Acute myeloblastosis and several forms of tumor, including one case of lymphosarcoma occurred when avian myeloblastosis virus (BAI-A strain) was inoculated into newly hatched chicks (SPF). The homogenate of lymphosarcoma inoculated intraperitoneally into other newly hatched chicks induced a high incidence of erythroblastic leucosis. Electron microscopy did not reveal the presence of C-type virus particles in the tumor tissue. The relationship between avian myeloblastosis virus, lymphosarcoma and erythroblastic leucosis is discussed.</p

Hatase, Osamu

Ishii, Hideo

Kanzaki, Yoshito

Oda, Takuzo

Okayama University Digital Information Repository

Avian myeloblastosis virus-induced lymphosarcoma producing erythroblastic leucosis in chicks

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 29, Issue 5 1975 Article 7OCTOBER 1975Avian myeloblastosis virus-inducedlymphosarcoma producing erythroblasticleucosis in chicksYoshito Kanzaki∗ Osamu Hatase†Hideo Ishii‡ Takuzo Oda∗∗∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Avian myeloblastosis virus-inducedlymphosarcoma producing erythroblasticleucosis in chicks∗Yoshito Kanzaki, Osamu Hatase, Hideo Ishii, and Takuzo OdaAbstractAcute myeloblastosis and several forms of tumor, including one case of lymphosarcoma oc-curred when avian myeloblastosis virus (BAI-A strain) was inoculated into newly hatched chicks(SPF). The homogenate of lymphosarcoma inoculated intraperitoneally into other newly hatchedchicks induced a high incidence of erythroblastic leucosis. Electron microscopy did not reveal thepresence of C-type virus particles in the tumor tissue. The relationship between avian myeloblas-tosis virus, lymphosarcoma and erythroblastic leucosis is discussed.∗PMID: 179285 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 29,391-396 (1975)AVIAN MYELOBLASTOSIS VIRUS·INDUCED LYMPHO.SARCOMA PRODUCING ERYTHROBLASTICLEUCOSIS IN CHICKSYoshito KANZAKI, Osamu HATASE, Hideo ISHIIand Takuzo ODADepartment oj Biochemistry, Cancer Institute, Okayama UniversityMedical School, Okayama 700, JapanReceivedJor publication, May 16, 1975Abstract: Acute myeloblastosis and several forms of tumor, in-cluding one case of lymphosarcoma occurred when avian myelo-blastosis virus (BAI.A strain) was inoculated into newly hatchedchicks (SPF). The homogenate of lymphosarcoma inoculated intra.peritoneally into other newly hatched chicks induced a high incidenceof erythroblastic leucosis. Electron microscopy did not reveal thepresence of C-type virus particles in the tumor tissue. The relation.ship between avian myeloblastosis virus, lymphosarcoma and erythro.blastic leucosis is discussed.The BAI-A strain of avian myeloblastosis virus (AMV) induces not onlyacute myeloblastosis but also viscera11ymphomatosis, nephrob1astoma, andosteopetrosis (1). At present, little is known about the oncogenic mechanism.According to Burmester et ai. (2) inoculation with a high concentration of thisvirus induces erythroblastosis and inoculation with a lower concentration in-duces lymphomatosis. It is uncertain whether the manifested form of leucosisis dependent upon virus concentration or upon contaminated clonal virus speci·fic for the disease (1,2). To examine the factors determining the form ofleucosis, we used AMV (BAI-A strain) and induced various forms of leucosisin chicks (SPF). One case of lymphosarcoma was found in this sample. Thislymphosarcoma differed from other reported leucoses because electron micro-scopy did not reveal the presence of C-type viral particles in the tumor tissueand the homogenate of the tumor induced acute erythrob1astic leucosis athigh incidence. In the present study, we report on this newly found tumorand discuss the mechanism of avian tumor induction by AMV.MATERIALS AND METHODSVirus inoculation: The BAI-A strain of AMY was obtained through thecourtesy of Dr. T. Shimizu, National Institute of Animal Health, Japan Agri-Abbreviations: SPF, specific pathogen free; AMY, avian myeloblastosis virus; PBS (-),phosphate buffer saline, divalent cation free.3911Kanzaki et al.: Avian myeloblastosis virus-induced lymphosarcoma producingProduced by The Berkeley Electronic Press, 1975392 Y. KANZAKI, O. HATASE, H. ISHII and T. QDAcultural Ministry at Tokyo, as frozen dried blood plasma from myeloblastosischicks. This sample was resolved aseptically in minimum essential medium(MEM) and was inoculated intraperitoneally into chicks within 24 hours afterhatching. Chicks attacked by acute myeloblastosis were exanquinated by heartpuncture, and the blood plasma collected. This plasma was diluted with PBS(-), and the diluted plasma was injected intraperitoneally into the next seriesof chicks within 24 hours after hatching.Chicks: White leghorn (SPF) chicks were kindly supplied by Dr. T. Kan,Research Institute of Microbiology, Osaka University, Kannonzi. They werefree of leucosis virus in immunological investigations at the same institute (3).Leucosis or Marek's disease did not occur during the 150-day observation periodin the 30 chicks of the control group.Diagnosis of various forms of the disease: Onset of myeloblastosis waschecked by hematocrit examination, and the peripheral blood picture wasinvestigated microscopically after Giemsa staining. Tumors were examined atautopsy. Lymphosarcoma was diagnosed after staining by hematoxylin-eosinand silver impregnation, and observed by electron microscopy.Maintenance and observation of chicks: The inoculated groups and the con-trol group of chicks were kept in separate cages in separate rooms. All cageswere sterilized before use. Food and water were separated between the twogroups and never interchanged. The manifestation of leucosis was examinedon blood smears and by hematocrit readings semi-weekly from 3 days afterinoculation in all birds. ATPase activity of myeloblastic chick plasma wasmeasured by the method of Beaudreau and Becker (4). It was reported thatAMV containing plasma showed high ATPase activity (5-7). Postmortem ex-amination was performed on all birds. Various forms of leucosis occurred inthe experimental groups. A .leg tumor was found at 70 days after inoculation(Figs. I and 2). Postmortem examination was performed on this tumor-bearingchick. The tumor, liver, kidney, and bone marrow were fixed with 10% for-malin and stained with hematoxylin-eosin and silver impregnation method formicroscopic examination, as described previously. Electron microscopic ex-amination was performed on the tumor. The tumor was stored in a deepfreezer at -80°C, thawed at room temperature, and homogenized vigorously upand down ten times in PBS (-) with a teflon homogenizer. The homogenatewas injected intraperitoneally into chicks, and the chicks were observed for90 days and diagnosed, as described previously.Electron microscopy: The HU.llC electron microscope was used for obser-vations of virus partices in myeloblastic chick bone marrow, lymphosarcomaand other tumors. The samples were fixed in 2.5% glutaraldehyde and 1%osmium tetroxide in O.lM cacodylate buffer, pH 7.2 and dehydrated in serieswith ethyl alcohol and embedded in Epon 812. Ultrathin sections were stainedwith uranyl acetate and lead citrate.2Acta Medica Okayama, Vol. 29 [1975], Iss. 5, Art. 7http://escholarship.lib.okayama-u.ac.jp/amo/vol29/iss5/7Lymphosarcoma by Avian Myeloblastosis VirusRESULTS393In vivo infectivity of AMVThe intraperitoneal injection of AMV in newly hatched chicks inducedacute myeloblastosis, nephroblastoma, ovarian tumor and lymphosarcoma.Injection of the myeloblastosis chick plasma induced myeloblastosis andnephroblastoma in the next experimental series (Table I).TABLE 1. INDUCTION OF VARIOUS FORMS OF NEOPLASMA BY BAI·A STRAIN OF AMVSeries Chicks Myeloblastosis Nephroblastoma Other tumor(No.)1st 11 4 3 22nd 15 4 3 03rd 23 3 2 0Myeloblastosis chick plasma was diluted with PBS (-), and a 0.5 ml aliquot was injected tothe next series of chicks (150 days observation).Leg tumor (lymphosarcoma)Microscopic examination after hematoxylin-eosin staining and silver im-pregnation method confirmed that the tumor was lymphosarcoma (Fig. 3).Giemsa-stained peripheral blood smears of the tumor-bearing chick showeda normal pattern, and the ATPase activity of the blood plasma was .negli-gible. The liver, kidney, and bone marrow appeared normal both macro- andmicroscopically (Fig. 2). Electron microscopic examination of the IY.J;Ilpho-sarcoma showed degenerative changes in the cytoplasm. Endoplasmic reti-culum and mitochondria were not distinctly observable. C-type partieles ofavian leucosis virus were not detected in the intra-cisternal and intercellularspaces (Fig. 4).MyeloblastosisThis disease occurred between the second to fifth week after virus inocu-lation. The peripheral blood cells were predominantly immature white cellsand mainly myeloblasts. Hematocrit values of the peripheral blood ofattacked chicks were about 50% of the control values. A number of C-typevirus particles of approximately 100 nm in diameter were observed in the bonemarrow by electron microscopy (Fig. 5). Some cases were complicated withnephroblastoma. The ATPase activity of myeloblastosis plasma was higherthan that of normal plasma.Manifestation of newly induced erythroblastic leucosisThe homogenate of the lymphosarcoma induced erythroblastic leucosisat high incidence. The onset was 2-4 weeks after the inoculation of thehomogenate, and the peripheral blood cells were mainly erythroblastic cells(Fig. 6). The hematocrit values of chicks with erythroblastic leucosis were3Kanzaki et al.: Avian myeloblastosis virus-induced lymphosarcoma producingProduced by The Berkeley Electronic Press, 1975394 Y. KANZAKI, Q. HATASE, H. ISHII and T. QDAFig. 1. Gross appearance of AMV induced lymphosarcoma in the chick.Fig. 2. Gross appearance at autopsy of the lymphosarcoma-bearing chick. Part of thetumor was enclosed with hematoma and retroperitoneal organs were observed to be normal.Fig. 3. Microphotograph of the lymphosarcoma stained by the silver impregnationmethod (x 100) .Fig. 4. Electron micrograph of the lymphosarcoma cells. C-type virus particles werenot observed in tumor cells (x 9,000).Fig. 5. Electron micrograph of a myeloblastosis chick bone marrow cell. C-type virusparticles are observed in the cytoplasm (x 20,000).Fig. 6. Peripheral blood smear of chick erythroblastic leucosis induced by the lympho-sarcoma homogenate. Erythroblastic cells are round in shape. The cytoplasm is polychro-matic and the nuclei have a cartwheel-like appearance (x 4(0).TABLE 2. INDUCTION OF ERYTHROBLASTIC LEUCOSIS BY THELYMPHOSARCOMA HOMOGENATESeries1st2ndChicksINo.)1189Erythroblastic leucosis(No.)4334Acta Medica Okayama, Vol. 29 [1975], Iss. 5, Art. 7http://escholarship.lib.okayama-u.ac.jp/amo/vol29/iss5/7Lymphosarcoma by Avian Myeloblastosis Virus 395about 50% of the normal values. Table 2 showed the frequency of thisdisease after homogenate inoculation. The ATPase activity in the leucosisplasma was negligible. These results were consistent with the results of Bonaret al. (8). During the three month observation period, no other types ofleucoses were found in the tested chicks.DISCUSSIONIt was reported (11) that R str~in, BAI-A strain, and RPL strain of avianleucosis virus induced erythroblastic leucosis, myeloblastosis, and viscerallymphomatosis, respectively. However, Baluda et al. (l, 9, 10) reported thatclonal virus form BAI-A strain of AMV induced various forms of tumorsin chicks and that mesenchymal cells were target cells for this virus.On the other hand, Burmester's group (2) speculated that the induction ofdifferent types of tumor was dependent on the viral concentration used forinoculation. It is still unknown whether the virus strain is multipotent intumor induction or whether various types of virus are mixed in the BAI-Astrain. In the present study a solitary lymphosarcoma was induced in a chick(SPF) by BAI-A strain of AMV. Although C-type viruses were not detectedin the lymphosarcoma by electron microscopy, the homogenate of the tumorinduced erythroblastic leucosis in chicks. These results may suggest thatBAI-A strain of AMV was either contaminated by another strain of virus orinduced lymphosarcoma under usual' conditions. These results also suggestthat a small amount of virus (not observed by electron microscopy) waspresent or that oncogenic factors weore produced in the lymphosarcoma cellby recombination or phenotypic mixing. The oncogenic factors may haveinduced erythroblastic leucosis. The results of the present study show that aclonal virus may be altered by passage through a living system. Further in-vestigation is in progress on the relationship between AMV and this sarcoma.Acknowledgment: The authors are indebted to Dr. Masaki Ohmori, Department of Pathol-ogy, 2nd Division, Okayama University Medical School, for his diagnosis of lympho-sarcoma. This investigation was supported in part by a Grant-in-Aid for group study oftumor virus from the Japan Ministry of Education, Science and Culture.REFERENCES1. Baluda, M. A. and Jamicsen, P. P.: In vivIJ infectivity studies with avian myeloblastosisvirus. Virology 1'!, 33, 1961.2. Burmester, B. R., Walter, W. G., Gross, M. A. and Fontes, A. K.: The oncogenic spec-trum of two pure strains of avian leukosis. ]. Natl. Cancer Inst. 23, 277, 1959.3. Kan, T.: Personal communication.4. Beaudreau, G. S. and Becker, C.: Virus of avian myeloblastosis: X. Photometric micro-determination of adenosine triphosphatase activity. ]. Natl. Cancer Inst. 20, 339, 1958.5Kanzaki et al.: Avian myeloblastosis virus-induced lymphosarcoma producingProduced by The Berkeley Electronic Press, 1975396 Y. KANZAKI, O. HATASE, H. IsHU and T. ODA5. Sharp, D. G., Mommaerts, E. B., Eckert, E. A., Beard, D. and Beard, J. W.: Virus ofavian erythromyeloblastic leukosis. II. Electrophoresis and sedimentation of the plasmaparticles and the enzyme dephosphorylating adenosine triphosphate. ]. Natl. Cancer Ins/.14:, 1027, 1954.6. Mommaerts, E. B., Sharp, D. G., Eckert, E. A., Beard, D. and Beard, J. W.: Virus ofavian erythromyeloblastic leukosis. I. Relation of specific plasma particles to the de-phosphorylation of adenosine triphosphate. ]. Natl. Cancer Inst. 14, lOll, 1954.7. Green,!. and Beard, J. W.: Virus of avian erythromyeloblastic leukosis. VI. Propertiesof the enzyme associated with the virus in dephosphorylating adenosine triphosphate. ].Natl. Cancer Ins/. 15, 1217, 1955.8. Bonar, R. A., Beaudreau, G. S., Sharp, D. G., Beard, D. and Beard, J. W.: Virus of avianerythroblastosis. V. Adenosinetriphosphatase activity of blood plasma from chickenswith the disease. ]. Natl. Cancer Inst. 19,909, 1957.9. Baluda, M. A. and Goetz,!. E.: Morphological conversion of cell cultures by avianmyeloblastosis virus. Virology 15, 185, 1961.10. Baluda, M. A., Moscovici, C. and Goetz,!. E.: Specificity of the in vitro inductive effectof avian myeloblastosis virus. Natl. Cancer Inst. Monogr. 17, 449, 1964.11. Shimizu, T.: Leukosis viruses. In New Virology I, ed. N. Higashi and N. Ishida, AsakuraBook Co., Ltd., Tokyo, p.439, 1972.6Acta Medica Okayama, Vol. 29 [1975], Iss. 5, Art. 7http://escholarship.lib.okayama-u.ac.jp/amo/vol29/iss5/7

Avian myeloblastosis virus-induced lymphosarcoma producing erythroblastic leucosis in chicks

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