| .4'3" 5"I.'é.flv‘". £15 L: i‘i‘! 843.3“: Q .‘ at»: 0 fl 5 , hr; ‘0". {In ‘I’I‘; \1 \l‘ {P ‘0‘ ‘81! 4 V “I If r 1'! 6610 1‘13 5‘ ‘2‘? Q‘ n I [\f.‘ WESES LIBRARY Michigan State University .. 'i‘ A' - w—---- -w m ~ w ---. a... .. — .. -. .— c- .- _...— .. .. . .y . w 3 ‘u g r . l ”V. —l - k ’ I u' , I ~ I; . I“ 'n‘. ‘ nu J '- N. “T. o. '. - ~—-_ .2 :s PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES rotum on or before data duo. DATE DUE DATE DUE DATE DUE ll ,EH301‘ I W MSU Is An Affirmative Action/Equal Opportunity Institution cMmeS-DJ m *1 SUE-{ACT A sum or 1:3 inn: 12155101: 0? n VIRAL- IzrDUCED mm: Imnmonnssroxn (131$: rmnmozm Jillard G. illiter Tr ans Lnission studies of an unusual avian renal tumor reported previously as an adenocarciuoma were undertaken since the neoplasm occurred in chickens inoculated wiLh an avian leu.:osis strain (BAI-A) formerly believed to cause only granuloblastic leu.iemia. The tumor was successfully transplanted throu QM 12 serial pass- ages. In the majority of cases, a palpable tumor at the site of inoculation developed in about 2 weeks, growth was pr% ressive and was followed by mortality in 'bout 3 additional weeks. The leukemia J ‘ o L*nanifes ta tion cn'racLe‘is Lic of Luis strain was eliz;1inaLed a ‘ter nine tra isplant pas sages . Virala Hti Jity of three of the transplant passages was tested by inoculation of tumor fi trates in leukemia susceptible chickens. Those from the last transplant passage failed to induce leukemia but 1' did cause a lfi Dh incidence or renal tumors and a moderate incidence of visceral lynphonato sis ando meopetrosis. Histopatholog ical evidence was preserted which justifies con- sidering this tumor a nephroblastoma (embryonal nephrona) rather than an adenocarcinoma. Although the leukemia manifestation was elira naLed fr m the neo- plast'c spectrum of strain A in later transplant passages and in the filLrate test of one of them, these experiments are not conclusive Willard G. Walter evidence of a separate etiological agent for leukemia. The possibil- ity that avian renal tumors should be included asenladditional member of he group of diseases known collectively as the avian leukosis complex is considered. A STUDY OF THE TRANSMISSION OF A VIRAL-INDUCED AVIAN NEPHROBLASTOMA (meRYomAL NEPHROMA) By 3 l) w" {J 7 Willard GJ Walter A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1961 .ACKNOWLEDGMENTS The author expresses his sincere appreciation to Dr. Ben R. Burmester for constant guidance and council throughout the course of this study. For his advice and many helpful suggestions; gratitude is expressed to Dr. Charles H. Cunningham. The careful study of his- topathological specimens by Lt. Col. Fredll Maurerof the Armed Forces Institute of Pathology was most helpful. Thanks are due to Mr. Barclay Larkinszfinrhis competent technical assistance and to Mr. Philip Coleman for his painstaking photographic efforts. ii. TABLE OF CONTENTS Introduction - . . . . . . . . . . . Review of Literature . . . . . . . . Nephroblastomas in general- . . Nephroblastomas in fowl . . . . Artificially induced nephroblastomas in fowl. . materials and methods. - . . . . . . Cell suspensions- . . - - . . . Cell-free extracts. - . . . . . Inoculation . . . . . . . . . . Chickens. . . . . . . . . . . . Histopathology. Requ_tS o o o o o o o o o o o o o o 9 Transmission of tumors with nephroblastoma cell suspensions passed serially . . Ante-mortem observations . Mortality of chickens from Pathology. . . . . . . . . Tumor regression . . . . . Viral-induced neoplasia- . Cell-free transmission. . . . . Discussion . . . . . . . . . . . . . Summary and Conclusions. . . . . . . Literature Cited 0 O O O O O O C O O O O O O O O O O O O O O O O O O nephroblastomas iii. \O\O\OOD(I)ODU'I\J1W p O 10 10 11 12 13 1h 17 20 26 27 Table Table Table Table Table LIST OF TABLES Duration of primary nephroblastoma and time of mortality related to age of test chickens and donor birds. Incidence and time of mortality from secondary neoplasms in chickens inoculated with nephro- blastoma cell suspensions. Leukemia duration and related pathology in chickens inoculated with nephroblastoma cell suspensions. Filtrate induced mortality incidence of selected passages of transplanted nephro- blastomas. Time of mortality related to type of disease induced by filtrates. iv. Fig. 1. Fig. 2. Fig. 3. Fig. h. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig.10. LIST OF FIGURES Nephroblastoma-pectoral; cell transplant. Sagital section shows ldbulated tumor masses encased in thin remnant of pectoral muscle. Necrotic tissue prevalent in lower portion. Nephroblastoma-pectoral; cell transplant. Ana- plastic cells growing in an unorganized manner among remnants of isolated muscle fibers. Nephroblastoma-pectoral; cell transplant. Small tumor with minimal connective tissue septa and slight evidence of cell nest formation. Peri- pheral lymphocytic foci. Nephroblastoma-pectoral; cell transplant. Cell nests separated by abundant sarcomatous stroma; note formation of primitive thick-walled tubules. Nephroblastoma-pectoral; cell transplant. Well defined tubules with basement membrane and scant interstitial stroma. Nephroblastoma-pectoral; cell transplant. meta- plastic bone in interstitial stroma bordered by area of thin-walled tubules. Nephroblastoma—renal ; filtrate induced. Large lobulated encapsuled tumor attached to remnant of left kidney; smaller neoplasm on posterior aspect. Nephroblastoma-renal; filtrate induced. Primitive cells arranged in nests separated by sarcomatous stroma. ‘Within nests the formation of tortuous tubules is seen. Nephroblastoma—renal; filtrate induced. Tumor composed of well differentiated tubules which remarkably simulate renal tissue, including glomerulus-like structure. NephrOblastoma-renal; filtrate induced. Sarcoma- tous stroma composed of relatively mature fibrous connective tissue, with small area of cartilage. Tubules are thinawalled, well differentiated and in some cases show papillary structure. T (fflTW/V‘Nva‘u’fi ’T .54“ ”VD-LIC'LI Recent lonH-term e kperine ents (B mester et al., 1959b) designed to test the tumor inducing p1tential of two strains 01 avian leucosis (R of Engelbrcth-Holn, and 33115) considered pure (Beard, 1337a) , i.e., capable of pruducing only leukemia, showed that both strains caused visceral lynplc.’ csis as well as tne swecific typecfifleukcnia haractcristic cf eacn straig. in addition, strain BAI—A Cd‘sec steepetros is and a tumor of the renal parenchyma. The latter was considered at the time to be a renal adenocarcinona. ~, 5 --l-- l‘p1epare cell-free extracts. Experi- m ments were conducted in parall- 1, using tu.r or extiact as 011e scurcecu virus and plasma from leukemic birds as the other. lnree serial pas- sages were made. Chicks were inoculated in rave nous 1y whenQBdays old. Infectious plasma continued to cause 100$ riortality . Using exoracts O I ‘I ”I derived from tumors, the nor sality from leukemia decreased Iron 50% 7 to 20% and the incidence of nephroblastomas rose from h0¢ to 80% from the second to the third passage. 'Within limitations of the experi- ments other data suggest no detectable change in the pathologic expression of the virus. Plasma from a leukemic bird of the second tumor passage again produced 100% leukemia upon subinoculation of 3-day old chicks. MATERIALS AND METHODS Cell suspensions. Nephrdblastomas were harvested under aseptic conditions and macerated in an Olson tumor mincer (Olson, l9hl). Two parts of Simms' salt solution (Simms and Sanders, l9h2) by volume were added to one part by volume of the mince. The suspension was passed through a double layer of coarse sterile gauze and 1,250 units of penicillin G and 2.5 mg of dihydrostreptomycin per ml were added. Cell suspensions were prepared from freshly harvested tumors and pro- cessed as rapidly as possible so that inoculation into other chickens was accomplished within 2 to 3 hr. Cell-free extracts. Filtrates of transplanted tumors were pre- pared 1 day prior to inoculation from tumors which had been har- vested from several transplant passages. The tumors were collected aseptically, sealed in Pyrex tubes and stored in dry ice. One tumor from each of transplant passages 2, 8, and 12a which had been stored for 215, 118, and 32 days, respectively, was selected for test of viral activity. The thawed tumor was processed in a Virtis homo- genizer with 19 parts by volume of Simms' salt solution for each gram of tumor. The homogenate 'was clarified in an International PR-l refrigerated centrifuge at approximately 1,200 X.§. for 20 min at hC. The supernatant fluid was carefully pipetted into a graduated cylin- der and l g of Celite 512* was added for each 20 ml of extract. The mixture was further clarified by passing it through a pad composed of Celite and Simms' salt solution on No. l Whatman filter paper in a Buchner funnel. It was then passed through a Selas 02 filter under negative pressure which did not exceed 50 mmci’mercury. The filtrate *Johns-Manville Products Corporation 8 9 was shell-frozen in sealed vials and stored overnight in dry ice. Inoculation. The cell suspensions were inoculated into the pec- toralis muscle except for the first passage in which the intraperito- neal route was used. The filtrates were inoculated into the cubital vein. The inocula were 0.2 cc, using a l-cc tuberculin syringe fitted with a 20-gauge needle for the cell suspensions and a 26-gauge needle for the filtrates. All inocula were kept in crushed ice during pre- paration and inoculation. Both types of inocula were used without additional dilution beyond that used in preparation. Chickens. All studies utilized progeny of an inbred line of single-comb White Leghorn chickens (Line 151) which had been maintain- ed in isolation for many years in order to reduce natural infection (Waters, 19h5, 1951). The spontaneous incidence of both visceral lymphomatosis and neural lymphomatosis in this flock does not exceed 5% and no cases of leukemia or osteopetrosis have been reported. The age of the chickens at the time of inoculation was variable for dif- ferent cell suspension passages, but was uniform within each passage. All chickens inoculated with filtrates were 7 days old. Histgpatholggy. Blood smears were stained with May-Gruenwald and Giemsa stains. Tissues were fixed in Zenker-formol solution and stained with hematoxylin and eosin. RESULTS Transmission of tumors with nephroblastoma cell suspensions passed serially Ante-mortem Observations. The renal tumor was readily transmitted using cell suSpensions in serial passage. Growth at the site of inoculation occurred in the majority of cases, and was progressive. While rapid progressive growth with fatal termination was usual, no cases of unquestionably metastatic renal tumors were observed. Examinations were conducted three times a week to determine by palpation the time of onset of neOplasia. The data in Table 1 are an approximation of the time at which one-half the tumor-bearing birds developed a palpable mass. The time of development of the tumors was variable and ranged from 10 days to 19 days. Those of passage 5 developed later than the tumors of the preceding or succeeding pass- ages. This discrepancy may perhaps be attributed to the use of a young donor in an early stage of oncogenesis. The tumor used for passage 5 was harvested at 2 weeks postinoculation, in contrast to most of the others which were selected at 3 to 4 weeks. In spite of the decreased incidence of tumors when older more resistant chickens were inoculated (passages 9, 10, 11, and 12b), no delay in the clinical onset of neoplasia was noted. In those birds which developed a tumor, a palpable mass was detectable in 10 to 12 days. Within the limitations of these experiments, the major effect of inoculation of an older chicken was a decreased incidence of mor- tality and not a delayed onset of neOplasia. Whether or not the time of mortality is delayed in older tumor bearing chickens cannot be definitely determined from the data. The mean time ofonset of palpable lO 11 tumors for all 12 passages was 13 days. The mean time of mortality, 33 days, occurred about 3 weeks later. Mortality of chickens from nephrdblastomas. The mortality rate in each passage is shown in Table 1. The age of the host has a direct bearing on both the incidence and the rate of development of many experimentally induced tumors. In these experiments, the age at the time of inoculation was unavoidably variable since passages were made with freshly harvested tumor and it was impossible to have birds of a constant age available for all passages. Older birds were deliberate- ly selected beginning with the 7th passage so that this factor could be appraised in relation to the alterations it might induce in'both the type of neoplasia and magnitude of the response. Eckert et a1. (1955) reported on the susceptibility of chickens of different ages to the development of granuloblastic leukemia, and results showed the youngest age tested (3 days) to be the most susceptible to BAI strain.A. Resistance increased progressively with age. Chickens up to 15 days of age proved highly susceptible to the tumor transplant, with mortality associated.with the primary neoplasm ranging from 67% to 97%. With chickens up to 60 days of age the inci- dence of tumors decreased. This is best illustrated in passages 12a and 12b, since the same inoculum was injected into chickens of two different ages. In the 11-day old group, 92% deve10ped the primary tumor. In the 60-day old group, only 17% had primary tumors. In spite of the apparent increased resistance with age, a high response was obtained in passage 11 where 60% of the chickens developed a.pri- mary tumor after inoculation at 29 days of age. Comparison of the time range with the median time for mortality in sash passage 12 indicated an atypical rate of development by some individuals of a passage group. This may be an expression of the susceptibilities of individuals to the tumor transplant,and an indication of lack of homo- geneity in this highly inbred population. The median time of mortality ‘would seem.to be preferable to the mean as an indication of response because of the relatively wide range. The median time ranged from a high of #1.5 days in passage 10 to a low of 20.5 days in passage 12a. In most passages the median time of death occurred in 2 to h weeks after a,palpable tumor developed in 50% of the chickens, but in pas- sage 12a the interval was only 8.5 days. Pathology. During the early stages of development, the tumor contained a glistening, gray-white, friable parenchyma enclosed in a firmly adherent thinawalled capsule. As the tumor increased in size, additional islands of neOplastic tissue became evident and were pal- pable. The cut surface of an entire neoplasm.revealed a mass divided into many compartments by septa continuous with the peripheral cap- sule. Centrally, a large tumor often contained extensive areas of hemorrhage and necrosis (Fig. 1). Histopathological study revealed these tumors to contain a wide range of morphological patterns. Variations were observed among tumors as well as among different areas within the same tumor. The least differentiated tumors were composed of anaplastic, generally ovoid, vesicular cells containing finely clumped chromatin and having an indistinct cytoplasmic membrane. Growth ‘was both expansive and invasive with the cells forming a sheet and showing little tendency for organization (Fig. 2). Lymphocytes were fairly prominent in the early lesion. They were seen both as single cells 13 throughout the lesion and as small foci located generally at the peri- phery of the tumor. ThiS'was apparently one manifestation of the host's defense mechanism.against the invasion of the cells contained in the inoculum. Tumors showing slightly greater organization were composed of masses of cells arranged in nests separated 'by varying amounts of connective tissue stroma (Fig. 3 and h). 'Within cell nests, organoid changes were common. Canalization occurred and tubular structures were formed which were thick-walled initially, being composed of many layers of cells. From these emerged tubules of more definite struc- ture consistingcnftightly packed columnar cells and resembling pseudo- stratified columnar epithelium. Those showing greater maturation were composed of a single layer of columnar cells (Fig. 5). Iniflally, the connective tissue stroma 'was hof no greater interest than the limiting membrane of any encapsulated neOplasm. With continued Observation it became apparent that the stroma exhibit- ed considerable variation in'both the amount and degree of maturation. In some tumors this tissue had the appearance of a malignant sarcoma composed of fibroblasts showing little evidence of fibril formation. At the other extreme the stroma consistedcn?well maturated connective tismxawhich was quiescent in appearance with fibrils much in evidence. In a few cases, microscopic areas of cartilage was seen in addition'U> irregular shaped spicules of partially ossified ‘bone (Fig. 6). The tubules were thin walled and mature. The general lack of rapid activ- ity in this lesion would suggest that the cartilage and bone 'were of metaplastic origin. Tumor regression. Only four cases of regression were Observed, in one each in a chicken Addom a nOHSQdao pd 0mm owdummm \ endow op mafia smegma m a m m H m o o a m o m o a o v a o s H maoflmnmmmsu HHoo maopmdanonnmoa gawk dowdadoonw muomofizo ma ufimdamooc hhddaooom Roam hpfiadphoa mo ofiwp dad ooamBAQQH .N maflflfi am . .oapmaflmbd no: 898 d u: \ .doHHwMI M .deod Hindsd u d * macs ma neon AH anon 0H -- + + mma-wma o ems «mama a u- + + em-mm 6 am osmmm .. + + mm-wm e mm mmcm m 963 ...L nosed - + - mm-aw o mm maama w .Hnoammoa Anton ambwa + + + HmHuMHH A NNH m.NMB sosaa + + + om-ms a 0m samma ambwa + + + :MIQN A :m mema m .Hpoanmom asaopom- nu, + + mlnm: d m: ::NMB .Hnoaagoa Hdhopoom noefla + + h SM n w hm mxmoa .Hpoanmos Haaopoom Sonata + n u an A am ammoa : nonadfi snobfla + u: + u: 6 Ha Mmma .HQonmoa adaopoom aoanua.+ sob-mfi n u n In A mm mxmmg m nosed + -- m -- a em omsm: u: nu + I: 6 mm amnma x -- x -- + -- a mm maama m sounds-nosed + + a, x -- r em ooamm H amddmoms maofipoom groan announce doawdadd *npmod nomads Hogans pampflaoonoo odmmwa cooam mmoao mfiadeoq mo mafia pawn owdmmmm m p o n P o E o H p a o a m d a n downsadooswpmom awmn mnOAmGMMWSm . Hoo aeopmmanonnmos ape: dopmadooaw maoMowmu ma hwoaogpdo. decode dad so-sdadu dwfiousoq .m manna wM\wm mM\sm wm\m o mm\a o o Aoouoas uoosflaosv adoapsoo ~m\ms AM\m PM\m a.ma am\e FM\s m.sw sm\sm 0 RNA os\mm o oa\: a.sa os\a oa\ad a.mw os\mm oa\m m os\as o:\a os\m m.» oa\m oa\m 0.0m OR\NH os\om m .on .on .oa a .os .oa R .0s .0s obdmmnm Hmpoa whopabndm oflmfioommaoz mfimoapomoopmo .omfi> museummapoanmmz wwwopmmanoascnmo some 0 a m w a a m m n mmaopmmflponamo: dopadammsdhp mo momdmmdm dowooaom mo menopause mafiadpaofi voosdaw opmaaflwh .: manna Time of nor d- :7 it Wh— D a y s U 4. o s t i n o c u 1 a t i o n Tcs+ Granuloblastosis Nephroblastomas Visc. lympho. passage Radian Range Hedian Range Median ngc 2 32 2h-108 117 75-192 178 111-190 8 67.5 59-75 127 6h-210 151 121-210 12a 13h.5 65-210 160 126-203 37 U 12629 1 ~. Fig. 1. Nephroblastora-pectoral; cell transplant. Sagital section shows lobulated tumor masses enCased in thin remnant of pectoral muscle. Necrotic tissue prevalent in lever portion. 1 1 km Fig. 2. Nephroblastoma-pector'.; cell transplant. Anaplastic cells 3 in an unorganize. manner a on: remnants of isolated muscle fibers (arrows) ZthSS approxJ7. fi - , .: I? and. .- Fig. 3. Egghroblastoma-pectoral; cell transplant. Small tumor with minimal connective tissue sorta and slight evidence of cell nest formation. Peripheral lymphocytic foci (arrows) Z X85 approx 7. Fig. A. Nephroblastoma-pectoral; cell transplant.O?ll nests separatni by abundant sarcomatous stroma; note formation of prim tive thick- walled tutu es ZleTS aporox;7. defined cll V 9 i I. O t splan ral; cell tran O J wrest L Nephroblastoma- stroma interstitial scant basement membranes and Z_X175 approxL7. tubules with (‘f‘o tic E 1% .E‘ 14;. f”. '18 rausulant. Cell f toral; . 1 .. Ieohroblastoma-ne I f’ O Fis- tubules i n-x-nlled tr area O? by ‘ rota tordered .1. u ial s: interstit 1n F L. O ' n _7. X85 appro> .; ‘V 3 ,. \g. i, ‘ I TH ‘1' 7.1 Fig. 7. Nephroblastoma-renal; filtrate induced. Large lobulated encapsulated tumor attached to remnant of left kidne'; smaller neoylasm on_posterior aspect. Nephroblastoma-renal; filtrate induced. Primitive cells arranged in nests separated by sarcomatous stroma. Within nests the fornsoion of tortuous tubules is seen Z§22O approx7. Fig. 9. Nephroblastoma-renal; filtrate induced. Tumor composed of well differentiated tubules which remarkably simulate renal tissue, including glomerulus-like structure (arrow) [£175 approx7. Fig. 10. Nephroblastoma-renal; filtrate induced. Sarcomatous stroma composed of relatively mature fibrous connective tissue) with small area of cartilage (arrow). prules are thin-walled, well differentia- ted and in some cases show :ajillary structure [7175 anprof7. willw.wwawwv 3 1293 020