EXPERTMENTAL CANINE. MALIGNANT LYMPHOMA: TRANSMTSSTON STUDTES AND ISOLATION SE A CANTNE HERPESVIRUS Thesis for the Degree of Phc. D. MiCHiGAN STATE UNIVERSITY THOMAS JOHN. KAKUK 1968 mm IIIWWWW WWWWWWWWWI 3 1293 01001 6750 This is to certify that the thesis entitled Experimental Canine Malignant Lymphoma: Transmission Studies and Isolation of a Canine Herpesvirus presented by Thomas John Kakuk has been accepted towards fulfillment of the requirements for M;— degree in M (yd/2&1?me Major professor Date MM— 0-169 mu m av HDAB & SON? 800K NNDERVINC. ‘ LIBRARY BlNDEHS ‘ ”HIM”. menu; ABSTRACT EXPERIMENTAL CANINE MALIGNANT LYMPHOMA: TRANSMISSION STUDIES AND ISOLATION OF A CANINE HERPESVIRUS by Thomas John Kakuk Canine malignant lymphoma (CML) was transmissible to the Beagle neonate in 2 serial passages, and a canine herpesvirus, designated as canine herpesvirus-Kakuk (CHV-K), that was pathogenic for the germfree Beagle neonate was isolated from a dog with CML. The clinical, hemato- logic, macrosc0pic, and microscopic findings of experimentally induced CML and a septicemia in Beagle neonates produced by CHV-K were described and discussed. Two serial passages of CML were accomplished with suspensions of viable CML whole cells in 2 litters of Beagle neonates. Definite evi- dence of malignant lymphoma developed in 3 of 11 dogs inoculated with a single dose of a cell suspension by 53, 54, and 78 days postinoculation, respectively. Hematologic results indicated that leukemia was present in 1 dog and subleukemia was present in 2 dogs between 41 and 52 days. Two dogs were anemic, whereas all 3 dogs had thrombocytopenia. Two dogs with overwhelming CML were preirradiated with x rays, whereas 1 dog was not so irradiated. Successful transmission of CML without pre- treatment of total body irradiation was considered noteworthy. Clinical and pathologic findings were similar to those reported for naturally occurring malignant lymphoma of dogs and cats. Organs and tissues Thomas John Kakuk having neoplastic involvement included: lymph nodes, thymus, liver, lung, kidney, spleen, bone marrow, gastrointestinal tract, and muscle. Malig- nant lymphoma did not occur in the third serial passage, although the inoculated animals had enlargement of the superficial lymph nodes. Biopsies of these enlarged lymph nodes revealed lymphocytic hyperplasia. The CHV-K was isolated from 18 germfree Beagle neonates inoculated with either cell suspensions prepared from a dog with CML or with extracts prepared from puppies with the septicemic disease. Two contact control puppies died from similar septicemic conditions, whereas the 3 non- contact control puppies remained healthy. The production of a fatal septicemic disease in Beagle neonates inoculated with material prepared from a dog with CML and the isolation of the virus from the kidney of this dog indicated that the virus came from the donor dog. A.fatal septicemic disease occurred in colostrum-deprived, germ- free Beagle neonates 6 to 16 days after inoculation with the virus. The fundamental histOpathologic lesion was necrosis in the liver, lung, kidney, heart, skeletal muscle, pancreas, and adrenal gland. A few intranuclear basOphilic and/or acidophilic inclusion bodies were seen in cells adjacent to the areas of necrosis. Splenomegaly and generalized lymphadenOpathy were commonly seen which microscopically consisted of marked lymphocytic and reticular cell hyperplasia. The marked hyper- plasia produced by this virus resembled neoplasia, although obvious neoplasms were not induced. Results of cross-serum neutralization tests indicated that the virus was closely related, or possibly identical to, the canine herpesviruses isolated from young puppies (Carmichael's strain:F205V and Stewart's Thomas John Kakuk strain:SLl8HLV). However, this was the first report in which a CHV was isolated from a dog with malignant lymphoma, and also the first report concerning the isolation of a CHV from an adult dog which was pathogenic for puppies. Results indicated that age at time of exposure is important in re- producing the disease in puppies. Newborn, germfree Beagles inoculated prior to 8 days of age were highly susceptible and nearly all died. One of 18 recipients has survived the infection. If puppies were not inoculated until after 8 days, there were clinical manifestations of the disease, but few died. Factors of importance in establishing a diagnosis included: (1) age of the puppies; (2) clinical signs; (3) marked thrombocytoPenia; and (4) pathologic findings. Pathologic changes in affected puppies were characteristic. Widespread hemorrhages were apparently related to the marked thrombocytOpenia consistently observed in herpes-infected pup- pies. Necrotic and hemorrhagic lesions in the liver, lung, and kidney of dead puppies suggested this viral infection. Focal renal hemorrhages have not been reported in dogs infected with infectious canine hepatitis or distemper viruses. Occasional intranuclear inclusions were seen in recently infected cells adjacent to areas of necrosis. The virus caused characteristic CPE in 12 to 16 hours when grown in dog kidney cell or thymic cell tissue cultures. EXPERIMENTAL CANINE MALIGNANT LYMPHOMA: TRANSMISSION STUDIES AND ISOLATION OF A CANINE HERPESVIRUS By Thomas John Kakuk A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1968 .»- / f‘ (gigs/4m? Dedicated to my wife Martha ii ACKNOWLEDGEMENTS The author wishes to express sincere appreciation to his major professor, Dr. R. F. Langham, in the guidance of this study, for teach- ing him cellular pathology and in reviewing this manuscript. My sincere thanks and appreciation is expressed to Dr. G. H. Conner, Leukemia Research Project Director, Department of Veterinary Surgery and Medicine, for his generous assistance with various aspects of this study, and for supplying the germfree Beagle neonates neces- sary to carry out this investigation. The gratitude of the author is expressed to the following guidance committee members: to Dr. C. C. Morrill, Chairman of the Department of Pathology, for advice and for reading the manuscript; to Dr. S. D. Sleight, for helpful suggestions in preparing and reviewing this manu- script; to Dr. R. W. Hinz, for preparing and giving advice concerning electronmicrosCOpy and reviewing the manuscript; and to Dr. R. W. Van Pelt, Jr., for reviewing this manuscript. Special appreciation is extended to Dr. J. R. Mitchell, Michigan Department of Public Health, for assistance in identifying the canine herpesvirus, and to Mr. C. A. Bowles for growing the canine herpesvirus in tissue culture. To Dr. J. A. Meore, the author extends sincere appreciation and thanks for advice and assistance concerning the germfree Beagle neonates. The author gratefully acknowledges the technical assistance of Mrs. Janet Sharon, Miss Simone Paillet, and Miss Judi Lee. iii For awarding a special Postdoctoral Fellowship, the author extends his appreciation and thanks to the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. iv TABLE OF CONTENTS INTRODUCTION. . . . . . . . . . REVIEW OF THE LITERATURE. . . . Spontaneous Canine Malignant Lymphoma History. . . . . . Definition . . . . Classification . . Clinical Findings. Incidence . Age . . . . Sex . . . . Breed . . . Duration. . Signs . . . Hematologic Findings Pathologic Findings. Gross . . . MicroscoPic . Diagnosis . Transplantable and Transmissible Canine Venereal Sarcoma . Oral Papilloma . . Anaplastic Thyroid Carcinoma Mast Cell Leukemia . V O Neoplasms Page 11 ll 12 l4 l4 l4 Malignant Lymphoma . Herpesvirus Infection in the Dog. History. . . . . . . Natural Disease. Distribution . . . Etiology and Properties of the Clinical Signs . Lesions. . . . . Species Susceptibility . Immunity . . . . . . MATERIALS AND METHODS . Virus Donor Dogs of Canine Malignant Lymphoma . . Experimental Animals. . . . Experiment I . Experiments II, III, Rearing of Germfree Puppies Transmission Technic. . Tissue Culture Procedure. . Fecal Examination . . . . . Hematology. . . . . . . . . Pathology . . . . .-. . Gross Procedures . . IV, and V . Microscopic Procedures . . . . Ultrastructure. . . . Electronmicroscopy . vi Page 15 l6 l6 l6 17 17 18 l9 19 20 21 21 22 22 22 29 30 30 31 31 32- 32 32 32 32 Page EXPERIMENTAL TRANSMISSION OF CANINE MALIGNANT LYMPHOMA TO THE BEAGLE NEONATE: EXPERIMENT I RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Clinical Signs. . . . . . . . . . . . . . . . . . . . . . . 33 Hematology. . . . . . . . . . . . . . . . . . . . . . . . . 34 Pathology . . . . . . . . . . . . . . . . . . . . . . . . . 34 Cross. . . . . . . . . . . . . . . . . . . . . . . . 34 Microscopic. . . . . . . . . . . . . . . . . . . . . 41 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 A CANINE HERPESVIRUS ISOLATED FROM A DOG WITH CANINE MALIGNANT LYMPHOMA PATHOLOGIC FOR THE BEAGLE NEONATE: EXPERIMENTS II, III, IV, V RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Tissue Culture Findings . . . . . . . . . . . . . . . . . . 51 Herpes-infected Puppies. . . . . . . . . . . . . . . 51 Donor Dog 10074. . . . . . . . . . . . . . . . . . . 52 Cross-Serum Neutralization Tests. . . . . . . . . . . . . . 52 Clinical Signs. . . . . . . . . . . . . . . . . . . . . . . 52 Microbiologic Examination . . . . . . . . . . . . . . . . . 55 Hematology. . . . . . . . . . . . . . . . . . . . . . . . . 55 Experiments II, III, IV. . . . . . . . . . . . . . . 55 Experiment V . . . . . . . . . . . . . . . . . . . . 57 Pathology . . . . . . . . . . . . . . . . . . . . . . . . . 57 Cross. . . . . . . . . . . . . . . . . . . . . . . . 57 Microsc0pic. . . . . . . . . . . . . . . . . . . . . 63 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Experiment I. . . . . . . . . . .'. . . . . . . . . . . . . 95 Experiments II, III, IV, V. . . . . . . . . . . . . . . . . 96 vii Page REFERENCES 0 O I O O O O O O O O O O O 0 O 0 O O O O O O O O O O O 98 APPENDICES O O I O O O O O O O I I O O O O O I O O O O O O O O 0 O 104 VITA. O O O O O O O O O O O O O I O O O O O O O O O O O O O O I O 118 viii Table 10 11 LIST OF TABLES Canine ne0plasms known to be transplantable and transmissible in the dog. . . . . . . . . . . . . . . . Experiment I. Serial passage of canine malignant lymphoma (CML) to 3 litters of Beagle neonates. . . . . . . Experiment II. Colostrum-deprived, germfree Beagle neonates inoculated with a fresh cell suspension pre- pared from a dog with malignant lymphoma (ML) . . . . . . Experiment III. Colostrum-deprived, germfree Beagle neonates inoculated with a stored cell suspension prepared from a dog with malignant lymphoma (ML). . . . . Experiments IV and V. Colostrum-deprived germfree Beagle neonates inoculated with cellular and cell- free extracts prepared from canine herpesvirus infected dogs . . . . . . . . . . . . . . . . . . . . . . . Transmissibility of malignant lymphoma following serial passage of cell suspensions and cell-free extracts into Beagle neonates . . . . . . . . . . . Pre- and postinoculation sequential hemogram values taken from Appendices l and 2 on Dogs 30030 and 30032; representing the lat serial passage of induced malig- nant lymphoma . . . . . . . . . . . . . . . . . . . . . . Pre- and postinoculation sequential hemogram values taken from Appendices 3 and 4 on Dog 30089; repre- senting the 2nd serial passage of induced malignant lymphoma and Control Dog 30088. . . . . . . . . . . . . . . Organ and tissue distribution of lymphocytic infiltra- tion demonstrated by histologic examination . . . . . . . . Cross-serum neutralization tests between canine herpes- virus-Kakuk (CHV-K) and Carmichael's canine herpesvirus (FZOSV) O 0 O O O O O O O O O O O O O 0 O O O O O O O O I 0 Platelet counts taken from 11 herpes infected dogs and 3 non-contact control dogs from Appendices 5 and 6. . . . ix Page 13 24 26 27 28 35 36 37 42 54 56 Table 10 11 LIST OF TABLES Page Canine neoplasms known to be transplantable and transmissible in the dog. . . . . . . . . . . . . . . . . . l3 Experiment I. Serial passage of canine malignant lymphoma (CML) to 3 litters of Beagle neonates. . . . . . . 24 Experiment II. Colostrum-deprived, germfree Beagle neonates inoculated with a fresh cell suspension pre— pared from a dog with malignant lymphoma (ML) . . . . . . . 26 Experiment III. Colostrum-deprived, germfree Beagle neonates inoculated with a stored cell suspension prepared from a dog with malignant lymphoma (ML). . . . . . 27 Experiments IV and V. Colostrum-deprived germfree Beagle neonates inoculated with cellular and cell- free extracts prepared from canine herpesvirus infected dogs . . . . . . . . . . . . . . . . . . . . . . . 28 Transmissibility of malignant lymphoma following serial passage of cell suspensions and cell-free extracts into Beagle neonates . . . . . . . . . . . . . . . 35 Pre- and postinoculation sequential hemogram values taken from Appendices l and 2 on Dogs 30030 and 30032; representing the lst serial passage of induced malig- nant lymphoma . . . . . . . . . . . . . . . . . . . . . . . 36 Pre- and postinoculation sequential hemogram values taken from Appendices 3 and 4 on Dog 30089; repre— senting the 2nd serial passage of induced malignant lymphoma and Control Dog 30088. . . . . . . . . . . . . . . 37 Organ and tissue distribution of lymphocytic infiltra- tion demonstrated by histologic examination . . . . . . . . 42 Cross-serum neutralization tests between canine herpes- virus-Kakuk (CHV-K) and Carmichael's canine herpesvirus (FZOSV) O O O O O O 0 O O O O O O O O 0 I O O 0 O O 0 O I O 54 Platelet counts taken from 11 herpes infected dogs and 3 non-contact control dogs from Appendices 5 and 6. . . . . 56 ix Table 12 13 Page Pre- and postinoculation sequential platelet counts taken from Appendices 7 and 8, Experiment V . . . . . . . . 58 Percent incidence of histopathologic lesions in 18 herpesvirus infected dogs . . . . . . . . . . . . . . . . . 64 Figure LIST OF FIGURES Page Iliac lymph node from initial donor dog (10019), a 20-month-old Doberman Pinscher. Notice the monotonous uniformity of neoplastic lymphocytes. H & E stain. x 680 O O O O O O O O O O O O O O O O O I O O O O I O O O O O 23 Mesenteric lymph node from Dog 30089. Notice the lack of demarcation between cortex and medulla, and the homogeneous whitish color . . . . . . . . . . . . . . . . . 38 First passage of cellularlyinduced malignant lymphoma in Dog 30032. At birth the dog was pretreated with 60.8 r total body irradiation. It was then inoculated with a cell suspension prepared from Donor 10019 (Figure l). (A) Liver, notice the nodular areas which protrude above the surface and the enlarged hepatic lymph nodes. (B) Kidney and enlarged renal lymph node. (C) Markedly enlarged mesenteric lymph node. (D) Pancreas. . . . . . . . . . . .p. . . . . . . . . . . . . . 39 Dog 30089, representing the second serial passage of malignant lymphoma induced by inoculating a cell sus- pension prepared from 30032 (Figures 3 and 5). Subject was inoculated at 2 days of age without radiation pre- treatment and was killed when 53 days old. Notice the enlarged liver (A) and the thickening of the central veins and portal triads (arrow). (B) Kidney and renal lymph node. (C) Mesenteric lymph node. (D) Portion of thymus. (E) Portion of omentum . . . . . . . . . . . . . . 39 Dog 30032. Notice neoplastic infiltration into subcu- taneous tissues and intercostal muscles (A) and the markedly enlarged thymus (B). . . . . . . . . . . . . . . . 40 Mesenteric lymph node obtained from Dog 30089. Notice the invasion and proliferation of lymphocytic cells in the perinodal tissue. H & E stain. x 130. . . . . . . . 40 Higher magnification of Figure 4. Notice monotonous distribution of lymphocytic cells. H & E stain. x 680. . . 43 Thymus. Malignant lymphoma. Hassall's corpuscles (arrow) which was invaded by neoplastic lymphocytes. H 8 E stain. x 680. O O I O I O I O O O O O O O O I I O I O 43 xi Figure 10 ll 12 l3 14 15 l6 l7 l8 19 20 21 Page Liver from Dog 30089. Accumulation of neoplastic lymphocytic cells at portal triad. H & E stain. x 130 I I I I I I I I I I I I I I I I I I I I I I I I I I I 45 Liver from Dog 30032. Notice massive lymphocytic cell infiltration; only a few hepatic cells remain. H & E StainI x 68OI I I I I I I I I I I I (I I I I I I I I I I I I 45 Pancreas. Malignant lymphoma. Overwhelming replace- ment of pancreatic parenchyma by neoplastic lympho- cyteSI H & E stainI x 375I I I I I I I I I I I I I I I I I 46 Massive accumulation of neoplastic lymphocytes in interstitial tissue of renal cortex. H & E stain. x 680. . 46 Spleen. Malignant lymphoma resembling the reticulum cell type. Notice the degree of undifferentiation. H & E Stain. x 680I I I I I I I I I I I I I I I I I I I I I 47 Section taken from intercostal muscles of Dog 30089. Invading lymphocytic cells have proliferated and replaced many muscle fibers. H & E stain. x 130 . . . . . . . . . . 47 Donor Dog 10074. Notice hyalinization of the glomeru- 1us and invasion of renal cortex by neoplastic lymphoid ce118I H & E stainI x 680 I I I I I I I I I I I I I I I I I 53 Herpetic erythematous rash in inguinal region of a lI-dfly-Old puppy in00u1ated With CHV—Kc o o o o o o c o o o 53 Dog 30270, inoculated with a cell suspension prepared from Donor 10074. Notice the lungs with a frothy exu- date, enlarged mottled liver, splenomegaly, and petechi— ation of gastrointestinal tract. A canine herpesvirus was isolated from dog kidney tissue culture cells inocu- lated with extract prepared from affected tissue. . . . . . 59 Lung from Dog 30243. Notice the petechial, ecchymotic and suffusive hemorrhages and the frothy exudate. . . . . .I 59 Higher magnification of Figure 17. Notice the focal areas of necrosis in the liver and the hemorrhages along the gastrointestinal tract. . . . . . . . . . . . . . 51 Focal hemorrhages in kidney of Dog 30242, that was inocu- lated with a cell suspension prepared from Donor 10074. Hemorrhagic areas represent necrotic foci packed with erythrocytes. . . . . . . . . . . . . . . . . . . . . . . . 62 Sagittal section of kidney in Figure 20. Notice that the hemorrhages extend from the capsule to the cortico- medullary junction. . . . . . . . . . . . . . . . . . . . . 62 xii Figure 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Page Skin section from Dog 30243. Notice the hyperplasia of prickle and basal cell layers with hydropic degenera- tion and necrosis of the prickle cell layer. H & E stain. x 140. . . . . . . . . . . . . . . . . . . . . . . 65 Higher magnification of an area in Figure 22. Notice the marked hydropic degeneration and necrosis of the prickle cell layer. H & E stain. x 350 . . . . . . . . . 65 Fibroblastic proliferation resembling a "fibroma" in the corium produced by CHV-K. H & E stain. x 275 . . . . . . 66 Higher magnification of Figure 24. H & E stain. x 680. . 66 Lung section from puppy inoculated with CHV-K. Necrotic alveolar wall is dilated and contains an acidOphilic fibrillar material. H & E stain. x 140 . . . . . . . . . 68 Higher magnification of Figure 26. Notice the intra- nuclear inclusion body (arrow). H & E stain. x 720 . . . 68 Section of lung taken from a puppy inoculated with CHV-K. Notice the marked reticular cell proliferation which obliterated pulmonary architecture. H & E stain. x 300. . . . . . . . . . . . . . . . . . . . . . . 69 Higher magnification of Figure 28. H & E stain. x 660. . 69 Focal necrosis of myocardium. Puppy was inoculated with CHV-K, and died 6 days postinoculation. H & E stain. x 142 I I I I I I I I I I I I I I I I I I I I I I I I I I 70 Higher magnification of area in Figure 30. Notice the numerous Anitschkow myocytes (arrow) adjacent to necro- tic zone. H & E stain. x 540 . . . . . . . . . . . . . . 70 Marked phagocytosis in thymus of a canine herpesvirus infected puppy. Note "starry sky" pattern. H & E stain. x 600 I I I I I I I I I II I I I I I I I I I I I ~I I I I I 71 Focal herpetic necrosis in liver of CHV-K inoculated puppy. Note the vacuolar appearance of the surrounding hepatocytes and the obliteration of hepatic sinusoids. H & E stain. x 165. . . . . . . . . . . . . . . . . . . . 71 Higher magnification of Figure 33. Notice the oval to irregular shaped intranuclear inclusion bodies in hepato- cyteSI H 8 E stainI x 480I I I I I I I I I I I I I I I I 72 Higher magnification of Figure 34. H & E stain. x 800. . 72 Subcapsular hemorrhage, damage of glomerular tufts, and tubular necrosis in renal cortex of CHV-K inoculated puppy. H & E stain. x 142. . . . . . . . . . . . . . . . 74 xiii Figure Page 37 Cellular destruction and hyalinization of glomerular tuft taken from an infected puppy. Note tubular necro- 818I H & E stainI x 620I I I I I I I I I I I I I I I I I I 74 38 Glomerular tuft with less damage than Figure 37. H & E Stain. X 6200 c c o o o o o o o o o o o o o o o o o o o o o 75 39 Intranuclear inclusion body in parietal epithelial cell of glomerular tuft. H & E stain. x 736 . . . . . . . . . . 75 40 Notice focal necrotic area in duodenal crypt. Puppy was inoculated with CHV-K and died 6 days postinoculation. H & E stainI x 3OOI I I I I I I I I I I I I I I I I I I I I 76 41 Focal necrotic area in pancreas of a puppy that was killed 7 days postinoculation. H & E stain. x 480 . . . . . . . . 76 42 Higher magnification of Figure 41. Notice intranuclear inclusion body in pancreatic acinar cell. H & E » stainI x 1200 I I I I I I I I I I I I I I I I I I I I I I I 78 43 Section of spleen taken from a lZ-day-old puppy inocu- lated as a newborn with a lymphocytic tumor preparation from Donor 10074. Notice the undifferentiated cells and mitotic figures. H & E stain. x 680. . . . . . . . . . . . 73 44 Section of spleen taken from non—contact control Puppy 30277I H & E stainI x 68OI I I I I I I I I I I I I I I I I 79 45 Section of mesenteric lymph node taken from Puppy 30241, that was inoculated with a cell suspension prepared from Donor 10074. Notice the marked lymphocytic hyperplasia. and numerous mitotic figures. H & E stain. x 350 . . . . . 79 46 Higher magnification of Figure 45. H & E stain. x 580. . . 30 47 Section of mesenteric lymph node taken from a puppy inocu- lated with CHV-K. Notice lymphoid depletion, necrosis and intranuclear inclusion body (arrow). H & E stain. x 640 . . . . . . . . . . . . . .'. . . . . . . . . . . . . 80 48 Section of severe skeletal muscle necrosis taken from a puppy inoculated with CHV-K. H & E stain. x 140. . . . . . 81 49 Higher magnification of area ianigure 48. H & E stain. x 700 I I I I I I I I I I I I I I I I I I I I I I I I I I I 81 50 Section of muscle taken from a puppy inoculated with CHV-K. Notice edema and proliferation of sarcolemmal cells. H & E stain. x 140. . . . . . . . . . . . . . . . . 32 51 Highmfmagnification of area in Figure 50. Notice the loss of striations. H & E stain. x 700 . . . . . . . . . . 82 xiv Figure 52 53 54 Page Section of cerebrum taken from a puppy inoculated with CHV-K. Notice glial cell accumulation. H & E stain. x 300 I I I I I I I I I I I I I I I I I I I I I I I I I I . 84 Intranuclear immature herpes-like particles (arrow) in ultrathin section of lymph node. Glutaraldehyde osmic- acid fixation, uranyl-acetate lead-hydroxide stain. x 65,000. . . . . . . . . . . . . . . . . . . . . . . . . . 85 Mature, membrane associated particle (arrow) in cyto- plasm of ultrathin lymph node section. Glutaraldehyde osmic-acid fixation, uranyl-acetate lead-hydroxide stain. x 50,000 . . . . . . . . . . . a . . . . . . . . . . 87 Appendix 1 LIST OF APPENDICES Pre- and postinoculation sequential hemogram values for Dog 30030 with induced malignant- lymphoma representing the lst serial passage, Experiment I. . . . . . . . . . . . . . . . . Pre- and postinoculation sequential hemogram values for Dog 30032 with induced malignant lymphoma representing the lst serial passage, Experiment I. . . . . . . . . . . . . . . . Pre— and postinoculation sequential hemogram values for Dog 30089 with induced malignant lymphoma representing the 2nd serial passage, Experiment I. . . . . . . . . . . . . . . . . Pre- and postinoculation sequential hemogram values for Control Dog 30088, Experiment I. . Pre- and postinoculation sequential hemogram values from canine herpesvirus infected dogs, Experiments II, III, IV 0 o o o o c o o '0 o .' Sequential hemogram values based on results from Appendix 5: Experiments II, III, IV . . Pre- and postinoculation sequential hemogram values from canine herpesvirus infected dogs, Experiment VI I I ‘I I I I .I I I I I I I I I I Sequential hemogram values based on results from Appendix 7: 'EXperiment V. . . . . . . . Page 104 105 106 . 107 108 110 , 112 _ 115 INTRODUCTION Canine malignant lymphoma (CML) has attracted considerable attention because of its histopathologic similarity to Burkitt's lymphoma of man (Basherville g£_§l,, 1966; Bras gtflgl,, 1965; Lukes g£_§l,, 1966). Malig- nant lymphoma of the dog has been used by several investigators in transmission studies and in morphologic investigations at the ultrastruc- tural level in an attempt to determine the etiologic agent and to compare or contrast the findings with those in leukemia of man. Cellular transmission of lymphoma in domestic chickens and rodents has been accomplished with relative ease by inoculating cells into the same species. Many of the murine and avian strains of leukemia virus have been obtained from in yiyg serial whole cell transmission studies (Eddy, 1964). Cellular transfer of lymphoma in domestic animals has, however, proved difficult. Recently, Moldovanu g£_§1, (1966) reported cellular transfer of malignant lymphoma to x-irradiated mongrel puppies. The research described herein was undertaken in order to attempt to transmit CML to the Beagle neonate by inoculating cell suspensions and cell-free extracts prepared from dogs with spontaneous malignant lymphoma, and to attempt to isolate a causative agent(s). In the foregoing report CML, which was found to be transmissible to the Beagle neonate in 2 serial passages, and a canine herpesvirus, designated as canine herpesvirus-Kakuk (CHV-K), was isolated from a dog with CML that was pathogenic for the germfree Beagle neonate. The clinical, hematologic, macrosc0pic, and microsc0pic findings of 2 experimentally induced CML and a septicemia in Beagle neonates produced by CHV~K are described herein. REVIEW OF THE LITERATURE Spontaneous Canine Malignant.Lymphoma History. Siedamgrotzky, in 1871, first reported lymphatic leukemia in a dog in which the lymph nodes and spleen were enlarged, and the ratio of white to red blood cells was 1 to 15. Soon after this, a number of authors described cases of malignant lymphoma, principally of the lympha- tic type, in dogs (Bollinger, 1874; Cadiot, 1892; Stockmann, 1893; Olt, 1899). The literature on canine malignant lymphoma (CML) is voluminous and thus many references which are essentially case reports are omitted. Definition. This is a highly fatal, malignant neoplasm characterized by the uncontrolled proliferation of neoplastic cells of the lympho- reticular system in almost any organ with a corresponding variety in clinical signs (Moulton, 1961; Smith, 1963). Classification. The literature reports on malignant lymphoma in dogs were confusing because of the different names applied to the syndrome. The most common names used were malignant lymphoma, leukemia, lympho- sarcoma, and leukosis, although many others, such as pseudo-Hodgkin's disease, aleukemic-leukemia, adenosarcoma, and reticulum cell sarcoma have been employed. Smith (1963) suggested that the term."1eukemic ne0plasia" be used as an inclusive term to cover: (1) those neoplastic diseases characterized by a great increase in the white blood cells, which was leukemia, (2) the few examples in which the disease is 4 recognized because of the presence of immature, anaplastic leukocytes in the blood stream, even though the total number of white blood cells were within or close to normal limits (subleukemia), and (3) those forms, the majority as far as the canine species was concerned, in which the disease is recognized because of tumorous masses of lymphocytic or reticular cells somewhere in the animal's body. However, Jarrett g£_§1, (1966) favored the terms lymphosarcoma and/or leukemia. They thought lymphosarcoma was the most acceptable term because the basic pathologic process was a malignancy of lymphoid_tissue. Yet the prefix would in- clude the lymphocytic series but would not include by definition the reticulum cell or histiocytic cell sarcomas. Also, Jarrett.gtugl, (1966) thought leukemia was an excellent descriptive term because it gave an indication of the prognosis; however, the aleukemic form is the most common type observed in the dog (Bloom and Meyer, 1945; Jarrett‘gtngl., 1966; Meier, 1957; Smith, 1963; Squire, 1964), and therefore, the term leukemia would not be descriptive. In this study, the term malignant lymphoma was chosen because it best described the clinicopathologic picture in the dog. The term was. coined by Gall and Mallory in 1942, who classified 618 cases of malignant lymphoma in man. This classification has been applied to malignant lymphoma in the dog by Bloom and Meyer (1945) and by Squire (1965), who did extensive cytologic studies on the disease. With this classi- fication the neoplasms were divided into 4 main types: histiocytic, lymphocytic, plasmocytic, and Hodgkin's. Clinical Findings Incidence. According to Bloom and Meyer (1945), Meier (1957), and Moulton (1961) the incidence of malignant lymphoma in the dog population 5 varies between 0.1 and 0.32. Recently, Dorn‘eenel, (1966) did a population- at-risk survey in 2 counties in California over 2-1/2 years, and found that the average annual incidence was 0.024%. In contrast, Jarrett‘ee .el. (1966) reported a 1.3% incidence of CML in England, and Backgren (1965) reported an 0.013% incidence in Sweden. .éfié- The peak incidence of CML was found to occur between 5 and 9 years (Bloom and Meyer, 1945; Born SE e1., 1967; Jarrett ££.§lna 1966; Meier, 1957; Moulton, 1961; Van Pelt and Conner, 1968), with an over-all spread of 6 months to 15 years. The peak incidence was best shown by Priester (1967), who did an extensive statistical study involving 237 cases of CML in 3 regions of the United States: Midwest, South, and West Coast. The data clearly indicated that the peak incidence was between 4 and 9 years. See, Some reports indicated a higher incidence of disease in male dogs (Bloom and Meyer, 1945; Irfan, 1961; Jennings, 1952; Priester [West Coast and Southern U.S.], 1967; Smith, 1963), whereas other reports indicated no sex differences (Dornee eI_L_., 1967; Jarrett e911,, 1966; Meier, 1957; Moulton, 1961; Priester [Mfldwest], 1967). One report indi- cated a higher incidence of this disease in females (Van Pelt and Conner, 1968). Bgeee, Some investigators claimed that this neoplasm was more com- mon in Scottish Terriers than could be accounted for by the p0pulation prOportion of this breed (Bloom and Meyer, 1945; Mulligan, 1949; White, 1946). According to Moulton (1961), this claim was not confirmed in a large series of CML studied at the University of California. In Priester's 6 (1967) statistical studies on CML, he found that the relative risk was, in every instance, significantly higher for Boxers than for all other purebreds. He did, however, find a high risk of CML for English Pointers in the South but risks were considerably lower for other regions (7.5 compared to 1.1). Some reports indicated that the incidence of CML was higher in purebred than in crossbred dogs (DOID.EEHélss 1967; Priester, 1967). Priester claimed that the higher incidence could have been accounted for on a genetic basis and/or because purebred dogs were more commonly licensed and were probably given more medical attention. Duration. In most instances, it was difficult to determine the exact duration because this was based on case histories provided by the owner. However, based on such information most reports indicated a dura- tion ranging from 7 to 434 days, the mean being 99 days (Bloom and Meyer, 1945; Jarrett SE e1., 1966; Meier, 1957; Moulton, 1961). Jarrett EEHEL- (1966) and Schalm (1966) had dogs with the disease that lived from 15 to 18 months after diagnosis. §$ge_, Reports indicated that bilateral peripheral lymphadenopathy was the most common clinical sign (Bloom and Meyer, 1945; Meier, 1957; Moulton, 1961; Smith, 1963; Van Pelt and Conner, 1968). However, Jarrett EEHEL: (1966), in their study of 122 cases, indicated that 50% of the dogs did not have peripheral lymphadenopathy; this was not in agreement with reports by other investigators, and thus this could be a unique finding of dogs with the ne0plasm in England. Enlarged lymph nodes (3 to 10 times normal size) were smooth, painless, well defined, rather firm, relatively mobile, and rarely adherent to the overlying skin or adjacent tissues. It was reported that 40% of the spleens were 7 enlarged enough to be palpated, and about 20% of the tonsils were enlarged and protruded from their crypts. Splenic enlargements could be verified with radiographic examination. Many clinical signs were nonspecific, such as inappetence, listless- ness, depression, lethargy, vomiting, weight loss, and polydipsia. More characteristically due to lymphadenopathy, there usually was interference with function, including: dyspnea, gagging and choking, coughing and diffi- culty in swallowing, ascites, hydrothorax, and local or generalized edema. Hematolggic Findings. Reports on the hematologic findings of CML were rarely diagnostic unless possibly a subleukemic or leukemic blood picture was found. However, in the dog, as previously mentioned, malig- nant lymphoma was characteristically aleukemic. According to reports in the literature, anemia, neutrophilic leukocytosis and, at times, thrombocytopenia were most frequently seen in later stages of the disease (Bloom and Meyer, 1945; Irfan, 1961; Jennings, 1953; Meier, 1957; Schalm, 1966; Squire, 1964). It was suggested that anemia was due to bone marrow infiltration of neoplastic cells and to myelogenous hyperplasia (Bloom and Meyer, 1945; Irfan, 1961; Meier, 1957; Schalm, 1966). These investigators considered that marked absolute leukocytosis was attributed to tissue destruction, secondary infections, and toxicosis. Pathologic Findings Gross Lygph nodes. Generalized involvement of both visceral and superficial lymph nodes was commonly reported with this ne0plasm. According to most reports the visceral lymph nodes were affected as 8 frequently as the superficials, although one investigator (Jarrett 22.21:: 1966), in a study of 122 cases of CML in England, claimed that either the visceral lymph nodes or superficial lymph nodes were enlarged, but not both. Bloom and Meyer (1945) and Cotchin (1954) indicated that the lymph nodes that were involved first were those of the throat and neck but men- tioned that their location could make them more easily noticed. Lymph nodes involved in CML in order of frequency were: the mandibular, cervical, retropharyngeal, prescapular, mediastinal, mesenteries, sub- lumbar, axillary, inguinal, bronchial, tracheal, iliac, and pOpliteal (Mbulton, 1961; Smith, 1963). Most of the literature reports indicated that the peripheral lymph nodes were frequently enlarged in dogs with malignant lymphoma, as compared to other domestic animals (Bloom and Meyer, 1945; Meier, 1957; Moulton, 1961; Smith, 1963; Squire, 1964). The mesenteric and sublumbar lymph nodes were the largest of the visceral lymph nodes (Meier, 1957; Mbulton, 1961; Smith, 1963; Squire, 1964). Often groups of lymph nodes coalesced (mesenteric, mediastinal) and formed enormous tumor masses. The cut surface was yellow, homo- geneously gray, pink-gray or cream colored; usually moist, and had bulg- ing cut surfaces. Some had necrotic centers, and others had hemorrhagic streaks. Demarcation between cortex and medulla was usually absent. Spleen. Splenomegaly, either moderate or severe, was present in a majority of dogs. The follicles appeared more numerous and promi- nent due to enlargement. At times, follicles fused forming large protrud- ing masses (Jarrett £3.2l3: 1966; Meier, 1957; MOulton, 1961; Smith, 1963). Liver. Hepatomegaly was common and had either a diffuse mottled appearance or nodular masses protruding above the surface form- ing whitish-yellow tumorous masses (Meier, 1957; Moulton, 1961; Squire, 1964). Hepatic tissue was yellow and friable. Other Organs. Crossly visible whitish-gray masses involved other organs, particularly the kidney and lung. Microscopic. A review of the histOpathologic changes was limited to the lymphocytic and reticulum cell types, since they were most com- monly found (Dorn.e§_el,, 1967; Priester, 1967; Smith, 1963). However, as pointed out by Bloom and Meyer (1945), Meier (1957), and Squire (1965), classification of malignant lymphoma was best made by employing imprint techniques. Using this method, these investigators concluded that CML could be classified into 4 types: histiocytic (reticulum cell), lymphocytic (divided into prolymphocytic, lymphoblastic, and lymphocytic types), plasmocytic, and Hodgkin's. Lyephocytic Type. According to literature reports, the lympho- cytic type was 4 times more common than the reticulum cell type (Dorn ._£._l-: 1967; Priester, 1967; Smith, 1963). In fresh imprints, the cell size ranged from 8 to 15/q: Cells generally were round, but irregular shapes occurred. BasOphilic cyto- plasm, at times, was more abundant depending on the type of cell, and the nuclei were round or oval. Chromatin patterns were stippled or pachychromatic. Nucleoli were usually multiple and the number of mitotic figures varied; however, they were more numerous in the lymphoblastic and prolymphocytic types. In contrast, cells in paraffin sections were 10 round or oval, with round or irregular vesicular to hyperchromatic nuclei. The cyt0p1asm was more abundant in the lymphoblastic and prolymphocytic cell types, and stained slightly basophilic with occasional azurophilic granules. I Reticulum Cell Type. In imprints the cells were larger (15 to 25 u) than most lymphocytic cells. The cyt0p1asm was more abundant but less baSOphilic. The nuclei were large and most commonly irregular in shape. Chromatin existedas coarse, unevenly divided, violet particles which were distinct from the colorless parachromatic spaces. There was no clumping of the chromatin. Nuclei were pale blue spheres embedded in the chromatineparachromatin network (Meier, 1957; Squire, 1965). In contrast, reticulum cells in paraffin sections were often larger and more pleomorphic than in the lymphocytic type. Cytoplasm was broad or indefi- nite and varied from slightly basophilic to acidophilic. Deposition of reticulum was often seen. The nuclei were large and frequently had folded, indented, or bizarre shapes. Nuclear membranes were distinct, chromatin was scarce, and nucleoli were evident and very large. Some reports indicated that Hodgkin's disease did not occur in the Adog (Feldman, 1932; Smith and Jones, 1966) and they classified it as an atypical reticulum cell sarcoma. Other reports indicated that Hodgkin's- like lesions did occur in the dog (Smith, 1963; Squire, 1965). gegan Involvement. The frequency at which the organs were invaded by tumor cells based on histOpathologic examination of dogs with malignant lymphoma has been reported (Dorn.ee_el,, 1967; Jarrett eene1., 1966; Meier, 1957; Smith, 1963; Van Pelt and Conner, 1968). According to these reports, the lymph nodes, spleen, liver, kidney, lung, and bone marrow were most frequently affected. The architecture was disrupted 11 in most of the organs infiltrated; however, the lymph nodes were most severely affected. Extramedullary erythro- and myelopoiesis were often present, occurring particularly in the liver and spleen (Jarrett eenel,, 1966; Moulton, 1961; Meier, 1957; Squire, 1964). It was reported that this, along with myeloid hyperplasia in the bone marrow, contributed to the frequent neutrophilia (Meier, 1957; Squire, 1965; Schalm, 1966). Diagnosis. Because of the wide range of clinical signs, a positive diagnosis of CML could be difficult. Since various infectious diseases caused enlargement of the lymph nodes in the dog, surgical biopsy with subsequent histOpathologic examination provided the best means for a positive diagnosis (Jarrett SE 31:: 1966; Meier, 1957; Moulton, 1961). In the absence of peripheral lymphadenopathy, exploratory_1aparotomy was the sole means of identifying the disease, and a biOpsy of the ne0plasm or lymph node permitted histOpathologic verification (Jarrett e£_el,, 1966). In patients with subleukemic or leukemic leukemia, the presence of anaplastic cells in the peripheral blood indicated a positive diag- nosis (Schalm, 1966). Unfortunately, the ne0plasm in the majority of dogs was extravascular (Moulton, 1961; Schalm, 1966; Meier, 1957; Squire, 1964). Transplantable and Transmissible Canine Ne0plasms According to Shimkin (1955), Novinsky, a Russian veterinarian, was the first to successfully transplant tumors in animals. After 44 unsuc- cessful attempts, Novinsky, in 1877, transplanted a venereal sarcoma from an adult dog to a number of puppies. Thus, he is given credit for being "the father of transplantable tumors". Soon after this, Ellermann and Bang (1908) and Rous (1910) not only transmitted tumors with whole cells 12 but were also able to produce tumors with cell-free extracts from these tumors, thus opening the new field of viral oncology. Since this time, a number of viral agents have proved to be the cause of neoplasms in birds and mammals (Eddy, 1964). Neoplasms known to be transplantable in the dog are presented in Table 1. It is seen that the papilloma (DeMonbreun and Goodpasture, 1932), venereal sarcoma (Karlson and Mann, 1952), anaplastic thyroid carcinoma (Allam eeue1., 1954, 1956), and mastocytoma (Lombard e£.e;,, 1963) have successfully been transmitted from dog to dog passage through 9 or more generations. In addition, there are a few reports of other canine tumors that were transplanted through 3 or less generations (Nielsen and Cole, 1961; Stewart 25.21:: 1959). Of the transplantable tumors, the papilloma (DeMonbreun and Goodpasture, 1932; M'Fadyean and Hobday, 1898) and mastocytoma (Lombard EEHEL': 1963) have been trans- mitted by cell-free filtrates. Venereal Sarcoma. An extensive review of the literature concerning this tumor was given by DeMonbreun and Goodpasture in 1934. Numerous experi— ments have shown that the venereal sarcoma can only be transmitted with viable whole cells. In nature, the tumor is transmitted to the geni- tals by coitus or through wounds of the skin by contact. Experimentally, this tumor has been passed through 40 generations (Karlson and Mann, 1952) of dogs during a period of 17 years (Table 1). During passage, there was no change in the ability of the tumor to become established and.no change in its histologic characteristics. The pathologic features of venereal sarcoma are well described by Moulton (1961) and Smith and Jones (1966). .GOHuouoaow owmmmoa nuHH nwsounu mHao pom: odomHuuou .mououuHHm ooHMIHHoo suHB wouuHamamuu mammHoooa ochmo hHoo .:0HumH=uoaHou cu oosaaH .oOHmmouwou mono "oaoumooumma was maoHHHdom “MSOHHHmoo was maooumo Hmouoco>n w o .doHuHawouou moouw ou aoHuoofich vdmloaoo $3 .JIAN 03me 8.8 N H 8808 EL 98 «songs: sesamfimz oomH ..mm:mm.a:m>ovHoz oequ N oHaHana oumaooa u wNqu.mw maoouomonmamg momH . .MM .qu vumoaoH omquH m H oumcooo ocoz vascumooummz ocoHooHovouo HomH .oHoo a domHon MH H H qqm H can maooummooumo oGOHomHavouo uoBSu HomH .oHou w somHon NHIOH H H omIoN H coo mumaama votz oGOHodevoum maocHoumo a. HomH .oHoo a oomHon mH H H oq H 000 locovm coHum>o l venomHuuoo maooHoomo ommH .qmmH ..Hm um anHH< 0H on H monooo u oomlomH vHou%£u oHuomHamq< mHmH .oeHum a Houuonzoz mNIHN N H monasm oooz maooumm locumolouwconu mmmH .oHSummmvooo a anounaozmm mmuom OH H monmsm ocoz me: .3321 a sausage «.13. N H ST? 282 ofimaoadamm NmmH .anoz a comHHMM Hm ow H monooa oaoz AmmmH .aHxaHgmv nme .hxwaH>oz III H H onlm mooz noaoouom Hoouoco> mucoumwom Amxmvv mooHumuoaow onoHuoondH Am%mwv ouammmoummom aoonooz cOHuoa owmmmom Hmoazz woumHnooaH IoasaaH we make mucouoq om< wow onu aH oHnHmmHaoamuu can oHoouaoHooaouu on On ozonx mamoHeooo ochoo .H oHooH 14 Oral Papilloma. This was the first tumor to be transmitted to the dog with cell-free extracts (M'Fadyean and Hobday, 1898), indicating a viral etiology. The incubation period of the experimental disease was between 28 and 42 days (Table l). The virus multiplies only in the oral or pharyngeal mucosa of the dog, with no autotransplantation to extraoral sites (Moulton, 1961). Pathologic features of the oral papilloma are described by DeMonbreun and Goodpasture (1932). Anaplastic Thyroid Carcinoma. Allam.eeuel., in 1954 and 1956, serially transplanted a spontaneous canine thyroid carcinoma through 30 generations in puppies (Table 1). Immunosuppressants used were x-irradiation and cortisone. After the 11th serial passage, 9 passages were accomplished without pretreatment, with 61% of the inoculated dogs having tumors. In contrast, the x-irradiated group had a tumor incidence of 86%. Metasta- sis to the regional lymph nodes was more common in the irradiated group. Regression of tumor occurred frequently in both groups. Histopathologi- cally, the tumors appeared similar through all serial passages. Mast Cell Leukemia} Lombard.eene;,, in 1963, serially transplanted to Beagle neonates a spontaneous mast cell leukemia through 9 generations using fresh tumor tissue in the first 7 and frozen tissue in the last 2 passages. Also, they were successful in transmitting mast cell leu- kemia with cell-free extracts through 3 generations. The latent period varied from 14 to 150 days, with a mean of 60 days. However, the latent period was longer for the first 3 passages as compared to subsequent passages. The histoPathologic features of cellularly induced and cell- free induced tumors were similar. Widespread metastasis was common in 15 dogs with either whole cell or cell-free extract induced neoplasms. Hematologic examinations revealed large numbers of mast cells in the peripheral blood smears. Malignant Lymphoma. Nielsen and Cole, in 1961, attempted homologous transplantation with 17 different canine ne0plasms using high doses of x-irradiation and prednisolone as immunosuppressants. Of the 17 neo- plasms attempted, 3 were successfully transplanted by 10 to 15 days postinoculation (Table 1). There were transplantation attempts with 9 reticulum cell sarcomas and 7 lymphomas. Therefore, they were unsuc- cessful in transferring canine malignant lymphoma by inoculation of cells into puppies. In 1966, Moldovanu e£_el, reported transmission of malignant lymphoma to newborn mongrel puppies. Puppies were x-irradiated with 84.5 to 128 r of total body radiation immediately after birth. The puppies were inoculated with whole cells or cell—free filtrates subcutaneously in the nape of the neck and intramuscularly in the leg 10 to 24 hours after irradiation. Repeated inoculations were given at weekly intervals. Fresh cell suspensions were made from biOpsy specimens obtained from dogs with spontaneous malignant lymphoma. After trypsinization, the trypan blue stained cells were counted. It was found that 65% of the cells remained viable. Cell filtrates were kept frozen at -80 C. during the interval between inoculations. They found that most dogs had lymph node enlargement 26 to 58 days postinoculation. However, surgical biOpsies of these nodes with subse- quent histopathologic examination revealed lymphoid hyperplasia. Three dogs at 20, 26, and 40 days postinoculation had malignant lymphoma, although 16 they did not include any histOpathologic verification of their findings. The second serial passage was based on a l4-day-old puppy that died of generalized malignant lymphoma 14 days postinoculation. Again, there was no histOpathologic verification of malignant lymphoma presented. In the third passage attempt, all puppies died from a nondescript "intercurrent infection". Herpesvirus Infection in the Dog Histogy. Carmichael and co-workers (1964) first described a fatal septi- cemic disease in infant puppies which they believed to be caused by a pleuropneumonia-like organism (PPLO). However, Stewart EEHEL- (1965a) found that a virus was responsible for this hemorrhagic disease of puppies and that the virus belonged to the herpesvirus group based on morphologic characteristics. Later, Carmichael _£_el, (1965a) also found that the septicemic disease was caused by a herpes-like virus and that their tissue cultures were contaminated by PPLO. Natural Disease. Carmichael and associates (1964 and 1965a) isolated a cytOpathogenic agent with dog kidney cell (DKC) cultures from blood, lungs, livers, spleens, and kidneys of 3 Springer Spaniel puppies that died between 2 and 3 weeks of age. All the remainder of the litter of 12 also died. The only sign of illness had been continual crying that began 8 to 12 hours before death. The isolate was designated as strain F-205V, and was used for experimental studies. The aforementioned iso- late was obtained from a New York kennel in 1961. Carmichael e£_el, (1964 and 1965a) obtained another isolate from puppies in Illinois in 1962, designated as strain A-l. 17 Stewart EEHEL- (1965a) reported deaths and runted pups among 2 litters of "close to term" fetal pups obtained from apparently healthy bitches by cesarean section. In 1 litter, 2 of 8 pups were diseased. Three of 8 pups in the second litter were infected, as shown by virus isolation from their kidneys. Virus also was isolated from the kidneys of the apparently healthy littermate fetuses. This strain of virus has been designated as SL18HLV. Distribution. Herpesvirus has been isolated from young puppies in 4 states and 1 District: New York, Illinois (Carmichae1.eenel., 1964), Washington, D.C. (Stewart, 1965a), Georgia (Schwartz and Martin, 1966), and Michigan (Carter, personal communication, 1967). Recently, the virus has also been isolated from young puppies in the United Kingdom (Cornwall eee_l_., 1966; Prydie e_t__a_l;., 1966). This apparently indicates that canine herpesvirus is widespread in the dog population. Etiologygand Properties of the Virus. The disease is caused by a herpes- virus which produces a cytOpathic effect (CPE) in DKC cultures. Char- acterization and serological studies of the virus indicated that it was a new member of the herpesvirus group (Carmichael E£.§l:’ 1965b; Spertzel eene;., 1965). Electronmicroscopy studies by Strandberg and Carmichael (1965) indicated that virus particles in thin sections of dog kidney cells had an average diameter of 142 me, The particles contain a DNA core surrounded by 2 membranes. The protein coat was composed of 162 sub- units, a characteristic shared by other herpesviruses. Carmichael and co-workers (1965b) found that the virus was inactivated by chloroform and ether, and was destroyed in less than 4 minutes at 56 C. Also, virus titers were maintained for months at -70 C. in virus stocks that 18 contained 10% serum. Infectivity was lost below pH 4.5 after 30 minutes. The virus was not related serologically to infectious canine hepatitis, distemper, infectious bovine rhinotracheitis, B-virus, equine rhino- pneumonitis, avian laryngotracheitis, or herpes simplex viruses (Carmichael t 1., 1965b; Spertzel e_t_:_a_l_., 1965; Stewart et al., 1965a). Clinical Signs. Incubation period varies between 3 and 8 days in puppies inoculated by intranasal instillation or by intraperitoneal injection (Carmichael eeue1., 1965a). Route of inoculation and virus dose did not appear to be related to the time of onset of signs or severity of illness. To the present, the natural disease has been recognized only in puppies less than 1 month old. Fatal illness occurred in those less than 1 week of age, whereas puppies older than 2 weeks did not become manifestly ill following inoculation but developed neutralizing anti- bodies (Carmichael £5.2l3: 1964). Carmichael and associates found that signs in older dogs inoculated with virus were limited to mild rhinitis or vaginitis. Recently, Binn.ee_e;, (1967) recovered herpesviruses from 2 of 16 adult dogs with upper respiratory diseases by tissue cul- ture means. Motohashi and Mbsanari (1966) also reported isolating a herpesvirus from a "diseased adult dog"; however, they were unable to reproduce disease in young puppies with the agent. Carmichael egel. (1964) and Stewart e_t_a_l_. (1965a) found that illness in puppies may start between the 5th and 18th days after birth. Principal signs included a soft yellowish-green, odorless stool, anorexia, labored breathing, abdominal pain, and crying. Carmichael and co-workers found that inoculated puppies generally appeared normal until 1 or 2 days before they died. After the onset of overt illness, they found death usually occurred between 24 and 48 hours. l9 Lesions. Pathologic findings have been described by Carmichael EEJEL° (1965a) and Stewart ££.§lc (1965a). They are characteristic: lesions in inoculated and naturally infected puppies consist of disseminated focal necrosis and hemorrhages. These lesions may be found in virtually all organs. Especially noteworthy changes occur in the kidneys, where subcapsular hemorrhages appear as bright red spots on a gray background of necrotic cortical tissue. The lungs are diffusely pneumonic. There is marked hyperemia, edema, and often there is froth in the air passages. Necrosis of alveolar walls with exudation of fibrinoid material in the alveolar spaces is a common finding. Some bronchial epithelial cells contain oval acidophilic intranuclear inclusion bodies. Focal necrosis and hemorrhages are also frequent in the liver, intestinal tract and adrenal glands. Spleens and lymph nodes are enlarged. Microsc0pic examination may reveal occasional cells in areas of necrosis with faintly acidophilic intranuclear inclusions. Such inclusions are not common. Inclusions are seen most commonly in sections of kidney, liver, and lung. Olander (1966) has reported encephalitis in puppies inoculated intracerebrally with canine herpesvirus. Species Susceptibility. So far as is known, only dogs are susceptible, and fatal infections have been reported only in puppies less than 1 month of age. Carmichael e5 El: (1965) found that suckling and weanling mice, chick embryos, rabbits, ferrets, and cell cultures derived from a variety of species were refractory to infection. Mild rhinitis and vaginitis were the only signs of illness in older dogs inoculated with virus. There was no evidence that CHV is pathogenic for man. 20 Immunity. Only limited information was available concerning immunity to this viral disease. Limited serologic studies indicate that anti- body to the virus is widely distributed in the dog population (Carmichael £5,213: 1965b; Spertze1.eeue;,, 1965). The author found that low neu- tralizing antibody titers developed in adult dogs inoculated with virus. The following findings on immunity were reported by Carmichael ££.él: (1965b): antibody levels reached maximal titers 4 to 5 weeks following intranasal or oral inoculation and gradually declined until they were no longer measurable after 6 months. They found that 2 bitches, whose naturally infected pups died, gave birth 1 year later to normal pups. Both bitches came from kennels in which the disease was known to be enzootic. Likewise, they found that when susceptible bitches were inoculated intravaginally with virus, they gave birth to puppies that all died within 2 weeks. Yet puppies from similarly inoculated bitches that had neutralizing antibody at the time of inoculation did not be- come ill. The puppies acquired maternal antibody and did not become ill following inoculation with virus at 1 week of age. MATERIALS AND METHODS Donor Dogs of Canine Malignant Lymphoma Two dogs with spontaneous canine malignant lymphoma (10019 and 10074) were used as donors (Tables 2, 3 and 4). Since 1 of the dogs used had typical lesions of CML which are well documented in the literature and are cited in the review of the literature, a description of lesions will not be repeated. The other dog (10019) had the thymic type of malignant lymphoma, which is not commonly seen in the dog. The lesions for the donor dog with the thymic type of CML will be described herein. The donor dog with thymic involvement was a 20-month-old, female, purebred Doberman Pinscher (10019, Table 2). Clinical history and signs included a sudden onset, dyspnea, anorexia, listlessness, and hydrothorax. Radiographs of the thoracic cavity revealed a tumorous mass. A hemogram indicated anemia and lowered platelet count, both of which have been common findings in over 65 spontaneous CML cases examined in this laboratory. In the thoracic cavity, there was approximately 200 m1. of fluid which contained numerous ne0plastic lymphocytes. The tumor mass observed at necropsy was located bilaterally in the anterior half of the thoracic cavity. The tumor appeared to involve the thymus and anterior mediastinal lymph node and extended to the trachea and esOphagus dorsally and the lungs posteriorly. Many of the superficial and deep body lymph nodes were enlarged and edematous. The liver and spleen were markedly enlarged. 21 22 Microscopically, the affected lymph nodes and anterior thoracic tumor mass consisted of a uniform distribution of neoplastic lymphocytes (Figure l). The lymph node architecture was obliterated with proliferat- ing lymphocytes. Neoplastic lymphoid cells were characterized by promi- nent, round to ovoid nuclei rich in chromatin, conspicuous cyt0p1asm, prominent nucleoli, and mitotic figures. Other organs affected with lymphocytic infiltration (Table 9) had a similar cell type as mentioned above with variable replacement of tissue parenchyma. Experimental Animals Experiment I. Conventionally raised purebred Beagle neonates of both sexes, varying in age from birth to 3 days, were used (Table 2). There were 3 serial passages of CML attempted, utilizing 3 litters of Beagle neonates (17 dogs). Using single doses of inoculum, 11 dogs were given viable whole cells and 4 were given cell-free extracts. Two were contact controls (Table 2). After weaning (5 weeks), they were transferred to isolation rooms Operated under a barrier system and were maintained on a pathogen-free diet.a gaperiments II, III;_IV, and V. Cesarean-derived, colostrum-deprived, germfree Beagle neonates of both sexes, varying in age from birth to 8 days, were used. Four litters of puppies were used in 4 transmission trials as outlined in Tables 3, 4 and 5. Experiment II involved the inoculation of 7 puppies with a fresh whole-cell suspension prepared aRalston Purina Co., Checkerboard Square, St. Louis, Mo. 23 ~ I f ,7 w Nana-‘3 fs' . ”II ' Figure l. Iliac lymph node from initial donor dog (10019), a 20—month-old female Doberman Pinscher. Notice the monotonous uniformity of neoplastic lymphocytes. H & E stain. x 680. 24 .Hs m.o om moo x ~.o .Ha m.H oH unease 02 z some n oooom Houuaoo oz a memo m oooom uomuuuno .Ha m.o >H sashes oz 2 memo m kooom .Ha m.o om mod N N.o .Ha n.H mH seesaw oz a memo m oooom N Nmoom .Ha m.o om moo x m.o .Ha m.H oH masses oz 2 ouuam mmoom .Ha m.o om moo x m.o .Ha m.H oH masses 02 o eaten «moon .Ha m.o om mos x m.o .Ha m.H oH masses 02 o euuao mmoom .3 no om on moo x m.o .Ha m.H oH masses wow 2 cacao «moom .Ha m.o om mod x m.o .Ha m.H oH moasea mo» 2 eunam Hmoom .Ha n.o om on moo x m.o .Ha m.H oH mosses mm» o euuom omoom H oHooH Aozmvv A.Ha\oHHo0v nuanced vmuoHsoooH «noun Now wouoHaoo ouoonHoom aOHumuocow 420 no OQOHuoaHauoH .mucnoo HHoo a mousom monomHH tHvouuH tcH ow< owmmmom nocon monocooo onmom mo muouuHH m on AHzov maonoaxH uooowHHma oaHamo mo owooooo HmHuom .H udoaHuooxm .N oHooH 25 .mnuHmon aHuaoummam woo vHo momma «\HIN ouo mwov Honuo osu “voHHHx ouoa mwoom van .mmoom .omoom owono mHmnoco>ouuaH I >H .szaoocMusonsm I om .mHHmodouHuoomuudH I mHo .Auw.oov oudouom mm .AINNooo N>m Aasafiasto N< .aa m .A>Moo muHo>ONNM ammo ooN "aoNSmNomnuN Noon Nagoya .Hs m.o om moon soaoa oN x N.N .Ha o.N NH o mosses oz 2 memo N NoNoN o .Na n.o om moon eoaaa ooN x N.N .Ha n.H oH o moasoa oz 2 mono N ooaom Houuaoo oz m ammo N quom .Na n.o om moon ooaaa ooN x N.N .Na m.H mo o «usage mu» 2 memo N ooNom HUNHUNO .Na m.m >H usaaea mow o memo N moflom uumuuxo .Na m.m >N gasses may 2 some N NsNom m oooom Homhuxm .Ne m.o >H usaaea 02 m some n oooom Ammmvv A.Ha\mHHooV nuanced wouoHoooaH ovouo xom woumHsoo muaoHoHoom aoHumuonom 480 we UGOHuoaHEMoH mucsoo HHoo I w mouoom moomoHH ImeuuH IaH ow< owoooom Hoaon voaaHuoooIIN oHoma - w ‘ M n1 . n . I - fie: ~ ‘5: I F I ha. Cf.i \ I.- IFICL ‘ d’HF‘c ~ II-HU H v U . flifl-II I A- I Ill . .I E .Q — III — u I . .H _ ~U . f I —H M. v V ~ u E. 5U 6‘ n a” AU“ 26 .astuodH HoucoaHHooxo mo ouooHoHooH no oQOHmoH 08mm ofiu no: new ooHHmm OHwOHOHomno .Ha m.~ :uHB woumHaoocH mm3 Houuaoo acounoo day I ummomo .mHHmoaouHuoomuuaH .Ha n.H woo .mHmaooamuaopso .Ha m.o .hHumHooosamuuaH .Ha n.o "mounou mcHsoHHom onu mo ooHHmmVOHwOHOHohno nuHe HNH wouoHHv .ESHoooaH «0 .HS m.N co>Hw ouoB ousoHoHoou wouoHaoocH HHHuoovIaouuo0Hoo .HH unoaHuooxm .m oHooH 27 .0 OBI um vououo mos HoHuousz . MQUQOH voaoHuaoaoHomo ozu oH> oaHHmo onoHOHmhzo mo .HE m.~ nuHs woumHooodH ouoB woo adHooodH HouooaHuooxo mo muaoHoHoou Scum HouoHomH ououmoom m dH voodoo ouoz oHouuaoo uomuoooIaoz I unmom was mmmomo .mHHoonouHuomoHudH .HE m.H was .hHooooamusonam .HE m.o .hHHdHaooaaouuoH .Ha m.o “mouaou wnH3OHHom onu mp oaHHMm UHmoHOHmmnm nuHa HuH wounHHw .ESHaoooH .HE m.N oo>Hw ouo3 muaonHoou woumHouoaH HHHHm I.mmm oomuooolnoz m H ennmom III mood smahH 8:3 I o HmflHHooo .H H NNNom iooH ovoa neahH BHHE I o HmBHHooo .H H HNNom iooH ovoa AmahH omHHHH I o HmBHHooo 2 H oNNom flooH Amhmvv aoHumcHeuoH “ovouoHaoonH New Ammovv wouoH omuaoHoHuom a: mo HoHHoumz InuoaH ow< Hocon AHZV oaosoahH uamcwHHma suHa mow m we ovoo noahH scum voumooum QOHmcoomno HHoo wououm o :uHs woumHoooaH mouoaooo onoom.oouwahow vo>HunovIaduumOHou .HHHeunoaHuooxm .q oHnma .cooHom coo hoovHx scum wounoouo uomuuxo ooHMIHHooo .Ho>HH woo .owoc :oahH .coonm .hooon mo mouooowoaon Scum ooumooum uomuuxo HMHsHHoUv .hnuHoos vooHoaou mocHHmm onOHOHmhno .Ha m.~ suHe voumHoooaH Houudou uomudoOInoz I omNomo .MHaoUHuaom mouH>ooouon scum voHo woo ocHHoo UHwOHOHomno .Ha n.~ auHa woumHouocH Houuoou uoouooo I momomo .hHHmooouHuoomuuoH .Ha m.H wad .hHooooomuoonso .HE m.o .hHHdHaoooaouuaH .HE n.o umfluDOH onaoHHom onu mo ooHHom UHwOHOHohno nuHB HNH wouoHHv .ESHaoooH mo .Ha m.N Go>Hm one? ouaonHuou HHHHo I HNH Houucoo uomuqootooz z m common III o>HHo I HmH uoouuxo oouwIHHoo z m mqmom memom voHv I o noouuxo ooumIHHou z m momom quom o>HHo I HmH uumuuxo moumIHHuo z w mouom nqmom .8 o>HHo I HmH uumuuxo oouwIHHoo z o ocmom Humom > oz voHv I o uomuuxo HoHsHHoo m H momom Homom onHHx I n uuouuxo HoHnHHmo 2 H momom Hemom voHv I OH Houuooo uomuooo 2 H Amomom III voHHHx I o oomuuxo HoHsHHoo x H Homom Hemom voHHHx I n uomuuxo HMHoHHoo 2 H mmmom quom o>HHo I mom oomuuxo uoHoHHoo E H Hmmom quom >H Amhwvv GOHuocHahoH woumHooonH o.vHoHuoumz xom Amhmov woumH omuooHoHoom >mo mo uaua IoooaH ow< Hosea IHuooxm mwov wouoomnH A>m0v mouH>moouon oanou mo monomHu Scum wounoonm mucouuxo ooHMIHHoo vow uoHoHHoo nuHs woumHaoooH mouooooa onmom ooumaouw uo>HuoovIasuumoHou .> one >H ouaoaHuooxm .m oHomH 29 from a dog with malignant lymphoma, and l was a contact control. Experi- ment III was a duplication of Experiment II, except that 2 puppies were used as non-contact controls and the inoculum for the 4 recipients was not a fresh cell preparation but was stored in a freezer8 at —70 C. (Table 4). In Experiment IV, puppies were recipients of a cellular ex- tract prepared from puppies in which canine herpesvirus-Kakuk (CHV-K) was isolated. For this experiment, 4, l-day-old puppies were inoculated and l was used as a contact control (Table 5). In Experiment V, 4, 8-day-old puppies were inoculated with CHV—K, while 1 puppy was a non- contact control (Table 5). Each inoculated puppy was given 2.5 ml. of inoculum diluted 1:1 with physiologic saline by the following routes: 0.5 m1. intramuscularly, 0.5 ml. subcutaneously, and 1.5 ml. intraperi- toneally. All control puppies (contact and non-contact) were injected‘ with 2.5 ml. of physiologic saline via the aforementioned routes. Rearinggof Germfree Puppies The germfree puppies were reared according to the methods described by Griesemer and Gibson (1963), which involves the feeding of a sterile dietb every 4 hours with a baby bottle. After weaning, they were trans- ferred to an isolation room and maintained on a pathogen—free diet, as previously mentioned. Also, oral and fecal swabs were taken periodi- cally to insure that a bacteria-free environment existed. No bacteria were isolated from puppies while maintained in germfree isolators. aRevco, Inc., Industrial Products Div., Deerfield, Mich. bVaramel, Baker Laboratories, Inc., Cleveland 15, Ohio 30 Transmission Technic Cell suspensions were prepared from thymus for the first and second serial passages and thymus and mesenteric lymph nodes for the third pas- sage (Experiment I, Table 2). After mincing, the tissue was homogenized in a homogenizera at a moderate speed with equal volumes of sterile physiologic saline solution at 5 C. Homogenization was done at time intervals which totaled a period of 1 hour. The cells were sedimented by centrifugation at 1000 rpm, resuspended in physiologic saline, and counted in a hemacytometer. Preparations of whole cells for other experiments (II and III) were carried out in a similar manner. Cell- free extracts were prepared according to the method of Moloney (1953). The amount of material inoculated, route, pretreatment, number of cells, and tissues inoculated are presented in Tables 2, 3, 4 and 5. Tissue Culture Procedure The tissue culture procedure described by Mitchell eevel, (1967) was followed. This procedure employed the use of primary dog kidney cultures and canine thymus. The nutrient and maintenance media used, number of cells used per tissue culture bottle, and inoculation procedures are described elsewhere (Carmichael et_ el” 1965b; Mitchell et_ el” 1967; Spertzel eeee;,, 1965). Virus isolation and identification were done in dog kidney cell cultures as described by Carmichael ££.§l: (1965b), Mitchell EEHEA- (1967) and Spertzel 23.3}: (1965). aVirTis Model-45, VirTis Research Equipment Co., Gardiner, N.Y. 31 Fecal Examination Using a qualitative sugar concentration method (Benbrook and 81088, 1961), fecal samples were examined at periodic intervals for parasitic ova. Those dogs infested with hookworms, ascarids, and Isospora bigemina were treated with diSOphenol,a piperazine citrate,b and nitrofurazone,c respectively, according to recommended dosages. Hematology Blood samples for hematologic procedures were taken by jugular veni- puncture at various daily intervals as noted in Appendices l, 2, 3, 4, 5, 6, 7, and 8. Blood was collected in vials containing 0.05 ml. of a 30% aqueous solution of tripotassium ethylenediametetraacetate mono- hydrate;d hemoglobin content (Hb.) was determined by the cyanmethemo— globin method; packed cell volume (PCV) was determined by using micro- hematocrit tubes. Total erythrocytic and leukocytic counts were made with an electronic counter.e Platelet counts were obtained with a hema- cytometer.f Differential leukocytic counts were made by observing 100 cells in a blood smear stained with Wright's stain.g aD.N.P., American Cyanamid Co., Princeton, N.J. bPipcide, Haver-Lockhart Laboratories, Kansas City, Mo. cFuradex, Eaton Laboratories, Div. of Norwich Pharmicals Co., Norwich, N.Y. dB-D Vacutainer, Becton, Dickinson and Co., Rutherford, N.Y. 8Coulter Counter, Model B, Coulter Electronics, Hialeah, Fla. fB-D Unopette Method, Becton, Dickinson and Co., Rutherford, N.J. gNational Aniline Division, Allied Chemical Corp. , New York 32 Pathology Gross Procedures. Lymph node specimens were obtained by surgical biopsy of enlarged lymph nodes where a diagnosis was warranted. The dogs that died and those that were tranquilized with promazine hydrochloride,a and subsequently exsanguinated by cardiac puncture, were subjected to a gross pathologic examination. Microscopic Procedures. Representative specimens were selected and immediately fixed in 10% buffered formalin, Bouin's solution, and; Zenker's solution. All specimens were paraffinb embedded, sectioned at 6qu and stained with Harris' hematoxylin and eosin Y (H & E). Selected specimens were subjected to special stains following the pro- cedures as outlined in the Armed Forces Institute of Pathology Manual of Histolpgic and Special StainingyTechnics (1960). Ultrastructure ‘Eeectronmicroscopy Procedures. Specimens of spleen, liver, lung, lymph node, kidney, and thymus were prepared for ultrastructural studies as described by Strandberg and Carmichael (1965). This technic employs the use of glutaraldehyde-osmic acid fixation and uranyl-acetate, lead- hydroxide staining. a Sparine, wyeth Laboratories, Inc., Philadelphia, Pa. bParaplast, Scientific Products, Evanston, Ill. EXPERIMENTAL TRANSMISSION OF CANINE MALIGNANT LYMPHOMA TO THE BEAGLE NEONATE EXPERIMENT I RESULTS Canine malignant lymphoma developed in 3 of 11 Beagle neonates inoculated between 1 and 3 days of age with the cell suspensions at 54 and 78 days postinoculation in the first passage and 53 days in the second passage. Two of the 3 dogs with malignant lymphoma were pre- irradiated, whereas 1 dog was not irradiated. In no case was CML transmitted using cell-free extracts. Even though CML did not occur in the third serial passage, dogs inoculated with either a cell suspen- sion or a cell-free extract had enlargement of the superficial lymph nodes. Surgical biOpsies of these enlarged lymph nodes with subsequent histologic examination revealed lymphocytic hyperplasia but not malig- nant lymphoma. Clinical Signs. Clinical signs included sudden onset with listlessness, extreme weakness, loss of weight, inappetence, diarrhea, dyspnea, and distention of the abdomen. Radiographs revealed an opaque mass in the anterior mediastinal region and a fluid line in the abdominal cavity. Paracentesis disclosed fluid in the peritoneal cavity which contained anaplastic lymphoid cells. Masses were palpable in the abdominal cavity. In Dogs 30030 and 30032, there were nodular masses in the abdominal and thoracic musculature and subcutaneous tissues. In Dog 30032, the super- 33 34 ficial lymph nodes were somewhat enlarged, whereas in Dogs 30030 and 30089, the superficial lymph nodes were normal in size. Enlarged lymph nodes were also observed in dogs inoculated with either cellular suspen- sions or cell-free extracts; however, malignant lymphoma did not develop in these dogs (Table 6). Hematology. Results of hematologic examinations are given in Appendices 1, 2, 3 and 4 and Tables 7 and 8. It was found that 2 of the 3 dogs with malignant lymphoma had anemia, all 3 had a left shift, and all 3 had a markedly lowered platelet count (Tables 7 and 8), as compared to the control. One dog (30030) had an absolute lymphophilia (leukemia), and between 41 and 52 days, all 3 dogs had atypical neoplastic lympho- cytic cells with retained nucleoli in the peripheral blood. Pathology gaggye. Experimentally transmitted CML involved primarily the organs and lymph nodes of the thoracic and abdominal cavities. Typically, the thymus and mediastinal lymph nodes were so greatly enlarged that the heart was displaced posteriorly. In the abdominal cavity the liver was markedly enlarged, friable, and had discrete whitish nodules or diffuse mottling; the lymph nodes were extensively enlarged and had a homogeneous whitish color throughout (Figure 2); the pancreas had a soft texture and resembled lymphatic tissue; and the omentum was diffusely thickened (Figures 3 and 4). There was infiltration of grossly visible ne0plastic tissue through the intercostal and flank muscles to the subcutaneous tissue of the body (Figure 5) and, in 1 dog, into the skin. 35 .Hz o>m£ “on vHo mono mwoo moo nH houmooaovosma%H uconamHu one muonen .mHouueoo woumHoooaHo: How owHom How mm3 momma H .m was N maoHumuooow owmmoom oHo m\m m\o o m mwoom «\N «\H m N «moon o\¢ o\N o H mHooH ucmEomuoHao Hz :qu mHma mwov mo oaOHuohoaowA. A: no Amoco enema IHcm mo .02 .oo Hoooa commemom Hooon IHHoo was chHmooamam HHoo mo owmmmom HmHHom wcHsoHHom AHZV mouooooo onoom ouoH mucouuxo oouw maonoahH uaoanHma mo muHHHAHmmHaonmuH .o oHooH .voOHo Houoanuoa onu cH HHooHooc vochuou nuHs mHHoo oHuhoozmahH HmoHohuo «Hos muonu .hHo>Huoommou.ANmoomV whom mm was HomoomV He uuz .nm eoe x mm aaeHeanHe .aao\om3 muqdou UHu%00£mE%4.ou=Homn< moHuom oHuhoounuhum wouoouuou II III I maoamahH uamcwHHoa noonvaH mo omomooo HMHHoo umH onu onunooouoou “Nmoom one omoom mwoo no N was H moochooo< scum noxou oosHo> aouwoao: HoHuoonvom aOHumHoooaHumoa woo Imam .n oHooH 37 .ommo Hm odd mH doosuoo mGOHumumomaH Buosxoon ououoooa on: mwov zoom .oooHo HouonoHuoa one GH HHooHoso oochumu nuHB mHHoo oHuhoonmahH HoOHohuo once cameo .oOHuoHsoocHuooo omoo on you a .oHoaoo vOOHo GOHumHoooaHonm com omm.o cowaq one mm Ho <.HH o.om om.q oooanmo oomamH mm NNH oNa.o ooo.m mNN Hm NH o.oH m.Nm oN.a ooo.oom oNN.NH HN o omm.o oo~.m omq Hm on o.o o.om mo.¢ ooo.mo~ ommamH mm mom wom.m omo.q woo Hm mo o.o m.w~ HN.¢ ooo.won ooq.¢H mm moo own.m Hoo.m coo om oa o.o n.- nm.~ oooaomq omoanH oHN oNN.N «Ho.o omo.m eoN mm moH o.oH o.oe oN.a ooo.oNN eNN.aH eo omoom won Houuooo om Nmm.N moe.m o NN HN o.o o.oH Hm.N ooo.oNH oNo.o mm oq mOH.m mow.H mum mm Ho o.o o.om «m.~ ooo.mnH nwm.m me o Hon.HH Non.H MHB mm Hm o.o o.om mm.m ooo.oo¢ omuaeH 0mm omm nom.w mmHam mum on mOH o.o o.om ow.~ cocammm HemaqH oHN omo.~ Hom.mH cmm.n mHn.H on ma H.oH o.oq nn.¢ ooo.nm~ om~.mm mo . mmoom woo oHHzooaHmom mHHnmoHuaoz moumoonmaeou moumoonoz I A5 3.93 3.55 A5 .aao\ oH .aao\ ooOHo moo 0mm: >02 .om >om x mm muoHouon .aao\om3 muaoou UHuhoonoahg ouoHoon< ooHHom UHuhoounuwnm concounoo wwoom woo Houunoo one maonoahH,uomanHma voodooH mo «wommma.HMHHoo cam onu waHuoooouoou Homoom woo co e was m mooHoaooo< scum dogma mooHo> aouwoaos HoHuaoavoo GOHumHsoonHuooo can Ioum .o oHooH 38 v-fla.v.ove ...... .. .....H.---V--fi_.--.—ov-—o~—fi Figure 2. Mesenteric lymph node from Dog 30089. Notice the lack of demarcation between cortex and medulla, and the homogeneous whitish color. 39 gure 3. First passage of cellularly malignant lymphoma in Dog 30032. At birth the dog was pretreated with 60.8 r total body irradiation. It was then inoculated with a cell sus— pension prepared from Donor 10019 (Figure 1). (A) Liver, notice the nodular areas which protrude above the surface and the enlarged hepatic lymph nodes. (B) Kidney and enlarged renal lymph node. (C) Markedly enlarged mesenteric lymph node. (D) Pancreas. Figure 4. Dog 30089, representing the second serial passage of malignant lymphoma induced by inoculating a cell suspension prepared from 30032 (Figures 3 and 5). Subject was inoculated at 2 days of age without radiation pretreatment and was killed when 53 days old. Notice the enlarged liver (A) and the thickening of the central veins and portal triads (arrow). (B) Kidney and renal lymph node. (C) Mesenteric lymph node. (D) Portion of thymus. (E) Portion of omentum. 40 Figure 5. Dog 30032. Notice neoplastic infiltration into subcutaneous tissues and intercostal muscles (A) and the markedly enlarged thymus (B). Figure 6. Mesenteric lymph node obtained from Dog 30089. Notice the invasion and proliferation of lymphocytic cells in the perinodal tissue. H & E stain. x 130. 41 .MicroscoEic. The organ and tissue distributions of lymphocytic infiltration in the donor dog and the 3 experimentally induced malignant lymphoma dogs are presented and compared in Table 9. Although the super— ficial lymph nodes were not affected grossly in Dogs 30030 and 30089, they were affected microscopically. Superficial lymph nodes of Dogs 30030 and 30089, which did not appear enlarged grossly, had diffuse proliferation of lymphocytic cells that disrupted the architecture of the germinal centers, filled the sinusoids, and infiltrated the capsule. Enlarged superficial, thoracic, and visceral lymph nodes consisted of lymphocytic cells of monotonous uniformity (Figures 6 and 7). In many instances, the cells had invaded the nodal capsule resulting in complete obliteration of normal architecture (Figure 6). The nodes consisted of continuous sheets of neoplastic lymphocytes (Figure 7) without demarca- tion between cortex and medulla. Vessels, nerves, and surrounding peri- nodal fat were diffusely infiltrated with neoplastic lymphocytes. Lympho- cytic infiltration in the capsular and perinodal areas was so profuse that circulation was disturbed to a point of thrombus formation. Lympho- cytic cell populations invading the capsule and replacing normal histo- logic structures were substantial evidence of malignancy. Mitotic figures were observed in all parts of the node and atypical mitotic figures were often.present. Infiltrating neoplastic lymphocytes in the thymus were morphologically similar to those described for the lymph nodes. There was, however, such an extensive proliferation of neoplastic lymphocytes that Hassall's corpuscles (Figure 8) and interstitial connective tissue were invaded, resulting in architectural obliteration. In the liver the lymphocytic 42 maosoahH uooowHHma oaooomuaoom mo Manon muouaomoa .ESuaoao .omouoaom .ooHuoouoH agomaouo I wanna Ho o eon .oHomaa I moaooHu Hoouoo o Houseman .HmHnoooun .HmaHuomHooa I oHomHosa oHHooooooOHHH .umHHH HmooouoH .umoeaHoom .oHuounoooa .Hmoou .oHuooo: I HmaHaovo< ooHHH Hmououxo .HmouHHooo .%HMHHon .HmHsomomouo I HmuoomHHom HmoH>uoo .Hmowahuonoouuou .uoHaoHvaoa I comma + + + + + + + + + + omoom + + + + + + + + + + Nmoom + + + + + + + + + + omoom I .. + + + + + + + + HeoHooH omoameu ouomuu zouuma coonm %oavHx mesa uo>HH msahsa oHomuonH HmoHaooA< HmuoanHom. omom .oz woo uozuo Ho moon mooooa smahq aoHuwcHamxo onOHoumH: mo venouumaoaov GOHumuuHquH oHumuoneahH mo aOHuaoHuumHo oaomHu one cowuo .o oHooH Figure 7. Higher magnification of Figure 4. Notice mo— notonous distribution of lymphocytic cells. H & E stain. x 680. Figure 8. Thymus. Malignant lymphoma. Hassall's corpuscle (arrow) which was invaded by neoplastic lymphocytes. H & E stain. x 680. 44 proliferation was usually limited to the periportal areas (Figure 9) and, in some instances, there was destruction of hepatic tissue (Figure 10). Widespread infiltration was observed in the pancreas, which also re- sulted in extensive destruction of its parenchyma (Figure 11). Infil- tration of lymphocytic cells in the kidney was confined to the cortex (Figure 12), particularly near the corticomedullary junction. The spleen was not markedly enlarged, yet the capsule and the sinusoids contained extremely anaplastic cells which resembled reticulum cells (Figure 13). Many intercostal muscle fibers were destroyed and replaced by infiltrating lymphocytic cells (Figure 14). Other organs and tissues were also infiltrated with neoplastic lymphocytes (Table 9). DISCUSSION This investigation demonstrated unequivocally that cellular induced malignant lymphoma is possible in the dog without pretreatment of total' body radiation. In Experiment I, CML develOped in 3 of 15 attempts in 53 to 78 days after inoculation using a single dose of inoculum. A pre- vious report by Moldovanu.ee.el, (1966) indicated that cell-induced CML can be accomplished by inoculating irradiated mongrel newborn puppies repeatedly. Results of EXperiment I indicated that lymphadenopathy was most fre- quent in dogs of the third serial passage, yet based on clinical examina- tion and subsequent biopsied lymph node specimens, CML was not present. In agreement with the results, Moldovanu.ee_e;, (1966) found that dogs inoculated with either cell-free extracts or CML cell suspensions developed lymphadenOpathy. It is well known that, rather than being a pathognomonic indication of early CML, lymphadenOpathy may be initiated ‘.‘., Figure 9. Liver from Dog 30089. Accumulation of neoplastic lymphocytic cells at portal triad. H & E stain. x 130. Figure 10. Liver from Dog 30032. Notice massive lympho- cytic cell infiltration; only a few hepatic cells remain. H & E stain. x 680. Rg'é‘rz... Figure 11. Pancreas. Malignant lymphoma. Overwhelming replacement of pancreatic parenchyma by neoplastic lymphocytes. H & E stain. x 375. Figure 12. Massive accumulation of neoplastic lympho— cytes in interstitial tissue of renal cortex. H & E stain. x 680. 48 by several infectious agents. Since Moldovanu E£Hél° (1966) reported difficulty with "intercurrent" infection, it is possible that much of the lymph node enlargement they observed was related to an infectious agent. With the aid of a barrier system for housing dogs, distemper and infectious hepatitis have been controlled. There are 2 possible explanations for the development of cellularly induced CML: (1) the cells inoculated could have multiplied and metasta- sized, and hence acted similarly to an AEHZEZQ tissue culture system, or (2) an agent could have been associated with the inoculated cells which, while multiplying, supported the replication of the agent, thus resulting in more cell transformation and multifocal malignant lymphoma. An argument against filterable agents having produced CML in these transmissions is that cell-free extracts failed to induce lymphoma, yet all 3 dogs with malignant lymphoma had numerous intracytoplasmic crystalline structures (Kakuk eene;,, 1968), which have been associated. with Rous sarcoma virus induced tumors (Monroe ££Mélra 1964; Rabotti _e_t_;_ e_l_.., 1966), and in leukemia of man (Dmochowski egg. , 1967). Crystals were more numerous in experimental CML than in spontaneous CML. Also, the latent period (50 to 80 days) which malignant lymphoma manifested in these transmissions, along with the inability to obtain localized growth at the inoculation site, may rule out simple transplan- tation as previously described (Marshak.eene;., 1967; Nielsen and Cole, 1961; Stewart egg” 1959). Cell-free transmitted malignant lymphoma in the cat has been demon- strated (Jarrett ££nél:: 1964b; Kawakami eeNe;,, 1967); also, recently the Cornell group (Richard ££.él:: 1968, unpublished data) has induced ML in kittens inoculated with cell-free filtrates. Jarrett e_t_ _a_l_. 49 (1964a,b) has demonstrated, by electronmicrosc0py, virus-like particles in the tissues of cats with lymphoma. Recently, Kawakamd and co-workers (1967) identified C-type viral particles associated with cell-free transmitted ML of cats, whose density was similar to those associated with the mouse leukemias. Although cell-free extracts have not trans- mitted lymphoma in the dog, the presence of C-type virus-like particles in naturally occurring CML suggests a viral etiology (Chapman 25.5l3: 1967). It has been observed by electronmicroscopy that the number of viral particles is greater in feline malignant lymphoma than in CML and thus this might explain the relative ease in transmitting lymphoma to cats with cell-free extracts. Therefore, it is possible that inoculated cells could temporarily grow in the recipient, thus giving a viral agent a chance to mature and replicate, infect, and transform more cells, which then may result in a widespread malignancy. Temporary growth of the cells and replication of an agent could explain the transient lymphadeno- pathy noticed 25 to 45 days postinoculation. Biopsy specimens from dogs having transient enlargement of lymph nodes revealed lymphocytic hyperplasia but not lymphoma. It has been reported that, in spontaneous CML, lymphadenopathy was most frequent in the superficial and mesenteric lymph nodes (Bloom and Meyer, 1945; Jarrettieeuel., 1966; Moulton, 1961; Smith,.l963). The results indicate, however, that lymph nodes that appear normal in size may contain neoplastic cells (Table 9). The 20-month-old donor dog (10019) had neoplastic involvement of the thymus, which has been a rare finding in spontaneous CML. This could be related to age since, in most dogs, lymphoma occurs between 4 and 9 years of age (Bloom and Meyer, 1945; Dorn e£_el,, 1967; Meier, 1957; Moulton, 1961; Priester, 1967). 50 Results indicated that in transmitted CML the thymus, visceral organs, thoracic lymph nodes, and abdominal body organs were involved in all 3 dogs. Thus, the experimentally induced CML pathologically simulates spontaneous lymphoma of the cat, which is predominantly a visceral form with little or no peripheral lymphadenopathy (Holzworth and Nielsen, 1955; Nielsen and Holzworth, 1953). The thymic involvement in experi- mental lymphoma of the dog also resembles the thymic form described for the cat. Hematologic results indicate that Dog 30030 had a leukemic leukemia, and Dogs 30032 and 30089 had a subleukemic leukemia based on the classi- fication devised by Dameshek and Gunz (1958). However, in spontaneous CML, the disease is most commonly an aleukemic leukemia (Bloom and Meyer, 1945; Smith, 1963; Schalm, 1966) or, better described as an extravascular ne0plasm. In agreement with literature reports on CML (Bloom and Meyer, 1945; Irfan, 1961; Schalm, 1966; Meier, 1957; Jennings, 1955) anemia, neutrophilic left shift, and lowered platelet counts are probably re- lated to the massive infiltration of neoplastic lymphocytes into the bone marrow, necrosis, and myeloid hyperplasia. Several investigators have reported close histopathologic similarity of spontaneous CML to that of Burkitt's lymphoma (Basherville‘eenel., 1966; Bras gel” 1965; Lukes ega_l_., 1966). This has been based on the histologic "starry-sky" pattern, a nonspecific phenomenon which has been observed in spontaneous malignant lymphoma of dogs (Basherville _e_t_a_l_., 1966; Bras Sea” 1965; Lukes e_t_.a_l_., 1966; Smith, 1963), cattle (Smith, 1965) and cats (Squire, 1966). A CANINE HERPESVIRUS ISOLATED FROM A DOG WITH CANINE MALIGNANT LYMPHOMA PATHOLOGIC FOR THE BEAGLE NEONATE EXPERIMENTS II, III, IV, v RESULTS A herpes-like virus was isolated from Beagle neonates inoculated with p0p1iteal lymph node material prepared from a dog with spontaneous malignant lymphoma. Because tissue culture and virus isolation results, clinical signs, hematologic findings and pathologic lesions were so similar in these 4 experiments, they are described together. A total of 25 germfree puppies between 1 and 8 days old were used in 4 experiments (Tables 3, 4 and 5): 20 inoculated, 2 contact controls, and 3 non—contact controls. All dogs except 1 (30251, Table 5), inocu- lated between 1 and 3 days of age, died or were killed between 6 and 16 days postinoculation. Both contact control dogs died with herpetic septicemia, and the 3 non-contact controls remained healthy. Only 1 dog died (30048, Table 5) when inoculated after 8 days of age; the others had clinical manifestations of disease but survived. Tissue Culture Findingg Herpes-infected Puppies. An agent cytopathogenic for primary dog kidney cultures was isolated from the lungs, livers, spleens, lymph nodes, and kidneys of dogs having the septicemic disease. The transmissible agent, based on serum neutralization tests, was designated as a strain of canine herpesvirus (Canine herpesvirus-Kakuk: CHV-K). Cytopathic 51 52 effects (CPE) were noticed 16 to 36 hours following preparation of the tissue cultures. The agent produced a focal type of CPE with rounding of the cells and swelling of the nuclei. The agent could be readily transmitted to dog kidney cell cultures or dog thymic tissue cultures but did not produce CPE or multiply in human embryo kidney. Donor Dog¥10074. In primary dog kidney cell cultures, CHV-K was isolated from a section of kidney stored at -70 C. Donor 10074 was the dog in which the prepared cell suspensions produced a septicemic disease in germfree Beagle neonates (Tables 3 and 4); thus, it was the origin of CHV-K. Histopathologic examination of the kidney from Dog 10074 revealed interstitial hemorrhage, focal glomerular damage, vacuolation of the tubules and infiltration of the renal pelvis and cortex with neoplastic lymphocytes (Figure 15). Cross-Serum Neutralization Tests The results of cross-serum neutralization tests between the CHV-K isolate and Carmichael's canine herpesvirus (F205V) are presented in Table 10. Canine herpesvirus-Kakuk did not neutralize antiserum against infectious bovine rhinotracheitis, pseudorabies, canine hepatitis, and canine distemper viruses. Clinical Signs Incubation period was 4 to 10 days. The first sign was anorexia; puppies rejected food 5 to 24 hours before death. This was followed by severe abdominal pain; even slight pressure applied to the abdomen initiated cries. Breathing was rapid and shallow, and many of the puppies had periods of severe gasping. During this pneumonic stage a serous 53 Figure 15. Donor Dog 10074. Notice hyalinization of the glomerulus and invasion of renal cortex by neoplastic lymphoid cells. H & E stain. x 680. Figure 16. Herpetic erythematous rash in inguinal region of a l6—day—old puppy inoculated with CHV—K. 54 Table 10. Cross-serum neutralization tests between canine herpesvirus-Kakuk (CHV-K) and Carmichael's canine herpesvirus (F205V) Highest dilution of antiserum Virus vs. Antiseruma giving complete neutralization CHV-Kb F205V 1:320 F2osvc onsv 1:160 CHV-K CHV-K 1:80 F205V CHV-K 1:80 aAntiserum against CHV-K was produced by hyperimmunizing an adult male dog with 10 intravenous injections of viable virus grown in primary dog kidney cells. bCHV-K: canine herpesvirus-Kakuk cF205V: canine herpesvirus-Carmichael, Cornell University 55 exudate came from the nose and, at times, was accompanied by epistaxis. Puppies then went through periods of extreme weakness, listlessness and, finally, death. Some puppies had an erythematous rash, principally in the skin of the inguinal regions, whereas others had accumulations of fluid in the ventral regions of the body. Elevated body temperatures were not detected; however, in moribund pups, body temperatures were below normal. Microbiologic Examination Portions of spleen, lung, liver, kidney, and lymph node were sub— mitted for bacteriologic examination. Bacteria were not isolated. Hematology [Experiments II,AIII, IV. Pre- and postinoculation sequential hemogram values in dogs inoculated between 1 and 3 days of age are presented in Appendices 5 and 6. Blood values in the appendices only include those dogs.in which a blood sample was obtained before death or before the puppies were killed. It is seen that the platelet count draps markedly between 6 and 11 days postinoculation. This is better compared and con- trasted in Table 11, which includes a summary of platelet counts in her- petic dogs and non-contact control dogs. It is seen that the mean platelet count was 70,400 (range: 24,000—178,000) for the herpetic dogs, as compared to 386,000 for non-contact control dogs (Table 11). Four of 10 dogs had decreases in total erythrocytic counts, and 3 of 10 had leukopenia (Appendix 5). The differential counts, packed cell volumes, hemoglobin contents, erythrocytic indices, and absolute leukocytic counts were not significantly altered (Appendices 5 and 6). 56 Table 11. Platelet counts taken from 11 herpes infected dogs and 3 non-contact control dogs from Appendices 5 and 6 Platelet counts/cmm. Dog Number Infected Uninfected 30236 144,000 --- 30241 178,000 --- 30243 24,000 ___ 39245 32,000 --- 30248 32,000 --- 30250a --- 425,000 30270 62,000 --- 30271 5 100,000 --- 30272 57,000 --- 30273b --- 362,000 30276 83,000 --- 30277c --- 372,000 30262d 38,000 —-— 30263 24,000 --- (Range) 24,000-178,000 70,400 (Mean) 362,000-425,000 386,000 (Mean) a’b:cDogs 30250, d 30273, and 30277 were non-contact controls Dog 30262 was a contact control with herpetic lesions 57 ‘Experiment V. Preinoculation and subsequent sequential hemogram values (Days 0, l, 4, 8, 10, 14, 22, 29, and 44) are presented in Appendices 7 and 8. Notice that 2 of 4 dogs (30246 and 30247) had left shifts between 4 and 10 days, as compared to Dogs 30049 and 30050 (Appendix 7). Dog 30048, that died, had marked erythropenia and neutropenia. Notice, too, the drop in platelet counts in inoculated dogs between 6 and 22 days as compared to the control (Table 12). Otherwise, the packed cell volume, hemoglobin content, erythrocytic indices, and abso- lute leukocytic counts were not significantly affected. Pathology Gross Skin. Five of 18 puppies had an erythematous rash (Figure 16). Also, 7 of 18 herpetic pups had subcutaneous edema, particularly in the ventral regions. Several puppies had petechial and/or ecchymotic hemor- rhages in the subcutis. Nasopharynx and Trachea. Petechiae and ecchymoses were observed rather frequently throughout various areas of the nasopharynx. Both the nasOpharynx and trachea commonly contained a frothy exudate. grgans of the thoracic cavity. The lungs were edematous, had gray and red mottled areas, and a frothy fluid exuded from the bronchioles and bronchi when pressure was applied (Figures 17 and 18). Subpleural petechial, ecchymotic, and suffusive hemorrhages were commonly observed. Some lungs were rather firm, indicating consolidation, but they floated in 10% buffered formalin. Usually, hearts appeared pale, valves were 58 Table 12. Pre- and.postinoculation sequential platelet counts taken from Appendices 7 and 8 4:— Platelet counts Dog Number Day [cmm. 30246 0 300,000 1 486,000 4 229,000 8 251,000 10 175,000 14 289,000 22 300,000 29 271,000 44 313,000 30247 0 336,000 1 489,000 4 211,000 8 205,000 10 261,000 14 290,000 22 126,000 29 300,000 44 23,000 30248a 0 428,000 1 378,000 4 244,000 6 32,000 30249 0 518,000 1 275,000 4 278,000 8 115,000 10 250,000 14 57,000 22 207,000 29 220,000 44 229,000 30250b 0 375,000 1 549,000 4 425,000 8 420,000 10 400,000 14 300,000 22 342,000 29 414,000 44 362,000 aDied 6 days postinoculation bUninoculated — non-contact control 59 Figure 17. Dog 30270, inoculated with a cell suspension prepared from Donor 10074. Notice the lungs with a frothy exu- date, enlarged mottled liver, splenomegaly, and petechiation of gastrointestinal tract. A canine herpesvirus was isolated from dog kidney tissue culture cells inoculated with extract prepared from affected tissue. 6 7 9 1 l0 H 13 i 1 1 . 1 1 ' ' ""1 'Y7 V' 11' T YV Y vvyv‘vxvvr y YYT TYTY Figure 18. Lung from Dog 30243. Notice the petechial, ecchymotic and suffusive hemorrhages and the frothy exu- date. ’ ‘ 60 swollen, and frequently there were subendocardial and epicardial hemor- rhages. Petechial hemorrhages and congestion were the most common macro- SCOpic lesions in the thymus. Organs of the abdominal cavity. Livers were enlarged, had numerous well-circumscribed small, red areas that measured between 1 and 3 mm. in diameter, and were yellowish (Figures 17 and 19). The kidney surfaces had a mottled appearance (Figure 20). Hemorrhagic areas were sharply demarcated from the adjacent pale portions. The cut surface revealed wedge-shaped to irregular areas of hyperemia and hemorrhage (Figure 21). The bases of the hemorrhages were located in the cortex and the apices extended into the medulla. There were a number of subserosal petechial and ecchymotic hemorrhages along the entire gastrointestinal tract in 10 of 18 pups. In 3 puppies, there was blood within the intestinal lumen. Feces had a richly mucoid appearance. The pancreas and adrenal glands appeared normal macrosc0pically. Spleens appeared enlarged and had a dark mahogany color. Upon cutting, the cut edges bulged. Lymph nodes. All lymph nodes were enlarged; some were also hemor- rhagic and hyperemia. Hemorrhages were, however, most commonly observed in the bronchial lymph nodes. Skeletal muscles. Whitish streaks were commonly seen in muscles of the pelvic limb but were also found scattered in other muscles. In 4 puppies there were petechial and ecchymotic hemorrhages distributed throughout the skeletal muscles. 61 Figure 19. Higher magnification of Figure 17. Notice the focal areas of necrosis in the liver and the hemorrhages along the gastrointestinal tract. 62 Figure 20. Focal hemorrhages in kidney of Dog 30242, that was inoculated with a cell suspension prepared from Donor 10074. Hemorrhagic areas represent necrotic foci packed with erythro- cytes. Figure 21. Sagittal section of kidney in Figure 20. Notice that the hemorrhages extended from the capsule to the corticomedullary junction. 63 Central nervous system. Hyperemia and hemorrhage were the only macroscopic lesions noticed. A few puppies had diffuse hemorrhages beneath the periosteum of the cranial bones. Petechial and ecchymotic hemorrhages occurred in many other organs and tissues, but with less frequency than the aforementioned. Microscopic. The most frequent histopathologic lesions.were necrosis, hemorrhage, and congestion, as shown in Table 13. It was also noticed that the microscopic picture is characteristic of an acute septicemic infection. Marked lymphocytic and reticular cell hyperplasia were also prominent features. Skin, The microSCOpic lesions were made up of clusters of vesicles containing an acidoPhilic fluid. There was hyperplasia, vesicle forma- tion, and necrosis of the stratum germinativum (Figures 22 and 23). In the necrotic areas, there was infiltration of neutrOphils. Marked fibro- blastic hyperplasia was located in the corium which surrounded the vesicle formations. These hyperplastic areas resembled fibromatous growths (Figures 24 and 25). NasOpharynx and trachea. Congestion, scattered hemorrhages, and hypersecretion of mucus were frequently observed in the nasopharynx. In addition, focal necrosis was seen in the nasal epithelium of a few puppies. The trachea contained an abundance of mucus. Organs of the thoracic cavity. The most prominent lung lesion was focal necrosis. These necrotic areas were characterized by swelling of the alveolar walls, loss of structure, and accumulation of a fibrillar 64 mmaounHw waHHnaomou mHauop mnu aH mHmmHmumehs oHummHnounHm voxumzm mHmmHauommn III III III III mm III III III III III III III Om NN oHuho IonmahH mHmmHauom III III III III III OOH III III III III III III III mN I»; Haas umHooHumm III III an III III III HH III III cc Nu OOH III OOH mamvm III III III OOH OOH HO III III III III III III «N OOH mHmou Imoommnm ammHmouho III III III III III III III III III III mm mm OOH III mo aoHu ImHonom> III III mm III III III III III III III cc mm OOH III wcHHHoBm Ho mm ON 50 NB OOH OOH no III mm on OOH OOH OOH mHaoHoemm ON cc cc mm mm III HO mm mm mm HO OOH OOH OOH mowmnuuoaom HH on mm OOH cc OOH ON cc HO on OOH OOH OOH OOH mHmouooz aHmum xahumam mstm mnahza mopos soonm uomuu Hmaouv< mmmuosmm «Homes unmom hoavHM uo>HH wasH mowssno Iommz amass Ho Hmuofloxm ufiwoaoeumm mwov OouomwsH mauH>mueumn maHsmo OH aH mnonoH onoHosummoumHn mo moaovHoaH unmoumm .MH oHan ' I.‘ , - Figure 22. Skin section from Dog 30243. Notice the hyperplasia of prickle and basal cell layers with hydropic degeneration and necrosis of the prickle cell layers H G E stain. x 140. Figure 23. Higher magnification of an area in Figure 22. Notice the marked hydropic degeneration and necrosis of the prickle cell layer. H G E stain. x 350. Figure 24. Fibroblastic proliferation resembling a "fibroma" in the corium produced by CHV-K. H & E stain. x 275. Figure 25. Higher magnification of Figure 24. H & E stain. x 680. 67 material which was acidophilic (Figures 26 and 27). The fibrillar material stained positive for fibrin, and the acidOphilic fluid in the alveolar lumens was PAS positive. Acid0philic staining oval inclusions were seen in the nuclei of some alveolar cells. A lacy exudate within the alveolar spaces was common. Alveolar macrophages were numerous and often vacuolar. Erythrocytes were within the necrotic lesions as well as in the alveolar lumens. Five of the puppies had marked reticuloendothelial cell prolifera- tions intermixed with lymphocytes and neutrophils (Figures 28 and 29). All hearts had variable degrees of interstitial focal necrosis (Figures 30 and 31) and, less frequently, areas of edema, congestion and myolysis. Associated with these necrotic areas were numerous Anitschkow myocytes (Figure 31) and, sometimes, hemorrhages. Several puppies had subendocardial and pericardial hemorrhages. Phagocytosis of thymic lymphocytes by macrOphages giving the "starry- sky" patteranas the most frequent lesion in the thymus (Figure 32). Other lesions seen microsc0pically were necrosis, hemorrhage, and hyperemia. Organs of the abdominal cavipy. In the livers, focal areas of ne- crosis scattered haphazardly throughout the parenchyma were a common histopathologic lesion. Characteristic of these necrotic areas were loss of structure, collapse of the reticular framework, and extensive hepatocellular damage (Figures 33 and 34). In most instances, there was no inflammatory reaction around the necrotic foci; however, there was lymphocytic hyperplasia in the periportal areas in 6 dogs. At the periphery of the necrotic foci, hepatocytes occasionally had a single, large intranuclear inclusion body (Figures 34 and 35). Other hepatocyte nuclei contained smaller irregular acidophilic inclusions (Figure 34). ._ 5‘ Figure 26. Lung section from puppy inoculated with CHV-K. Necrotic alveolar wall is dilated and contains an acidophilia fibrillar material. H & E stain. x 140. Figure 27. Higher magnification of Figure 26. Notice the intranuclear inclusion body (arrow). H & E stain. x 720. 69 o Irv- ' -' -. '" V ‘ .'rr . - IF... , $ . ' ' A R ‘ v, 0 > . I. "'4 ‘ ’%‘:H . k“ . 1., {44‘ «.7,‘#‘ I _.- ' ' 'I ,' 5"}: . 1' ' I _ P 7 .0 ' ,7 q ' 4 J. *2 I. ' - ° ‘ ,' O . ‘ a, I ’ fi- -. .' . ‘0 h D- J- ' _ 5"" - I .4! _ . a. a‘ " ~‘ a :J'.’ 4' ’I‘.£;‘::‘I::I -_ a»! "’ Figure 28. Section of lung taken from a puppy inoculated with CHV—K. Notice the marked reticular cell proliferation which obliterated pulmonary architecture. H & E stain. x 300. Figure 29. Higher magnification of Figure 28. H 5' E stain. x 660. 70 \ K - Kow- -,. g"\\. 3%.”. .< “Qt”??? Figure 30. Focal necrosis of myocardium. Puppy was inocu- lated with CHV-K, and died 6 days postinoculation. H & E stain. x 142. Figure 31. Higher magnification of area in Figure 30. Notice the numerous Anitschkow myocytes (arrow) adjacent to necrotic zone. H & E stain. x 540. , . a 928... Figure 32. Marked phagocytosis in thymus of a canine herpes— virus infected puppy. Note "starry sky" pattern. H & E stain. x 600. Figure 33. Focal herpetic necrosis in liver of CHV-K inoculated puppy. Note the vacuolar appearance of the sur- rounding hepatocytes and the obliteration of hepatic sinusoids. H & E stain. x 165. 72 -'~ 5 user. Figure 34. Higher magnification of Figure 33. Notice the oval to irregular shaped intranuclear inclusion bodies in hepatocytes. H & E stain. x 480. - . I- Figure 35. Higher magnification of Figure 34. H & E stain. x 800. 73 Inclusions were more numerous in some livers than in others; nevertheless, they could be found in most livers with considerable search. Hepatocytes surrounding the necrotic focinere markedly swollen (obliterating the sinusoids) and had vacuolated cytoplasm which did not contain fat. Lesions in the kidney were usually limited to the cortex, involving both glomeruli and tubules (Figure 36). Glomeruli were markedly swollen, obliterating Bowman's space, and were frequently acid0philic and struc- tureless. Hyaline-appearing material was PAS positive, although no fibrin could be demonstrated with Weigert's stain. Glomerular tufts often were hyalinized, and structural patterns were obliterated (Figure 37); however, there also were glomeruli with less damage (Figure 38). Intranuclear inclusion bodies were seen in the parietal epithelial cells of the glomeruli of most kidneys (Figure 39). They were usually oval, stained basophilic and the chromatin was marginated forming a rim around the nuclear membrane. Sometimes the inclusions were faintly acidOphilic; however, they were usually basOphilic. In no case were inclusions ob- served in the renal tubules. Leakage of blood appeared to occur in the vicinity of the capillaries. Erythrocytes thus were seen around glomeruli and adjacent tubules. Tubules had undergone various degrees of swelling, vacuolation and ischemic necrosis in the cortex (Figure 36). Hemorrhages were frequently located subcapsularly, around areas of necrosis, in interstitium of medulla and submucosa of the renal pelvis. Subserosal, submucosal and mucosal hemorrhages and hyperemia were commonly seen in the gastrointestinal tract. Focal areas of necrosis were limited to the gastrointestinal crypts (Figure 40), which many times led to hemorrhage into the mucosa. Peyer's patches appeared hyperplastic with some of the lymphoid elements undergoing necrosis. Goblet cells 74 Il . I: Figure 36. Subcapsular hemorrhage, damage of glomerular tufts, and tubular necrosis in renal cortex of CHV-K inocu- lated puppy. H & E stain. x 142. 'wK-t-r' Figure 37. Cellular destruction and hyalinization of glomerular tuft taken from an infected puppy. Note tubular necrosis. H & E stain. x 620. . . r ‘. .u.‘ a , a \‘ . ‘.V Figure 38 Glomerular tuft with less damage than Figure 37. H & E stain. x 620. .0 .I I k. VI 1') ‘ ‘ ‘. II. ‘ 5 Figure 39. Intranuclear inclusion body in parietal epi- thelial cell of glomerular tuft. H & E stain. x 736. Figure 40. Notice focal ne rotic area in duodenal crypt. Puppy was inoculated with CHV-K and died 6 days postinocu- lation. H & E stain. x 300. Figure 41. Focal necrotic area in pancreas of a puppy that was killed 7 days postinoculation. H & E stain. x 480. 77 appeared markedly dilated, resulting in hypersecretion of mucus, thus explaining the mucous-laden feces seen grossly. The pancreas and adrenal glands had focal areas of necrosis (Figures 41 and 42) along with a few intranuclear inclusion bodies adjacent to and within these necrotic zones. In the spleen the reticular cells had undergone diffuse hyperplasia, obliterating the normal histologic structures, as compared to the con- trol, and thus there was no demarcation between Malpighian corpuscles and red pulp. The spleen contained numerous erythrocytes which perhaps gave it the dark mahogany color seen grossly. Numerous megakaryocytes had undergone necrosis. Pyknosis, karyorrhexis, karyolysis, and erythro- phagocytosis were evident throughout the sections of spleens examined. The hyperplastic reticular cells appeared anaplastic (Figure 43) as compared to the control (Figure 44), yet the splenic capsule was not infiltrated. Lymph nodes. HistoPathologically there were 3 outstanding lesions: (1) diffuse lymphoid hyperplasia (Figures 45 and 46), (2) profuse hemor- rhages, especially in the bronchial lymph nodes, and (3) marked karyor- rhexis of lymphocytes in the lymph nodes of a few dogs. Occasional baSOphilic intranuclear inclusions were seen in lymphocytes (Figure 47). Skeletal muscles. The white streaks seen grossly consisted of various degrees of Zenker's necrosis (Figures 48, 49, 50, and 51). In many dogs, there was marked destruction of the skeletal muscle fibers, while in others congestion, hemorrhage, and edema prevailed. Figure 42. Higher magnification of Figure 41. Notice intranuclear inclusion body in pancreatic acinar cell. H & E stain. x 1200. Figure 43. Section of spleen taken from a lZ—day-old puppy inoculated as a newborn with a lymphocytic tumor prepa— ration from Donor 10074. Notice the undifferentiated cells and mitotic figures. H & E stain. x 680. Figure 44. Section of spleen taken from non-contact control Puppy 30277. H & E stain. x 680. 0“ it! .. A“. of" . "sw- ‘ . I- '5’. .‘, , . '_ 1"." "l ”9% , 9.12741, r, .. , ' ' " '1" .i. Figure 45. Section of mesenteric lymph node taken from Puppy 30241, that was inoculated with a cell suspension pre- pared from Donor 10074. Notice the marked lymphocytic hyper- plasia and numerous mitotic figures. H & E stain. x 350. Figure 46. Higher magnification of Figure 45. H & E stain. x 580. . .9. I , ' . la 4‘38 -J"‘“, ‘54 .' 'a'" ‘0‘?"’ - S ' I’I. . 43...“! l .‘ -‘Pe..‘..ll~";v' 2. Figure 47. Section of mesenteric lymph node taken from a puppy inoculated with CHV-K. Notice lymphoid depletion, necrosis and intranuclear inclusion body (arrow). H & E stain. x 640. 81 . . ’1 ‘ '° .\ _ I -I‘.. I Figure 48. Section of severe skeletal muscle necrosis taken from a puppy inoculated with CHV-K. H & E stain. x 140. Figure 49. Higher magnification of area in Figure 48. H & E stain. x 700. Figure 50. Section of muscle taken from a puppy inocu- lated with CHV-K. Notice edema and proliferation of sarco- lemmal cells. H & E stain. x 140. 1‘7""..4! 32. 33“” LA x . -- _ Figure 51. Higher magnification of area in Figure 50. Notice the loss of striations. H 8 E stain. x 700. 83 Central nervous system. Congestion was the most common histopath- logic lesion, followed by focal capillary hemorrhage, neuronal degenera- tion, and glial cell accumulation (Figure 52). ElectronmicrOSCOpy ElectronmicroscOpic examination demonstrated herpes-like virus particles in affected tissues. Immature intranuclear viral particles (Figure 53) measured 100 mu.in diameter and were envelOped by the nuclear membrane upon release from the nucleus into the cytoplasm. Mature par- ticles in the cytoplasm (Figure 54) which possessed outer envelopes were found in vacuoles and measured 175 mu,in diameter. DISCUSSION Cross-serum neutralization tests indicated that the virus isolated from a dog with CML (10074) is closely related to or possibly identical to the strain of canine herpesvirus (F205V) isolated by Carmichael‘s; al, (1965a) from young puppies. Likewise, the strains of canine herpes- virus isolated from young puppies by Stewart gtflgl. (1965a) and Spertzel .2£“2l3 (1965) also appear immunologically related. This is the first report, so far as the writer knows, concerning the isolation of a herpes- like virus from a dog with malignant lymphoma. There may be 4 different views as to the role of this virus in a disease process, as follows: (1) the virus passed the placental barrier as previously reported (Stewart 55“31., 1965) and thus the puppies of Experiment II were in— fected ignggggg; (2) the virus is responsible for a septicemic disease in newborn puppies and possibly can subsequently lead to malignant lymphoma; (3) canine herpesvirus infection was an incidental infection 84 Figure 52. Section of cerebrum taken from a puppy inoculated with CHV-K. Notice glial cell accumulation. H 8 E stain. x 300. 85 Figure 53. Intranuclear immature herpes-like particles (arrow) in ultrathin section of lymph node. Glutaraldehyde osmic-acid fixation, uranyl-acetate lead-hydroxide stain. x 65,000. 87 Figure 54. Mature, membrane associated particle (arrow) in cyt0p1asm of ultrathin lymph node section. Glutaraldehyde osmic- acid fixation, uranyl-acetate lead-hydroxide stain. x 50,000. 89 to malignant lymphoma in the donor dog; and (4) the virus, because of its latent potentialities, could trigger another agent(s) [virus(es)] to produce malignant lymphoma in dogs.. These 4 views are discussed herein. In this study, passage across the placental barrier appears not to be the source of virus. In Experiment II, all 7 of the inoculated pup- pies, as well as the contact control, had the septicemic lesions and thus, at this time, it was not known whether the virus was from the malig- nant lymphoma dog or from the bitch. Results of Experiment III, using non-contact controls, indicated that the origin of the canine herpes- virus was the dog with malignant lymphoma. The septicemic disease de- veloped in all inoculated puppies while the isolated controls remained healthy, which indicated that the virus was from the original ML donor dog. This was further substantiated when the virus was isolated, by tissue culture means, from the frozen kidney of the dog with CML. In agreement with Carmichael ggngl. (1964, 1965a) and Stewart's; al, (1965a), the results indicate that the virus does produce a fatal septicemic.disease in newborn puppies. In their experiments, convention- ally raised puppies were used to reproduce the disease, whereas in this investigation germfree Beagle neonates were utilized. It was found that age at time of exposure is very important in reproducing the disease. Newborn, germfree Beagles inoculated prior to 8 days of age are very susceptible, the condition being almost 1002 fatal 6 to 16 days post— inoculation. According to Carmichael it. grand Stewart 3; $1., the disease has not been seen in the field after puppies are 2 to 3 weeks old; thus, in agreement with results obtained in these experiments, susceptibility decreases with age. Only 1 recipient (30051) inoculated at 1 day of age and at this writing 295 days old has survived the infection. 90 If the puppies are not inoculated until after 8 days, there may be clinical and hematologic manifestations of the disease, but very few deaths occur. The abrupt resistance is possibly related to the tempera— ture control mechanism which does not develop until the puppy is about 1 week old (Fox, 1966). One recipient (30048) died when inoculated after 8 days; the clinical signs, hematologic findings, and pathologic findings were identical to those found in puppies inoculated before 8 days. Littermates of this dog are presently 171 days old and appear healthy. Transmission of canine herpesvirus infection appears to occur by contact as demonstrated by the results. Experimentally, Carmichael's; .21. (1965a) have shown, too, that conventionally raised puppies are susceptible when exposed to virus in aerosol chambers. Likewise, Carmichael and co-workers found that intravaginal inoculation of bitches with virus 1 to 2 weeks before whelping resulted in fatal infections of the newborn, yet illness was not observed in the bitches. It appears that the virus can cross the placenta, since infections have been seen in pups obtained by cesarean section (Stewart g£_al,, 1965a). Clinical signs were similar in germfree puppies and puppies raised conventionally by Carmichael £5.31, (1965a). However, emesis and greenish- yellow diarrhea observed by Carmichael and co-workers was not observed in these experiments. This may be due to secondary bacterial invaders in conventionally raised puppies. In germfree puppies anorexia was the first sign, followed by evidence of severe abdominal pain and finally dyspnea with severe gasping, and death 5 to 24 hours after rejection of food. 91 When comparing hematologic results of non-contact control dogs with those of inoculated and contact control dogs, it is seen that the latter had a markedly lowered platelet count. This may account for the wide- spread hemorrhage seen pathologically. Leukocyte counts were not sig- nificantly affected. Nevertheless, in germfree dogs inoculated with canine distemper virus, Gibson.g£“§l, (1965) observed a marked leukopenia. Thus it appears that the infectious agents alter the blood picture dif- ferently under germfree conditions. Focal hemorrhages covering the lung and kidney surfaces are gross diagnostic features of herpes infection compared to canine distemper and infectious canine hepatitis (ICH). Hemorrhages can, however, involve any organ and tissue, and thus ICH must be included in a differential diagnosis. In addition, splenomegaly, hepatomegaly and lymphadenOpathy are frequently observed in CHV infections. The pathologic features of CHV infection in germfree puppies and conventionally reared puppies (Carmichael g£_§1,, 1965a) are similar. However, Gibson EENEA: (1965) found that the prominent lesions of canine distemper virus infections using germfree puppies are lymphoid depletion, reticular cell hyperplasia, and neuronal degeneration. In conventionally reared puppies, secondary bacterial invaders cause widespread lesions (DeMonbreun, 1937; Dunkin and Laidlaw, 1926), unlike those observed in germfree dogs. It is noticed, therefore, that canine distemper in its natural form is a synergistic interaction between virus and secondary bacterial invaders, whereas this does not seem to occur with CHV infections. Histopathologically, 2 types of lesions were observed: (1) focal necrosis of the kidney, lung, liver, heart, skeletal muscle and other 92 organs, and (2) reticular cell and lymphocytic hyperplasia. The necrotic lesions in the lung and kidney are diagnostic histopathologic features of this disease. In the lung, the alveolar walls are dilated and filled with a fibrillar acidophilic staining material that stains positive for fibrin. This lesion is surrounded by necrotic septal cells, some of which contain oval to irregular acidOphilic intranuclear inclusion bodies. In some cases inclusions are very difficult to find. It appears that l’ . the virus causes swelling of a number of glomeruli which is followed by "VIA ' x 1‘51 ischemic necrosis of the tubules. This observation is based on the histo- pathologic finding that only tubules in the renal cortex are affected. Variable degrees of degeneration are seen (cloudy swelling, vacuolation, 0.5- and necrosis), which apparently are related to the degree of pathologic alteration in the glomeruli. This observation is interesting since, if a dog recovers from herpesvirus infection, glomerulonephritis could be a sequela later in life. Lymphoid hyperplasia was so pronounced in the lymph nodes that normal histologic structures were obliterated. Lymph nodes consisted of lymphoblastic cells that had numerous mitotic figures, although the cap- sule and perinodal structures were not infiltrated. Lymphocytic hyper- plasia occurred frequently in the periportal areas of the liver and less commonly in the lung. There was marked reticular cell proliferation of the spleens; the cells appeared to resemble cells seen in reticulum cell sarcoma, but this did not appear to represent a neoplasm. The presence of lymphoid and reticular hyperplasia in the lungs along with focal areas of necrosis simulates the lesions seen in Marek's disease of chickens (Biggs, 1967), which is thought to be caused by a herpesvirus (Churchill and Biggs, 1967). 93 Canine herpesvirus infection could have been incidental to malig- nant lymphoma or the virus could be responsible, or in part be responsible, for the etiology of malignant lymphoma. Presently this cannot be answered, yet Hinz (personal communication) found that affected tissues examined contained herpes-like viruses in 252 of the dogs with ML examined by electronmicroscOpy. Isolation of these viruses by’igugiggg.technics was not accomplished. Several authors have reported the presence of herpes- like viruses in Burkitt tumor cells grown‘igugitgg, although isolation of an agent other than Herpesvirus hominis was not achieved (Epstein gtflgl., 1964; O'Conor and Rabson, 1965; Stewart.ggnal., 1965b). Henle and Henle (1966) have studied extensively 10 lines of Burkitt tumor cells and found that 7 of 10 had herpes-like viral particles as detected by electron- microscopy and fluorescent microscopy. Also, it was found that the viruses in Burkitt tumor cells did not react immunologically with anti- serum produced against 10 different viruses of the herpesvirus group. Canine herpesvirus, likewise, does not react with other viruses of the herpesvirus group; however, CHV-K isolates from a dog with malignant lymphoma and the CHV (Carmichael e_t a_l.., 1965a; Stewart 5331., 1965a) isolated from young puppies appears identical as demonstrated by cross- serum neutralization tests. In addition, CPE in dog kidney cultures and thymus tissue cultures were similar. _ Recently, Mitchell 35.21, (1967) isolated_a herpes-like virus from P3J Burkitt lymphoma cells superinfected with Moloney sarcoma virus that caused CPE in dog thymic cells. Stewart and Durr (1967) found that a cell-free extract prepared from SL1 Burkitt tumor cells and inoculated into newborn hamsters intracerebrally resulted in encephalitis with marked glial cell proliferation. Therefore, it appears that the 94 herpes-like agent found in Burkitt tumor cells can be pathogenic for the newborn hamster. Canine herpesvirus is not pathogenic for laboratory animals as reported by Carmichael ggugl. (1964) and Spertzel g£_§l, (1965). The author (unpublished data) has been unable to produce lesions in hamsters inoculated by various routes with CHV-K. It does appear clear, however, that canine herpesvirus is a good candidate virus to pursue in regard to its oncogenic potentialities. This is based on the facts that (1) the herpesvirus group is notoriously known for its latent state and (2) since Stewart g£_§l, (1965a) have data suggesting that CHV, too, has a state of latency, this might sug- gest also that CHV could play a role in triggering or causing CML. In addition, the recent finding by Churchill and Biggs (1967) that Marek's disease of chickens is likely caused by a herpesvirus suggests the possi- bility that a similar agent could be involved in malignant lymphoma of domestic animals and man. SUMMARY Canine malignant lymphoma (CML) was transmissible to the Beagle neonate in 2 serial passages, and a canine herpesvirus, designated as canine herpesvirus-Kakuk (CHV-K), that was pathogenic for the germfree Beagle neonate, was isolated from a dog with CML. The clinical, hema- tologic, macroscopic, and microscOpic findings of experimentally induced CML and a septicemia in Beagle neonates produced by CHV-K were described and discussed. I. Experimental Transmission of Canine Malignant Lymphoma to the Beagle Neonate Experiment I Two serial passages of CML were accomplished in 2 litters of Beagle neonates with suspensions of viable CML whole cells. In these experi- ments 3 of 11 dogs inoculated with a single dose of a cell suspension developed malignant lymphoma which was clearly recognized at 53, 54, and 78 days postinoculation. Hematologic results indicated that leukemia was present in Dog 30030 and that subleukemia was present in Dogs 30032 and 30089 between 41 and 52 days. Likewise, 2 dogs had an anemia, whereas all 3 dogs had lowered platelet counts. Two dogs with over— whelming CML were preirradiated with x rays, whereas 1 dog was not irra- diated. Thus, a significant feature of this work was the successful transmission of CML without pretreatment of total body irradiation. Malignant lymphoma did not occur in the third serial passage, although animals had enlargement of the superficial lymph nodes. Biopsies of 95 96 these enlarged lymph nodes revealed lymphoid hyperplasia but not malig- nant lymphoma. The clinical and pathologic changes were similar to those reported for naturally occurring malignant lymphoma of dogs and cats . II. A Canine Herpesvirus Isolated from a Dog with Canine Malignant Lymphoma Patholggic for the Beagle Neonate Experiments II, III, IV, V. The canine herpesvirus (CHV-K) was isolated from 18 germfree Beagle neonates inoculated with either a cell suspension prepared from a dog with CML or extracts prepared from puppies with the septicemic disease. Two contact control puppies died from similar septicemic conditions, whereas the 3 non-contact control puppies remained healthy. From these results it was concluded that the CHV-K could be transmitted by direct contact and artificial inoculation. The production of a fatal septicemic disease in Beagle neonates inoculated with material prepared from a dog with CML and the isolation of the virus from the kidney of this dog indicated that the virus came from the donor dog. This virus was responsible for a fatal septicemic disease in colostrumrdeprived, germfree Beagle neonates 6 to 16 days postinoculation. The fundamental histOpathologic lesion was necrosis in the liver, lung, kidney, heart, skeletal muscle, pancreas, and adrenal gland. A few intranuclear basophilic and/or acidOphilic inclusions were seen in cells adjacent to the areas of necrosis. Splenomegaly and generalized lymphadenopathy were commonly seen which microscopically consisted of marked lymphocytic and reticular cell hyperplasia. The marked hyperplasia produced by this virus resembled neoplasia, although obvious ne0plasms have not been induced. 97 Results of cross-serum neutralization tests indicated that the virus was closely related, or possibly identical to, the canine herpesviruses isolated from young puppies (Carmichael's strain: F205V, and Stewart's strain: SL18HLV). However, this was the first report in which a CHV was isolated from a dog with malignant lymphoma, and also the first report concerning the isolation of CHV from an adult dog which was patho- genic for puppies. Results indicated that age at time of exposure is very important. in reproducing the disease in puppies. Newborn, germfree Beagles inocu- lated prior to 8 days of age were highly susceptible, and nearly all died. One of 18 recipients inoculated at 1 day of age is presently 295 days old, having survived the infection. If the puppies were not inocu- lated until after 8 days, there were clinical manifestations of the disease, but few deaths occurred (Experiment V). Factors of importance in establishing a diagnosis included: (1) age of the puppies; (2) clinical signs; (3) marked thrombocytopenia; and (4) pathologic findings. Pathologic changes in affected puppies were characteristic. 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The Kakuk family moved to Stephenson, Michigan, June 1945, where the author attended the Stephenson public schools. While in high school, the author was president of the freshman and sophomore classes, received 4 letters each in football, basketball, and track, was captain of the basketball and football teams his junor and senior years, and was editor of the school paper his senior year. He entered Michigan State University in 1955, earning a 3.8. degree in wildlife management in June 1959. The author then entered veterinary school at Michigan State University, in 1959, receiving the D.V.M. degree in June 1963. In the summer of 1963, he entered the graduate school of the University of Wisconsin, Madison, where he was awarded an NIH postdoctoral pathology traineeship grant. Under this grant, he carried out research concerning fowl leukosis, receiving the M.S. degree in veterinary science and a minor in human pathology in 1965. In June 1965 he accepted a position on the canine leukemia research project, and also entered the graduate school of Michigan State University, majoring in veterinary pathology. In July 1966 he was awarded a special NIH postdoctoral fellowship concerning canine malignant lymphoma. The author married Martha K. Smith of East Lansing, Michigan, in 1963. They have 1 child, Robert, 2—1/2, and are expecting another child in May 1968. The author has written several scientific publications. 118 "‘mmm