/2 Unknown 3w and 4w 2/2# Unknown 5/5 Sheet 3/3 Sheet 4/4 Sheet 5w 115 s 0/3 15 0/5 3 1/5 * The numerator indicates the number of cultures in which cell proliferation occurred in one or more explants. The denominator indicates the number of cultures inoculated. t The numerator indicates the number of explants that showed cell proliferation. The denominator indicates the total number of explants that adhered to the glass. "Sheet" indicates that cultures were made from trypsinized cells which grew in sheets. :t The numerator indicates the number of papillomas that developed in response to injections of tissueculture fluids into the oral mucosa of dogs. The denominator indicates the total number of oral mucosa sites that were inoculated. w Fluids removed from cultures at the time indicated were pooled and injected into one site in the oral mucosa of each of five dogs. A papilloma developed at only one site. # Cultures were inoculated with living papilloma cells infected with N-211 strain of virus. Various procedures were carried out in trying to find a means of detecting virus in inoculated cultures. The possibility that the union of virus and antibody either on or within cells might cause degeneration of cells in inoculated cultures was considered. Neither antiserum nor lymphocytes from immune animals produced an observable effect on cultures of pup tissues previously inoculated with virus. The interference phenomenon was used as the basis of another approach to the problem of detecting and assaying canine oral papilloma virus. Several tests of the ability of the papilloma virus to interfere with the plaque-forming capacity of Western equine encephalomyelitis virus in mono- Table 3 show the variation in length of the incubation period in relation to the concentration of virus inoculated. The range in incubation periods after inoculation with 10-4, 10-5 and 10-6 dilutions of papilloma extract was -72 days. The range in incubation periods for 10-1, 10-2, and 10-3 dilutions was 8148 days. An attempt was made to recover virus during the incubation period. Three donor pups were each inoculated at ten sites with supernatant fluid from a 10 per cent suspension of papilloma tissue. The oral mucosa was removed from single inoculation sites from each pup at 3 days, 1 week, and at weekly intervals thereafter. Each tissue specimen was divided into three portions. One piece was

4 CHAMBERS AND EVANS--- Canine Oral Papillomatosis 1191 fixed for light microscopy and one piece for electron microscopy. The remainder of the specimen was cut into small pieces and frozen in 0.5 ml. of Locke's solution. Later, the frozen specimens were thawed, minced to small fragments, ground in a mortar with sterile glass particles, and diluted with 1.0 ml. of Locke's solution. The supernatant fluid was injected into the oral mucosa of pups to test for the presence of canine oral papilloma virus. The 3-day specimen from one of the three donor pups yielded virus. No virus was detected in tissue specimens removed from the three dogs 1, 2, and 3 weeks after inoculation or in specimens harvested from two of the dogs 4 and 5 weeks after inoculation. The third dog was eliminated from the study after 3 weeks because of illness. The 5-week tissue specimens from the two remaining dogs contained a few tiny papules that were considered early papillomas. These two dogs later developed tumors at other inoculated sites. The failure to detect virus in the 5-week specimens is puzzling. It may have resulted from dilution of virus or absence of papilloma cells in the fragment of tissue used for virus detection. Fach)rs influencing number, size, and appearance of tumors.--the number, size, and appearance of papillomas in the oral mucosa depended upon the concentration of virus in the inoculum, the method of inoculation, the number of sites inoculated, and the time at which the observations were made. The application of l0-1 to l0-3 dilutions of N-211 strain of virus to a scarified area usually produced many papillomas which were first observed as small individual growths. Many additional tumors frequently appeared during the following week. As the tumors increased in size, numerous short papillae were formed which gave the tumor a fungiform appearance. The continued growth and the elongation of papillae produced the filiform appearance which was characteristic of many large papillomas. As the tumors grew they became confluent in areas where papillomas were numerous and close together. Tumors that arose as the result of inoculation of various dilutions of virus into separate patches of scarified oral mucosa are shown in Figure 2. A confluent papilloma mass occurred at the site of the 10-3 inoculation. Several discrete tumors, separated by sufficient distance to prevent them from becoming confluent, are shown at the site of the 10-4 inoculation. One discrete tumor resulted from the 10-5 inoculation. When the inoculation was made by a single injection, the inoculum was confined to a small area ~b n,. t, :~ IO- 0 ~ T 8 Incubation Period (weeks) CHART 1.--Range in incubation period of canine oral papilloma virus. TABLE 3 RELATION OF VIRUS CONCENTRATION TO INCUBATION PERIOD IN DOGS INOCULATED WITH TWO OR MORE CONCENTRATIONS OF THE SAME PREPARATION OF VIRUS Doe DILUTION OF STOCK SUSPENSION OF PAPILLOMA TISSUE* lo-1 10-~ lo-a N-310 N-311 N-313 N-316 N-317 N-318 N ,,? ,, 31, 31, 45 31, 31, 31, , 5~ 45, 45, , * The same stock virus was used for all inoculations. Dogs N-310, N-311, N-313, and N-316 were given inoculations by injection of the virus preparation into the various sites of oral mucosa. Dogs N-317, N-318, and N-319 were given inoculations by application of the virus preparation to small scarified patches of oral mucosa. All animals were examined at weekly intervals after inoculation. t The three numbers indicate the number of days elapsing between inoculation and the first observation of papillomas at three inoculation sites.

5 1192 Cancer Research Vol. 19, December, 1959 and gave rise to papillomas that were close together. As these papillomas grew they often became confluent, and it was not clear whether the fully developed growth had arisen from one or from several tumors. Papillomas that arose at three sites that were injected with a 10-4 dilution of virus and at one site (arrow) that was injected with a 10-6 dilution are shown in Figure 1. A variation of the injection method consisted of injecting inoeula into the oral mucosa to form a bleb and then making approximately five to fifteen needle punctures into or around the bleb. The tumors that resulted from this multiple-puncture method of inoculation were usually more numerous that those resulting from the simple injection. They often became confluent over a small area and were then described as discrete tumors. The ultimate size of the tumor tissue that developed was apparently dependent on the concen < I0 II 12>12 Tumor Growth Period (weeks) C~ART ~.--Growth period of canine oral papillomas in 57 dogs that had been inoculated with stock virus. tration of virus in the inoculum, the method of inoculation, and on the length of the growth period. Examination of the oral mucosa of dog T-105 on the 33d day after multiple-puncture inoculation with a 10-1 dilution of virus revealed three or four beginning papillomas. One week later there were approximately 31 small papillomas, and the following week all papillomas had regressed. The development of the papilloma tissue in this dog was limited by the onset of regression. In dogs N-34~ and N-343 which were given inoculations of a dilution of papilloma extract, the papilloma growth periods were 11 and 14 weeks, respectively. In spite of these long growth periods, the maximum amount of tumor tissue that developed in these dogs was very small, apparently because the concentration of virus in the inoculum was low and because the single-injection method of inoculation exposed relatively few cells to the virus. Factors influencing the time of regression..--the growth period is designated as the interval between the first appearance of papillomas and the first definite signs of spontaneous regression. The sloughing of any portion of the filiform material, or decrease in the size of small, fungiform tumors, was regarded as definite regression. The length of the growth period of tumors in 57 dogs is presented in Chart 3. The growth period ranged from 10 days to 31 weeks, with 33 per cent of the dogs showing regression within the first 4 weeks, 56 per cent during the second 4 weeks, and 31 per cent 9 weeks or more after appearance of tumors. Dogs whose tumors showed unusually short or unusually long growth periods are of particular interest. Data on the six dogs that exhibited unusually early regression of papillomas are presented in Table 4. Tumors regressed within 33 days from the time of their appearance in these dogs. It is notable that all these dogs had been given inoculations of the relatively high concentrations of virus. The amount of tumor tissue that had developed in the dogs' mouths at the time regression occurred was small to moderate. The possibility that the concentration of virus in the inoeulum might affect the duration of the growth period was considered. Thirty-two of the 57 dogs represented in Chart 3 had received a 10-1 dilution of N-311 stock virus, and one dog had received a 10-1 dilution of N-313 virus. Twelve dogs had received 10-2 to 10-5 dilutions of N-311 virus. A comparison of the tumor growth periods for these dogs is presented in Chart 3. There was a tendency toward delayed regression of tumors in dogs that were inoculated with tumor extracts diluted to 10-2 or more. The possibility that delayed regressions were related to the size of tumors was also considered. A large amount of tumor tissue was present in the mouths of six of the eight dogs that received the 10-2 to 10-5 inocula and whose tumors regressed after a growth period of 8 weeks or more; the other two dogs had one or two small tumors. A large amount of tumor tissue was also present in the mouths of two of the four dogs that received the 10-1 inoculum and whose tumors regressed after a growth period of 8 weeks or more; the other two dogs in this group probably had small to moderate tumors, although our data on tumor size in these two dogs are incomplete. Of the four dogs that received the 10-2 to 10-5 inocula and whose tumors regressed early, three had moderate to large tumors and one had several small tumors. No significant relationship between tumor size and late regression could be deduced from these data. The regression period.--the growth of papillomas terminated with the first definite signs of regression. Once regression was established, it proceeded rapidly in most instances until all filiform

6 CHAMBERS AND EvANs--Canine Oral Papillomatosis 1198 and fungiform papilloma tissue had sloughed, leaving an elevated papilloma base. In two-thirds of a group of 54 dogs that had no treatment other than virus inoculation, this phase of regression was completed within a period of approximately 1 week. Of the remaining eighteen dogs, fifteen required ~ weeks for this phase of the regression period, and three required 3 weeks. The second phase of regression involved the resorption of the papilloma base and the return of the appearance of the mucosa to normal with no scar remaining. The small amount of data collected on this phase indicates that a period of a few days to 3 weeks usually elapsed before the mucosa resumed a normal appearance. In a few instances this phase was prolonged to 4-7 weeks. When papillomas regressed from pigmented areas of the mouth, the apparently normal mucosa sometimes remained without pigment for several weeks. Usually all papillomas in the mouth of any given dog regressed at the same time regardless of their size, location, or time of appearance. This was true of secondary papillomas as well as of primary papillomas. Secondary papillomas.--a second crop of tumors sometimes appeared in dogs exhibiting late regressions. Of the seventeen dogs whose tumors regressed 8 weeks or more after their appearance as shown in Chart 2, seven developed secondary papillomas. The number of such tumors ranged from one or two to a great many. Figures 3 and 4 show the numerous secondary papillomas that developed in uninoculated sites in dog N days after inoculation and 64 days after the first of two primary tumors appeared at an inoculated site. The second primary tumor developed at a site of inoculation in the lower left oral mucosa and is not shown in the photograph. The secondary papillomas occurred on the floor and the roof of the mouth, the tongue, the gingival mucosa, and the outer lip. Data concerning dogs that developed secondary papillomas are presented in Table 5. The secondary papillomas appeared 7-9 weeks after the primary papillomas were first observed. In view of this fact, it is evident from the data in Chart that papillomas usually start to regress before one would expect secondary papillomas to appear. It is possible that we failed to observe secondary papillomas in a small proportion of animals since ~15- (5 ~3,. 10" q~ 4:1 INOCULUM 9 IO-Idilution tumor extract [] I0-2"- 1(~5dilution tumor extract I Tumor Growth Period (weeks) CHART 3.--Relation of growth period to concentration of virus in the inoculum. TABLE 4 DATA ON DOGS EXHIBITING UNUSUALLYEARLYREGRESSION OFP.~ILLOMAS DoG VIRUS INOCULUM Stock Diluor s,urce tion ~ETHOD OF INOCULATION INCUBATION PERIOD G ROW TH PERIOD REGRESSION PERIOD APPROX. NO AND SIZE O1~' PAPILLOMAS N-182 T-68 T-77" ~-101 N-317t N-351 N N-211-S 10 -x N t N N-~ll r N-~ll Scarification and injection and puncture and puncture Scarification Scarification Between 20 and Between 10 and < small Numerous small Numerous small 1 moderate, 12 small More than 13 confluent Moderate * Dog T-77 was one of five litter-mates given inoculations of papilloma virus at the same time. The other four pups were included in immunization experiments. Papillomas regressed early in all five pups regardless of treatment. t Dog N-317 was used in a virus titration and received an inoculation of one dilution of virus every 2 or 3 days for 19 days. Inoculation of virus dilutions began with the 10 -r dilution and proceeded to the l0-1 dilution. Papillomas developed at sites inoculated with 10-1, 10-2, l0-3, and 10-4 dilutions.

7 1194 Cancer Research Vol. 19, December, 1959 they might easily be overlooked in dogs with large primary tumors. The fact that 7-9 weeks passed between the appearance of the primary and the secondary papillomas suggests that the virus shed by the primary tumors soon after they appeared may have been responsible for the secondary infections. This period of 7-9 weeks may be comparable to the incubation period in the natural infection. The case of dog N-314 provides information on the natural incubation period. This dog was not inoculated but was allowed to run in the paddock with inoculated dogs. Papillomas developed in its mouth 63 days after they were first observed in the mouths oculated with a 5 per cent or 10 per cent concentration of tumor extract, all seven dogs that developed secondary papillomas had been inoculated with 10-2 to 10-5 dilutions of tumor extract. Thus it appears that the more dilute inocula favored the development of secondary papillomas. No relation was found between the development of secondary tumors and the length of the incubation period or the growth period, or the size of the primary tumors. DISCUSSION The assay of canine oral papilloma virus was not described in the earlier studies of this disease. TABLE 5 TIME OF APPEARANCE AND REGRESSION OF SECONDARY PAPILLOMAS DoG DILUTION OF N-~ll STOCK YIRUS* METHOD OF INOCULATION INCUBATION PERIOD, PRIMARY TUMORS INTERVAL BETWEEN APPEARANCE OF PRIMARY AND SEC- ONDARY PAPILLOMAS Primary tumors (days) GROWTH PERIOD Seondary tumors (days) N-311 N-322 N-342 N-S43 N-458 N-459 N _10- s 10-~ Scarification Scarification * Virus used as inoculum. of the inoculated dogs. Another pup, T-98, was inoculated at the same time as several other dogs with which it was housed. Papillomas failed to develop in T-98 until 64 days after they were observed in the mouths of the other dogs in the same paddock. It appears that dog T-98 did not receive an infective exposure to virus at the time of inoculation but was infected by the virus shed from the papillomas in the mouths of infected dogs in the same paddock. The relationship of the concentration of inoculated virus to the development or failure of development of secondary papillomas was analyzed. Whereas six of ten dogs in the delayed-regression group without secondary papillomas had been in- This presented an imposing problem at the outset of these studies, largely because the virus will not infect tissues other than the oral mucosa of dogs. It was suspected that virus would spread freely in the saliva, and hence inoculation of multiple sites in the mouth would yield confusing results. Fortunately this was not the case. As many as twelve injections of graded doses of virus could be made in the mouth of each dog. The likelihood that virus will spread from one injection site to another is probably fairly well represented by the results of control inoculations cited in Table 1. A papilloma developed in only one of twelve control injection sites. This means that, if a given specimen is tested for virus in duplicate in two different dogs and is Fro. 1.--Discrete lesions produced by the simultaneousinjection method of inoculation. The three tumors shown in the upper mucosa were the result of injection of a 10-4 dilution of stock virus at three different sites. The arrow points to a small lesion in the lower part of the mouth that appeared in response to injections of a 10-8 dilution. FiG. 2.--Papillomas that appeared in response to inoculation by the staggered-scarification method. The dilution of the virus inoculated at each site is indicated. FIGs. 3 and 4 (Dog N-458).--Numerous secondary papillomas that developed in uninoculated sites 98 days after inoculation and 64 days after the first primary tumor appeared. This tumor developed at the anterior site of inoculation of the upper right oral mucosa and is one of the tumors shown at this site in Figure 4. All other tumors in Figures 4 and 5 are secondary tumors. At the time the photographs were taken the first primary tumor was 128 days old and the secondary tumors were 64 days old.

8 FIG. 3a.--A 6"2-year-old male with mycosis fungoides. Appearance of hands before treatment with epoxypiperazine. FiG. 3b.--Appearance after treatment. Downloaded from cancerres.aacrjournals.org on July 3, American Association for Cancer

9 CHAMBERS AN'D EvANs--Canine Oral Papillomatosis 1195 positive in both, the probability that this result was due to spread of virus from another specimen inoculated simultaneously in the same dogs is not more than one in 100. The data in the present study were analyzed in an attempt to find a common factor in either very early regressions or in very late regressions. Our data suggest that concentration of virus in the inoculum may influence the time of regression in an inconstant manner. Late regression of papillomas was observed more frequently in dogs that were given inoculations of 10-2 to 10-5 dilutions of tumor extract than in dogs that were given inoculations of a 10-1 dilution. The higher dilutions also seemed to favor the development of secondary papillomas. Late regressions of tumors that developed in dogs given inoculations of 5 per cent or l0 per cent tumor extract were not accompanied by secondary tumors. Since relatively few dogs have been given inoculations of small infective doses of virus, the significance of these findings remains to be determined. Limited data suggest that the incubation period in the natural infection is approximately the same length as the interval between the appearance of primary papillomas and secondary papillomas in experimental infections. This is longer than the usual incubation period in the experimental infection. The long incubation periods that occurred in a few dogs following inoculation with 10-4 to 10-6 dilutions of tumor extract suggest that secondary papillomas and the natural infection may result from the relatively small amounts of virus that reach susceptible cells. More than 57 dogs with canine oral papillomas were observed for several months. Papillomas regressed spontaneously in every instance. The longest interval from the time tumors appeared until regression occurred was ~1 weeks. No signs of malignancy or metastasis were observed in any of the dogs. SUMMARY Multiple simultaneous injections of graded doses of virus into the oral mucosa of young dogs were found to give useful assays of canine oral papilloma virus. The Infective Doses0 of a typical stock virus preparation injected in a volume of 0.05 to 0.1 ml. was approximately A study of the experimentally produced disease revealed incubation periods of 4-8 weeks in 89 of 90 dogs. The growth period, extending from the first apperance of papillomas to the first sign of regression, ranged from 4-8 weeks in 37 of 57 dogs and 9-~1 weeks in twelve dogs. All tumors in each dog regressed simultaneously. In most dogs all filiform papilloma tissue sloughed within 1 week after regression was first evident. Resorption of the papilloma base required a few additional days to several weeks. The time of regression did not appear to be related to tumor mass or to length of incubation period but appeared to be related to the concentration of tumor tissue inoculated. There was a tendency toward early regressions in dogs that were given inoculations of a l0-1 dilution of tumor tissue and a tendency toward delayed regressions in dogs that were given inoculations of 10-2 to l0-5 dilutions. REFERENCES 1. DEMONBREUN, W. A., and GOODPASTURE, E. W. Infectious Oral Papillomatosis of Dogs. Am. J. Path., 8:43-56, 198s ~. ~knd~y, G. M. A System of Bacteriology, 7:~5~-58. 1st ed. London, England: His Majesty's Stationery Office, GOODLOW, R. J. Canine Oral Papillomatosis. Thesis, University of Minnesota, Minneapolis, Minn., M'FADYEAN, J., and HOBBAV, F. Note on the Experimental Transmission of Warts in the Dog. J. Comp. Path. & Therap., 11: , PENBERTHY, J. Contagious Warty Tumours in Dogs. J. Comp. Path. & Therap., 11:363-65, RUSTIGIAN, R.; JOHNSTON, P.; and REIHART, H. Infection of Monkey Kidney Tissue Cultures with Virus-like Agents. Proc. Soc. Exper. Biol. & Med., 88:8-16, 1955.

10 Canine Oral Papillomatosis I. Virus Assay and Observations on the Various Stages of the Experimental Infection Velma C. Chambers and Charles A. Evans Cancer Res 1959;19: Updated version Access the most recent version of this article at: alerts Sign up to receive free -alerts related to this article or journal. Reprints and Subscriptions Permissions To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at To request permission to re-use all or part of this article, use this link Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.