ABSTRACT CLINICO-PATHOLOGIC OBSERVATIONS OF HOG CHOLERA IN GNOTOBIOTIC PIGS by Kenneth Duane Weide Clinical and pathologic signs found in field cases of hog cholera often present a problem in diagnosis. Concur- rent infections, infestations, and nutritional deficiencies make diagnosis difficult. In this study, investigations were made into the effects of the hog cholera virus itself, utilizing gnotobiotic pigs obtained by technics used for rearing germfree laboratory animals. It was hoped that true, uncomplicated hog cholera could thereby be reproduced. Twenty-one l4-day-old pigs from 3 litters, obtained and raised under gnotobiotic* conditions, were each in- oculated intramuscularly with 750,000 lethal doses of hog cholera virus. Six uninoculated pigs in separate isolators served as controls. The clinical course of the disease was followed by ob- serving rectal temperatures, total and differential leuko- cyte counts, hemoglobin and hematocrit values, food con- ‘sumption and weight gains. Gross lesions were determined at time of death; histologic studies were made of nearly all tissues. Kenneth Duane Weide Ten cholera-infected and 4 control pigs were success- fully maintained free of any demonstrable bacteria throughout the experiment. Bacillus subtilis was isolated from the re- maining pigs. Differences could not be detected between the monocontaminated pigs and those found to bebacteria-free. Both groups were, therefore, evaluated together. Rectal temperatures were diphasic following virus admin- istration, reaching 2 mean peaks of 106 F. and 106.3 F. Total circulating leukocytes decreased nearly to one-third their preinoculation levels within 24 hours. There was a re- duction in hemoglobin and hematocrit levels, followed by a rise in numbers of circulating nucleated erythrocytes. Food consumption decreased several days before death, the greatest inappetence occurring 24 hours prior to death. All 21 in- fected pigs died within a period of 4 to 15 days after inoculation. Consistent gross lesions were limited to hemorrhages in the lymph nodes and kidneys. Histologically, hemorrhages were found in the tonsils, tongue, diaphragm, lung, heart, lymph nodes, meninges, urinary bladder, kidney, liver and tubular digestive tract. Hemorrhages and edema were most extensive in the internal lymph nodes. Necrosis was found in the gall- bladder, lymph nodes, and tubular digestive tract. Leuko- cytio cellular infiltrations were found in the liver and associated with necrosis in the gallbladder and portions of the tubular digestive tract. Anitschkow myocytes were found Kenneth Duane Weide in large numbers within the myocardium. Lymphoid depletion was common in the lymph nodes and spleen. Degenerative changes were present in the smaller blood vessels of the major tissues. These included endothelial proliferation, hyalinization and vacuolar changes in the vessel walls, and thrombosis. Frequently the blood vessel alterations in in- fected pigs were subtle, enabling no definitive conclusions to be reached. Livers of infected pigs were characterized by glycogen depletion and absence of extramedullary hemopoietic areas. Correlation could not be made between the number of organs showing hemorrhage and/or the severity of these hemorrhages, with the elevated temperatures, leukopenia, survival time, or other clinical signs. Lesions were somewhat independent of the clinical signs; death, in turn, was not dependent on the severity of either clinical signs or gross lesions. The signs and lesions found in infected pigs in this study were attributed to the effects of the virus itself, and served to emphasize the marked variation in the response of individual pigs to the hog cholera virus. Both control and infected pigs were kept in plastic iso- lators within the same room, and no precautions other than those routinely employed in rearing germfree pigs were used. Cross infection between isolators did not occur. This, in Kenneth Duane Weide itself, was significant and suggested that the technics used may be a useful tool in the study of infectious disease of domestic animals. *Gnotobiotic refers to animals reared under germfree conditions or in the presence of known microbial flora. CLINICO-PATHOLOGIO OBSERVATIONS OF HOG CHOLERA IN GNOTOBIOTIC PIGS by Kenneth Duane Weide A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Veterinary Pathology 1962 ACKNOWLEDGMENTS Acknowledgment is gratefully extended to Dr. Glenn L. Haxler, research director, who contributed freely of his time, energy and skill in helping to secure and raise experimental animals used in this work, and for his invaluable assistance in the histopathologic studies. Appreciation is extended to Dr. C. C. Morrill, academic advisor, for his advice, criti- cism and assistance in major portions of this work. The con- tinuous constructive criticism of Dr. C. K. Whitehair through» out the term of graduate study and the duration of this exp perimental work is greatly appreciated, as well as the time and physical energy which he so freely donated. Collabora- tion with these three individuals has indeed been a reward- ing experience. Dr. W. D. Pounden, Chairman, Department of Veterinary Science, Ohio Agricultural Experiment Station, is to be ex- tended my sincerest appreciation for his continuous encour- agement and his many efforts on my behalf. The allotment of time to pursue graduate studies was obtained entirely through his labors, to say nothing of the provision of numerous supplies and equipment for completion of this research. To my good friends, Dr. Kenneth K. Keahey and Dr. Edward J. Bicknell, Michigan State University, and Dr. vance L. Sanger, Ohio Agricultural Experiment Station, I extend grateful acknowledgment for their many efforts incident to this thesis. 11 To my wife and daughter, who have so graciously and patiently endured the rigors of graduate study, this dissertation is affectionately dedicated. iii TABLE OF CONTENTS I. INTRODUCTION. . . . . . . . . . . . . . . II. REVIEW OF LITERATURE. . . . . . . . . . . A. Clinical Signs. . . . . . . . . . . . B. Cross Lesion. . . . . . . . . . . . . 0. Microscopic Lesions. . . . . . . . . l. Digestive System. . . . . . . . . 2. Respiratory System. . . . . . . . 3. Genito-Urinary System . 4. Cardio-Vascular System. 5. Lymphatic System. . . . . . . . . 33 6. 7. 8. 9. Nervous System . Musculo-Skeletal System Integument. . . . Summation of Microscopic Lesions. D. Current Status of Hog Cholera . . . . III. MATERIALS AND METHODS . . . . . . . . . A. Germfree Technics . . . . . . . . . 3. Experimental Procedures . . . . . . IV. RESULTS . . . . . . . . . . . . . . . . A. Clinical Signs. . . . . . . . . . . B. Gross Lesions . . . . . . . . . . . iv ‘OODCD'Q'N‘ICMU'IWUWHF F4 id P’ k4 rd o C) C) 12 12 15 l9 19 30 V. VI. 0. Microscopic Lesions. . . . . . . . . l. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Skin, Lip, Check and Ear . . . . Tonsils. . . . . . . . . . . . . Tongue and Diaphragm . . . . . . Epiglottis, Trachea and Lung . . Salivary Glands and Pancreas . . Aorta and Heart. . . . . . . . . Spleen and Lymph Nodes . . . . . Endocrine Organs . . . . . . . . Meninges . . . . . . . . . . . . Urinary Ducts and Bladder. . . . Kidney............. Liver and Gallbladder. . . . . . Esophagus and Stomach. . . . . . Small Intestine, Cecum and Colon Reproductive Organs. . . . . . . DISCUSSION 0 O O O O O O O O O O O O O 0 SUMMARY AND CONCLUSIONS. . . . . . . . . LIST OF REFERENCES . . . . . . . . . . . 35 35 35 36 41 41 46 49 62 62 65 65 66 78 81 86 87 95 97 LIST OF TABLES my. I. Summary of Reported Gross Lesions of Hog Cholera. . . . . . . . . . . . . . . . . II. Summary of Maximum and Minimum Body Tempera- tures of Infected Pigs After Inoculation with Virus . . . . . . . . . L . . . . . . . III. Hemocytometer Leukocyte Counts of Infected and Control Pigs . . . . . . . . . . . . . . IV. Relationship of Hemocytometer Leukocyte Counts to Actual Numbers of Circulating Leukocytes in Infected Pigs. . . . . . . . . V. Hemoglobin Levels of Infected and Control Pigs . . . . . . . . . . . . . . . . VI. Food Consumption of Infected Pigs. . . . . . VII. Body Weights of Infected and Control Pigs. . VIII. Summary of Gross Necropsy Findings in 21 Infected Pigs . . . . . . . . . . . . . . IX. Summary of Microscopic Lymph Node Lesions in 21 Hog Cholera-Infected Pigs. . . . . . . page . 6 . 20 . 23 . 25 . 27 . 29 . 31 . 33 8c 34 . 57 10 11 12 13 14 15 16 LIST OF FIGURES Surgical and Transfer Isolators . . . . . Rearing IBOlatoro o o e e e e e e o e e 0 Mean Rectal Temperatures Following Inocuation With Virus. 0 e e e e o e e c Total Mean Circulating Leukocytes Following Inoculation with Virus. . . . . Total Mean Circulating Nucleated Erythrocytes Following Inoculation with Virus. Mean Hemoglobin values for Infected and Control Pigs. . . . . . . . . . . . . Mean Hematocrit values for Infected and Control Pigs. . . . . . . . . . . . . Mean Food Consumption of Infected Pigs Prior to Death . . . . . . . . . . . Time of Death of Infected Pigs Following Inoculation with Virus. . . . . . . . . . Variations in Kidney Hemorrhages Observed in Infected Pigs. . . . . . . . . . . . . Hemorrhage in Tonsillar Area. . . . . . . Hemorrhage, Edema, and Thrombi Underlying Tonsillar Area. . . . . . . . . . . . . . Endothelial Proliferation in Blood Vessel of Tongue. . . . . . . . . . . . . Proliferation of Endothelial Cells in Blood Vessel of Tongue . . . . . . . . Hyalinization of Blood Vessel Wall 1nT°n8ueeeoeoeeeoeeooeeo Pyknosis of Endothelial Cells of Blood Vesse1.in Tongue. , . . . . . . . . vii 21 21 24 24 28 28 32 32 38 38 38 38 40 no 2O 21 22 23 24 25 26 27 28 29 3O 31 32 33 34 35 36 37 38 Hemorrhage in Papilla of Tongue. . . . Hemorrhage and Edema of the Diaphragm. Perivascular and Intralobular Hemorrhageinxlungeoeeeeoeee Hemorrhage in Alveoli of Lungs . . . . Hemorrhage and Edema of Visceral Pleura. . Proteinaceous Fluid in Alveoli of Lung Interstitial Edema of Lung . . . .-. . Hemorrhage in Salivary Gland . . . . . Hemorrhage in Myocardium . . . . . . . Epicardial Hemorrhage. . . . . . . . . Subendocardial Hemorrhage Associated With Parkinje Fibers e e e e e o e e e Anitschkow Myocytes Associated with Myocardial Hemorrhage. . . . . . . . . Anitschkow Myocytes in Myocardium. . . Anitschkow Myocytes in Myocardium. . . Anitschkow Myocyte in Heart of Control Spleen of Control Pig. . . . . . . . . Spleen of Infected Pig . . . . . . . . Hemorrhages on Surface of Spleen . . . White Pulp of Spleen of Infected Pig . White Pulp of Spleen of Control Pig . Blood Vessel in Spleen Occluded with Endothelial Cells. . . . . . . . . . . Hyalinization of Wall of Blood Vessel in Spleen. O O 0 fl. 0 O O O O O O O O 0 viii 4O 40 43 43 43 43 45 45 45 45 48 48 as 48 51 51 51 51 54 52+ 54 54 Figure 39 4O 41 42 43 45 46 47 48 49 50 51 52 53 54 55 56 Vacuolar Degeneration of Blood Vessel inSpleen............-... Edema in Cortex of Lymph Node . . . . . . Lymph Node of Control Pig . . . . . . . . Lymph Node With Moderate Peripheral HemOrrhageoeeeeeoeeeeooee Lymph Node With Extensive Peripheral Hemorrhage............... Lymph Node With Moderate Diffuse Hemorrhage Lymph Node With Extensive Diffuse Hemomhageooceeeeeeeeeeee Swollen Endothelial Cells in Blood Vessel OfLymthOdeeoeeeeoeeeeeee Hyalinization of Wall of Blood Vessel in Cortex of Lymph Node . . . . . . . . . Hyalinization of Wall of Blood Vessel in Medulla of Lymph Node . . . . . . . . macrophages in Lymph Node Laden with Erythrocytes . . . . . . . . . . . . Subcapsular and Intratrabecular HemorrhagelnThymuS.....oo.... Hemorrhage and Edema of Pachymeninges . . Hemorrhage and Edema in Propria Mucosae OfUrinaryBladder........... Hemorrhages and Dilated Tubules in Cortex of Kidney . . . . . . . . . . . Hemorrhage in Medulla of Kidney . . . . . Hemorrhage and Proteinaceous Casts incortQXOfKidneyeeeeeeeeeee Hyaline Changes in Blood Vessel Wall and Degenerationeof Endothelial Cells. Cortex OfKidneyoeeeeoeeeoeeeeeee ix I? 56 56 56 56 59 59 59 59 61 61 61 61 64 64 64 64 68 68 58 59 6O 61 62 63 64 65 66 67 68 69 7O 71 72 73 74 75 Large Amounts of Glycogen in Liver of ContrOI P18 0 O O O O O O O O O O O O O O O O Hemopoietic Foci in Liver of Control Pig. . . Absence of Glycogen in Hepatic Cells OfInfeCtede-geeeeeeoeeeeeeee Fatty Changes Near Central Vein of Liver. . . Coagulation Necrosis Near Central Vein of Liver of Control Pig . . . . . . . . . . . . Coagulation Necrosis in Liver of Control Pig. Focus of Lymphocytes in Interlobular Araaolever................ Focus of Cells, Predominantly Lymphocytes, in Interlobular Area of Liver. . . . . . . . . . Paravascular Infiltration of Lymphocytes Near Intel'lObularvelneeeeoeeeeeeeoe Paravascular Infiltration of Lymphocytes in Liver . . . . . . . . . . . . . . . . . . Mass of Lymphocytes Protruding Into an Interlobular Vein of the Liver. . . . . . . . Paravascular Infiltration of Lymphocytes inlliveroeeeeeeeeeeeeeeeeee Lymphocytic Infiltration Around Interlobular Bile Ducts in Liver . . . . . . . . . . . . . Capsular Hemorrhage of Liver. . . . . . . . . Edema of Gallbladder Mucosa . . . . . . . . . Focal Necrosis of Gallbladder Mucosa. . . . . Necrosis of Mucosa of Esophagus . . . . . . . Degenerative Changes in Wall of Blood Vessel. Smeucosa Of Esophagus. e e e e e o e e e e o Necrosis of Mucosa of Stomach.. . . . . . . . X 68 68 7O 70 7O 70 73 73 73 73 75 75 75 75 77 77 77 77' 80 mm -76 77 78 79 80 81 82 83 84 85 86 Necrotic Area of Stomach Mucosa With Leukocytic Infiltration e e e e e o e Necrosis and Hemorrhage of Stomach Mucosa. Hemorrhage in Propria Mucosae of Small Intestine. o e e e e e e e e 0 e e o e e e Hemorrhage in Peyer's Patch of Small Inte Stine O O O O O O O C C O O O O O O C O Thrombus in Blood Vessel of Propria Mucosae of Small Intestine . . . . . . . . _Necrosis, Hemorrhage, and Congestion of Mucosa of Small Intestine. . . . . . . . . Hemorrhage, Edema and Thrombi in Submucosa of Cecum . . . . . . . . . . . . Hemorrhage in Submucosa of Cecum . . . . . Hemorrhage in Propria Mucosae of Cecum . . Hemorrhage in Fold of Submucosa of Colon . Thrombi in Submucosa of Colon. . . . . . . xi Page 80 80 80 83 83 83 83 85 85 85 85 I. INTRODUCTION Hog cholera has been present in the United States for at least 150 years (191. Today, "it is unquestionably the most important disease of swine in the United States".(477. Although suitable prophylactic measures have been avail- able since 1905 (12), control of any disease cannot be ac- complished simply by maintaining the economic losses within acceptable limits. Actual control can only be accomplished by eradication of the disease. Eradication in turn is de- pendent upon knowledge of the disease including accurate diagnosis. Hog cholera presents a confusing picture, from both clinical and pathologic standpoints. Its incubation period, duration, clinical signs, and lesions are quite variable. Concurrent infections and infestations with other micro- organisms and parasites produce further variations and in- crease the difficulty of correct diagnosis. The literature on hog cholera is full of references to "changes due to secondary invaders". The problem of diagnosis, then, be- comes one of detection of signs and lesions due to the hog cholera virus alone. Investigations into the effects of hog cholera virus on the pig have been limited to animals living in an environ- ment abounding in microbial life. It is doubtful if truly "uncomplicated" hog cholera has existed. The clinical and 1 pathologic findings present in conventional hog cholera have been described and are summarized in several recent publi- cations (16,18,39,47). Adaptation of equipment and technics developed at the University of Notre Dame (49,50,51) to the rearing of farm animals in a microbe-free state provided a tool heretofore not available for basic studies in animal disease (41,53,54). With them, it is now possible to study the effects of a single disease entity on the individual animal. This report describes the results of a clinical and pathologic study of the effect of hog cholera virus on sus- ceptible pigs maintained in an otherwise germfree or mono- contaminated environment. "Germfree" refers to pigs reared in the absence of known microbial flora. "Monocontaminated" refers to pigs reared in the presence of one known microbial agent. Animals reared under germfree conditions or in the presence of known microbial flora have been called "gnotobiotes" (37). The purpose of this study was to produce truly un- complicated hog cholera in susceptible pigs, and to study the clinical signs and gross and microscopic lesions of the disease. It was hoped that this investigation would pro- vide the basis for a better understanding of the clinical signs and lesions of hog cholera and, in addition, provide the framework for further studies on clinically related conditions. II. —REVIEW OF LITERATURE The tremendous volume of literature which has accumulat- ed since 1900 concerning the various aspects of hog cholera makesa complete literature review beyond the scope of this dissertation. An attempt has been made, however, to review those publications pertinent to the substance of this study; i.e., the clinical signs and gross and microscopic lesions in hog cholera. A. Clinical Signs The recognition of hog cholera as a specific disease entity with characteristic signs and lesions was first recorded by Detmers and Law (9) in 1879. Salmon prepared a thorough report which was published in 1889 (40). Subse- quent reports (10,27,3l,36) established the classical signs present in conventional hog cholera. Recent summations of the clinical hog cholera picture (16,18,24,34,39,47,52) tend to place less emphasis on respiratory and intestinal signs attributing them chiefly to secondary bacterial invasion. The incubation period of the disease is from five to ten days following contact with contaminated material. The first signs observed are usually dullness, inactivity and anorexia. Inappetence is greatest near termination of the disease; animals may stagger and weave when walking. The disease is generally accompanied by diarrhea, though con- stipation may occur during the early stages. Sometimes 3 4 vomiting occurs. Body temperatures reach 106 F. to 107 F. with- in 2 to 6 days after infection. Convulsions and other signs of central nervous system involvement occur occasionally. In acute forms. death may occur in a few hours, often with few noticeable signs. In less severe attacks the affected ani- mals may live 5 to 15 days. Under natural conditions, the morbidity and mortality are close to 100%. If the course of the disease is prolonged more than 7 days. bacterial compli- cations may occur, consisting chiefly of pneumonia and ulcerative or necrotic enteritis (52). It was recognized in early investigations by Dinwiddie. and others, that leukopenia was a characteristic sign of hog cholera (11.27.31). This early and marked decline in cir- culating leukocytes was considered the most striking cellular change (4.7.26,29.30,45,48). The decline was very evident within 48 hours following contact with the virus (26.29.30. 45). Leukopenia usually preceded any temperature reaction, and reached a maximum 5 to 6 days after infection (7,26). Circulating lymphocytes, eosinophils and basophils were re- ported to decline and neutrophils to increase in some in- vestigations (26.39.45); other studies reported a neutrophil decrease (sometimes total) and a lymphocyte elevation (30); nonspecific variable changes were found by Dinwiddie to occur in differential leukocyte studies (11). A gradual decline in circulating erythrocytes has been a frequent observation with the appearance of a moderate number of nucleated erythrocytes in the blood stream (ll.26.29.30,451 B. 79522; Lesions Early studies on the gross lesions of hog cholera (10. 23.27.31.36) included the following as characteristic: erythema of the skin with purplish discoloration of the ven- tral portions of the body and areas of the head. Petechial hemorrhages were found on the serosal and mucosal surfaces of the stomach and intestines. with localized or diffuse necrosis of the mucosa. Discrete to numerous hemorrhages. varying in size from petechial to ecchymotic were found on and in the lungs. heart, liver, gallbladder. urinary bladder, pharynx. brain and skin. Hemorrhages in the kidneys were constantly observed, the cortex being more frequently and more exten- sively involved. Hemorrhagic infarcts were common in the spleen; peripheral hemorrhages were characteristic in the lymph nodes. A summation of the gross lesions found in 3 investigations is presented in Table 1. Recent summations by Dunne (l6), Runnells g; 9;. (39), and Smith and Jones (47) do not differ significantly from the foregoing. Co W M981 n Proescher and Seil (35).in.19l7. apparently made the first attempts to systematically record the microscopic lesions of hog cholera. Additional studies (2,8,10,13,14,15. 20,21.22,25.32.33) have provided supplementary information but the initial elucidation of the microscopic lesions of hog cholera are found in reports by Proescher (35.36) and Seifried (42.43.44). TABLE I. Summary of Reported Gross Lesions of HOg Cholera P ' n h'w Location of Lesion Hoskins 23 and Kernkamp Dunne Proesgher (36) (26) gt al. (13) Kidney 100 92.5 64.3 Urinary bladder 83.2 Lymph nodes 100 83.5 96.0 Spleen 34 60.2 27.4 Larynx 75 59.6 23.8 Lungs Hyperemia 44.9 Hemorrhage 2702 1507 Inflammation 40.4 58.3 Large Intestine Mucosal hemorrhage 24.6 10.7 Serosal hemorrhage 6.3 Inflammation 13.8 16.8 Button ulcers 20.0 Heart 29.0 13.1 Liver 6.2 10.7 Small Intestine Mucosal hemorrhage 20 9.3 1.2 Serosal hemorrhage 7.2 1.2 Inflammation 03 1505 Stomach Mucosal hemorrhage 40 8.7 8.3 Serosal hemorrhage 3.9 3.6 Inflammation 3.6 45.2 ‘The virus of hog cholera is thought to exert a direct ef- fect on the vascular system. the lesions resulting from degen- erative changes in the capillaries, precapillaries. arteries and veins (47). l. ‘Qigestiyg System In general, microscopic lesions have not been common in the liver. Occasionally infiltration of the interlobular 7 connective tissue with mononuclear cells has been seen. Congestion, cloudy swelling. and hemosiderosis were common. Focal necrosis of the mucosa of the gallbladder has been reported by Luedke and Dunne (32) as well as intranuclear inclusion bodies.(2)o The stomach and small intestine were generally free of microscopic lesions. though occasional serosal and mucosal hemorrhages were observed. The cecum and colon frequently had degenerative changes with accumulations of mononuclear cells around blood vessels. especially in the mucosa. Blood vessel lesions consisting of hyaline changes of the walls with endothelial proliferation and occlusion of lumina were commonly encountered. If extensive. this led to general necrosis of the mucosa, or. more often. accord- ing to Dunne 23,21. (14), focal necrosis resulting in so called "button ulcers". The latter was particularly true if the disease was of longer duration than average. 2. Respiratory §ystem Subpleural hemorrhages were commonly found in the lungs and increased numbers of mononuclear cells were re- ported in the alveolar walls and around blood vessels. vascular changes with thickened vessel walls and occluded lumina were present. 3. Genito-Urinary §y§tem Extensive changes have been reported in the kidneys of pigs with hog cholera. Subcapsular hemorrhages were 8 common, extending around uriniferous tubules and frequent- ly causing the tubules to be widely separated. Necrosis of tubular epithelium.was not uncommon. Extensive hemorrhage was most often present between the proximal and distal con- voluted tubules. Foci of blood frequently obliterated the renal tissue. The capillary endothelium was swollen. Capillaries of the glomeruli were often distended with erythrocytes, occluded with endothelial cells. or plugged with thrombi. The thrombi were reported as including rem- nants of ruptured capillaries. swollen endothelial cells and pyknotic nuclei. Necrotic endothelial cells were seen lining many capillaries. Perivascular infiltrations with macrophages and lymphocytes were seen. especially in more advanced stages of the disease. The infiltrating cells fre- quently showed degenerative changes. The urinary bladder. ureters and urethra had peri- vascular hemorrhages and accumulations of mononuclear cells. Frequently the hemorrhages extended deeply into the mucosa. 4. Cagdio-Vasculag System Frequently, the heart exhibited no microscopic changes. Occasionally subendocardial and subpericardial hemorrhages occurred; localized mononuclear cell infiltration and cloudy swelling were observed. 5 e LIZEEhatic S! Stem Infarcts of the spleen were frequent and arose from blood vessel lesions. namely endothelial degeneration and 9 hyalinization with resulting thrombosis. Focal necrosis of the spleen was widespread. In addition, lymphoid depletions of the splenic follicles. mononuclear cell in- filtration and hemorrhage with numerous phagocytized erythrocytes were frequently present. In all cases of hog cholera, various degrees of hemorrhage were present in one or more lymph nodes. Many times, all lymph nodes were grossly involved. Commonly these changes were peripheral, but medullary and diffuse hemorrhage was not infrequent. The blood vessels were found engorged, small vessels were thrombosed and phago- cytized erythrocytes were abundant. Infiltration of mono- nuclear cells was observed. Three groups of lesions in the lymph nodes have been described by Seifried (44): (l) swelling and hyperemia, (2) peripheral hemorrhage, and (3) dense, total hemorrhage. These 3 types of lesions have been found in varying degrees in most cases of hog cholera. 6. Nggzcus Sygtem The most striking microscopic lesion observed in the brain resulted from the accumulation of lymphocytes in the perivascular spaces around arteries and veins. Hemorrhages have been observed around blood vessels, especially in the cerebellum and spinal cord. Focal accumulation offimicrog- lial elements have been seen, but degenerative changes in the neurons were infrequent. IO 7. Musculo-Skeletal System Hemorrhages have been found in the muscles. especially the diaphragm. In acutely infected pigs. lesions have been reported by Dunne 23,21, (15) at the costochondral Junction of the ribs. These consisted of a mildly irregular epiphy- seal line with an adjacent narrow transverse band of hemorrhage e 8. nt en Intracytoplasmic inclusion bodies have been reported in epithelial cells of the conjunctiva (22). The skin was observed to be hyperemic with occasional hemorrhage near the hair follicles. Degenerative changes in blood vessels were also observed. 9. Summatigg 2: Microsgopig Lesions Vascular lesions were considered responsible in great part for the characteristic pathologic picture of hog cholera regardless of which organ or tissue was involved. These were endothelial proliferation, hyalinization and degener- ative changes in the vessel walls, and thrombosis. Hemor- rhage and necrosis were the results. Mononuclear and lymphocytic infiltration was not uncommon, especially peri- vascularly. The duration of the disease in general did not determine the type or severity of the lesions. Pathologic findings in the lymph nodes. kidneys and central nervous system were thought to be of special value in diagnosis. 11 Proescher and Hoffman (36) described hog cholera as an “acute, specific endangitis. combined with thrombo- angitis and endarteritis production". D. Current Status 2; Hog Cholera A great amount of research has been devoted to the various facets of hog cholera in the past half-century. The excellent work of past and recent investigators has helped to understand and to alleviate the effects of the disease, but the problems of hog cholera are not all solved, nor is the complete picture elucidated. Today, emphasis is being placed on eradication of the disease. Therefore, the possibility of reservoirs becomes important. In addition. attempts are being made to develop laboratory tests for simple identification of the disease. If eradication is to be accomplished, the effectiveness of the various vaccines becomes more critical. More study is needed on the chronicity of the disease. which has been re- cognized but its importance not ascertained. The effect of complications by bacterial and other organisms are under in- vestigation and as technics improve. "new" conditions are be- ing recognized which play a possible role in diagnosis. Thus the overall hog cholera problem is changing. Emphasis must be placed on the elucidation of problems which obstruct the final goal - eradication of hog cholera. III. MATERIALS AND METHODS A. Germfree Tgchnicg The development by Trexler (49.50.51) of equipment and procedures utilizing flexible plastic film has provided a practical and economical method of rearing animals in a germfree state. Adaptation of these technics by Waxler (53,54) and Schmidt (41) to rearing germfree pigs was utilized in this study. Female swine, 112 days pregnant and not vaccinated against hog cholera. were anesthetized by other or carbon dioxide and placed in lateral recumbency. A sterile surgical isolation unit (Fig. 1) was secured to the skin of the flank region by adhesive. Working through shoulder-length rubber gloves. the operators made an incision through the floor of the isolator and the abdominal wall, using cautery for the primary skin incision. The uterus was then brought through the incision and opened. The pigs were removed. the umbi- lical cords were ligated and severed, and the animals were passed into a sterile transportation isolator. They were then dried, weighed. and placed in individual stainless steel cages within a rearing isolator (Fig. 2). Autoclaved cows' milk with added vitamins and minerals (to compensate for those deficient in milk. or destroyed by heat and pressure) was fed every 3 hours from 8 A.M. to 11 P.M. daily. Initial- ly 60 ml. was fed each pig at each feeding. This was in- creased by 15 m1. every 5 days. Bacteriologic examination 12 13 Figure 1. Surgical and Transfer Isolators A. B. C. D. E. Air Supply and Filter Air Outlet Trap Surgical Isolator Table and Straps for Restraining Sow Transfer Isolator 14 Figure 2. Rearing Isolator F. G. H. I. Sterile Lock Supplies for Rearing, Bleeding and Culturing Rearing Cages Air Inlet and Outlet Equipment 15 of rectal swabs and waste material from the cages was made at selected intervals to determine the microbial status. Steri- lization of all equipment and supplies was by autoclave (250 F. for 30 minutes) or germicidal chemical (2% paracetic acid) (28.49.50.51). B. Experimental Procedures Twenty-one 14-day-old Yorkshire pigs (10 males. 11 fe- males) from 3 litters. obtained and raised under germfree con- ditions. were each inoculated intramuscularly with 1 ml. of standardized hog cholera virus“. Six uninoculated pigs (3 males. 3 females) in separate isolators served as controls. All pigs were free from known microbial flora when examined at 10 days of age. Rectal temperatures were taken twice daily both prior to inoculation and following administration of the virus. Similar- ly. total and differential leukocyte counts and hemoglobin and hematocrit values were determined (6). Pigs were Observed reg- ularly for signs of illness. and necrOpsies were performed ac- cording to routine procedures (38) as soon after death as possi- ble. The 6 control pigs were killed by barbiturate overdose between 10 and 15 days after inoculation of the exposed pigs. Room temperature was maintained at 75.F. to 80 F. Food consumption was determined daily for each pig. At the time of inoculation. daily food consumption was 540 ml. *750,ooo lethal doses of Station Virus 315 from the Hog Cholera Research Station. ARS. USDA. Ames. Iowa, obtained through the courtesy of Dr. J. F. Torrey. 16 per pig. This was increased to 630 ml. on the second day after inoculation. When anorexia appeared in inoculated pigs. the amount of milk not consumed was subtracted from the quantity fed and this adjusted amount was used in deter- mining the volume to be fed the control pigs. Body weights were determined at the time of death. Following necropsy, the following tissues from each pig were preserved in 10% neutral formalin: representative_ portions of the skin of the flank, back. sternum and thigh. the lip. cheek, and tongue (tip, middle. and posterior por- tion), and middle portions of the external ear. The follow- ing external lymph nodes were preserved: external inguinal, prefemoral, prescapular, parotid. mandibular and supra- pharyngeal. Both right and left tonsillar areas were pre- served as well as the parotid and mandibular salivary glands. Portions of the digestive system preserved included: 3 equi-distant portions of the esophagus, the esophageal, cardiac, fundic and pyloric portions of the stomach, the pyloric valve. 9 equi-distant levels of the small intestine. the ileocecal valve. the tip and body of the cecum, 8 equi- distant levels of the large intestine, and anal tissue. Pieces of all 4 major lobes of each liver were taken to- gether with the gallbladder. pancreas, thymus. thyroid, and 4 equi-distant levels of the spleen. Preservation was made of portions of the walls of all 4 chambers of the heart. a portion of the posterior aorta. the 17 epiglottis, trachea. diaphragm, and representative samples of all 7 lobes of the lungs. The pituitary, adrenals. testicles. spermatic cord. body of the uterus and 3 levels of the horns, oviducts. ovaries, body and neck of the urinary bladder. ureters. 3 levels of the pelvic urethra and 2 of the external urethra. prepuce, and portions of the anterior. middle and posterior parts of both kidneys were preserved. Internal lymph nodes taken included: mediastinal, gastric. splenic, hepatic. mesenteric. renal, cecal, colic and internal inguinal. Paraffin sections of each tissue were stained with hematoxylin and eosin according to routine laboratory pro- cedures (1).- Special stains were used- on selected tissues. The brain, spinal cord. bone marrow. ribs. and eye were preserved but are not included in this study because of time limitations. Bacteriologic examination of rectal swabs and waste material from the cages was made when pigs were 10 days of age and again at the time of death, samples being taken be- fore pigs were removed from isolators. Samples were streak- ed on blood agar plates which were then incubated both aero- bically and anaerobically at room temperature. 37 C. and 55 C. Thioglycollate medium.was also inoculated and incubated at these same temperatures. To detect the presence of the hog cholera virus in in- oculated pigs. fluid extracts of the spleen of 3 pigs were 18 frozen for subsequent inoculation into farm raised, hog cholera susceptible pigs. Six pigs were given the extracts only, and 6 pigs were given the extracts plus commercial anti-hog cholera serum. IV. RESULTS Ten cholera-infected and 4 control pigs were success- fully maintained free of any demonstrable bacteria through! out the experiment. Bacillus gubtilig was isolated from 11 infected and 2 control pigs. At no time were monocontam- inated and bacteria-free pigs found within the same isola- tion unit. When bacteria gained entrance to an isolator. all pigs in that unit became contaminated. Since differences in clinical signs. gross necropsy findings, and microscopic lesions between the monocontaminated and bacteria-free groups could not be detected. both.were evaluated together. A. Clinigal Signg A summary of maximum and minimum body temperatures is presented in Table II. Rectal temperature curves (Fig. 3) were diphasic. The first peak was reached at a mean time of 2.5 days (range. 2.0-3.5 days) and the second peak at a mean time of 6 days (range. 3.5-7.0 days). Near normal temper- atures were present between the peaks at a mean time of 4 days (range 3.0-5.0). The rectal temperatures at the first peak ranged from 104.8 F. to 107.0 F. (mean 106.0 F.) and temper- atures at the second peak ranged from 105.4 F. to 107.0 F. (mean, 106.3 F.). Following the second peak, the tempera- tures remained elevated and later considerable individual variation was recorded. Deaths occurred at various times after inoculation; the time of death did not cause variation 19 20 TABLE II. Summary of Maximum and Minimum.Body Temperatures of Infected Pigs After Inoculation with Virus Day of Peak 1 Day of Day of Peak 2 Fig First Temp. Low Valley Second Temp. No. High Temp. F. Temp. Temp._F. High Temp. F. 3 2.5 105.6 4.0 103.6 6.0 106.6 4 2.5 104.8 4.5 104.1 5.5 106.0 5 2.5 105.7 3.0 104.6 3.5 105.6 6 3.5 105.0 4.0 103.8 6.0 105.8 7 2.5 105.1 4.0 103.4 5.5 106.4 8 2.5 106.? 4.0 104.1 5.5 106.5 9 3.0 106.0 4.0 104.6 6.0 106.6 10 3.0 106.5 4.0 105.2 5.5 106.7 11 3.0 105.9 4.0 104.0 5.5 106.4 14 2.0 106.4 4.5 102.0 6.0 105.9 15 3.5 105.8 4.5 104.4 6.0 105.8 16 1.5 105.4 4.5 103.8 6.0 105.4 17 2.5 106.4 4.0 103.6 6.0 106.0 18 2.5 105.8 5.0 104.0 6.5 106.? 19 2.5 107.0 4.0 102.0 6.5 107.0 21 2.5 106.6 4.5 104.2 5.0 107.0 22 2.5 106.4 5.0 103.4 7.0 106.0 23 2.5 105.8 t.0 102.0 25 2.5 106.8 3.5 102.8 26 2.0 106.4 4.0 104.0 6.0 106.6 28 2.0 106.4 4.0 - 104.2 Mean 2.5 106.0 4.1 103.7 6.1 106.3 mg. 200-3e5 104.8-107.0 3.0-5.0 102.0-105.2 3e5-7e0 105e4‘10710 21 1 Cholera p188 — ) 1 6 Control pigs -I-I- O 105 l I l "a” 0: I 104 103 - I 102 7“.|‘ FAHRENHE IT TEMPE RA TURE 101 -3 -2 -1 0 1 2 3 u 5 6 7 8 9 10 11 12 . 1 DAYS POST-INOCULATION Figure 3. Mean Rectal Temperatures Following Inoculation With Virus. (Area between dotted lines represents normal range.) ‘—|-I-I-|-.-.-.-l-"|.l~.-." III-III' “gum“""'uo.....ufl“”“‘“""‘ é.“ wtuocvrousns § ‘3 LEUCOCYTES THOUSANDS/mm3 U1 IDJUS'I’ED Cholera pigs — Control pigs -l-I- -3 -2 —1 o 1 2 3 u 5 6 7 8 9 10 11 12 DAYS POST-INOCULATION u..— Figure 4. Total Mean Circulating Leukocytes Following Inoc- __..__~_—. . _ifi__. _— ulation With Virus. (Dotted line representing hemocytcmeter counts includes nucleated erythrocytes. Solid line repre- sents actual numbers of leukocytes.) 22 in the diphasic temperature curve, except for the fact that a subnormal temperature frequently developed a few hours be- fore death. A summary of total leukocyte numbers following inocula- tion with virulent hog cholera virus is presented in Table III. Total circulating leukocyte numbers (Fig. 4) were decreased to nearly one-third the preinoculation levels (mean. 7700/cmm.) within 24 hours. A mean low of 2347 cells was recorded 2‘ days after inoculation. Hemocytometer counts of leukocytes indicated a gradual rise to approximately two-thirds the pre- inoculation levels by the seventh day. However, subsequent examination of blood smears revealed this increase to be al- most entirely due to nucleated erythrocytes which appeared in large numbers by the fifth day after inoculation (Fig. 5). The nuclei of the red blood cells were not destroyed with the mature erythrocytes in the leukocyte counting procedure. Since these nucleated cells could not be distinguished from leukocytes in the hemocytcmeter count. their number was in- advertently included in the total leukocyte count. Adjust- ments of total leukocyte counts is presented in Table IV. At no time did total leukocyte counts change to indicate im- pending death of the infected pigs. Differential leukocyte counts remained constant in both control and cholera infected pigs. Lymphocytes varied from a mean of 62.1 to 65.3% and neutrophils ranged from a mean of 33.0 to 37.1%. Monocytes and eosinophils each represented 1% or less of the total differential leukocyte count and baso- phils represented one half per cent or less of the tetal count. 23 TABLE III. Hemocytometer Leukocyte Counts of Infected and Control Pigs (cells/cmm.) Pig De ter n culation No. 0 2 3 5 5 :7 Control 10550 8100 9100 8350 8800 8250 7950 7800 2 6600 7150 7750 9300 6250 9600 7850 6350 12 11250 10150 11650 10150 045? 10000 9950 11250 20 5450 6550 6950 7150 83;: 0900 7150 ' 6950 24 5300 7750 6950 7000 disc 7550 7700 7850 27 7200 7300 7750 8050 7311 7900 8650 8500 Infegjgg , 7400 2400 1950 2100 2;:: 3;“: 2100 4850 4 7500 2900 2200 2250 L450 23:: 5 7550 2500 2250 3950 5300 3100 1950 6400 6 6900 4400 3300 2000 1650 2000 1600 4500 7 3300 3400 1350 3450 2950 2900 2000 3250 8 8150 3100 2150 3950 4200 3600 5750 9 9000 2950 2100 3300 3150 4200 4500 7100 10 6750 3000 1950 4000 3350 5500 7050 6550 11 7050 2450 2200 5850 5150 5550 7550 9000 14 8550 2150 1900 2700 4250 4650 7700 15 6750 1950 3450 2850 4200 3550 5400 5750 16 7050 2100 1700 2150 3050 3400 3950 4000 17 9050 1550 2750 3700 1800 3650 4200 4000 18 7850 2500 1800 2650 2450 2900 4550 5000 19 7700 2750 2900 4400 2750 4650 7550 21 7950 7100 1450 3150 1550 3400 3950 6200 22 8100 800 1900 1550 3750 3600 3450 23 7750 1500 1750 4250 25 6900 3200 1900 2900 26 6950 2850 2550 2850 1500 3800 5500 6250 28 8050 1600 5900 3700 2350 24 100 r!) g 80 o 8 E 60 8 H 40 Cholera pigs— F1 Control pigs-I-e- d 20 E E. 0 I-I-l-I-l-I-l-I-I-I‘I-I-l-I-I-I-l-I-l-I-Il -3 -2 -l O 1 2 3 l; 5 6 7 8 9 10 11 12 DAYS POST-INOCULATION Figure 5. Total Mean Circulating Nucleated Erythrocytes Following Inoculation With Virus (cells/100 leukocytes). 7 12 "‘. I-I-|-'-'-'-l-I-l-I-I-'-'-'-'l-l-l-l-|-.‘ 10 D 0 § 8 \ / ChOlera p183 _ Control pigs -l-I- Genus/loo m1. 4:- DAYS P0 51‘ - IIIO C ULA TION Wm-——-——w———~~— 7‘“ - ———- . ‘ ----—-q_-—‘—-4-> ——_.." ‘— — “—T Figure 6. Mean Hemoglobin Values for Infected and Control Pigs. 25 RBC - erythrocytes ** WBC - leukocytes TABLE IV. Relationship of Hemocytometer Leukocyte Counts to 0 Actual Numbers of Circulating Leukocytes in Infected Pigs . (cells/cm.) , % Nucleated Mean Hemo- Mean No. RBC in Hemo- Corrected Mean Days After cytometer Nucleated cytometer Mean “30*” Control Inoculation Counts RBc*/100 wsc Counts Cow mac -3 7733 7733 8283 0 7679 7679 7725 l 2721 2721 7833 2 2383 1.7 1.5 2347 8358 3 3050 2.0 2.0 2989 8333 4 3055 2.6 2.1 2990 8325 5 3500 17.1 14.6 2989 8200 6 4471 50.1 33.4 2978 8208 7 5590 87.6 46.7 2979 8117 8 5644 88.8 47.0 2991 8492 9 5472 86.8 46.5 2928 7842 10 5721 83.1 45.4 3124 8392 11 5700 89.0 47.1 3015 8863 12 5825 82.0 45.1 3198 8900 13 5900 85.0 45.9 3192 9025 11. 6150 84.0 45.6 2301. .9500 ... * e-l_ 26 A summary of hemoglobin levels following inoculation with virulent virus is presented in Table V. Both hemoglobin and hematocrit determinations (Figs. 6 and 7) were closely parallel. A decrease in both occurred within 24 hours after inoculation with the virus. This decrease was temporarily stabilized until the sixth day when a second decline reduced levels to approximately 40% of their original values. Al- though circulating nucleated erythrocytes reached their peak on the seventh day after inoculation, they did not prevent this second decline. Food consumption decreased several days before death, with the greatest inappetence occurring 24 hours prior to death. Decreased vigor and interest in consuming milk were observed 2 to 5 days after inoculation with the virus. Table VI summarizes food consumption prior to death of in- fected pigs. The time required for complete food consump- tion increased from 2 to 3 minutes to nearly 3 hours. The anticipated food consumption (Fig. 8) was based on the usual increase in consumption encountered with previous groups of pigs raised under similar conditions. Diarrhea occurred 3 to 9 days after inoculation; the character of the feces changed from a dark, tarry consistency initially to a very fluid state just before death. Pigs dy- ing early in the experiment did not develop as severe a diarrhea as did those living a longer period of time. It was difficult to detect the exact onset of securing since the germfree control pigs had semisolid to fluid stools starting at about 1 week of age. (mg./100 m1.) t o Afte Inoc 27 Da Hemoglobin levels of infected and control pigs. TABLE V. Pig NO. 915 nun 579 e O O mum 065 5 Cal“ um... 005 O O O nun 688 uo.n 865 O O O mun 560. 111111 55.7. nun 915 e O O mun 0/314 0 e mum Control 1% 11.5 11.9 10.9 12.0 11.6 11.5 0 5 7 2 11 0 12 6 11 1 2 1 11.7 11.6 11.4 1 10.9 11.5 10.9 10.5 11.2 11.4 1 . 11.2 10.9 12.0 11.5 11.3 10.9 1 . 10.9 10.8 11.5 20 2h 27 7.6 8.0 8.1 Infected ‘122 O O . #55 658 see 655 571.. F’s/Din” 020 e e e 888 016 e e 10 11 15 16 17 18 98 14!.» nu.u,o . O O 555 ockeo, O O O ,o,o,o oznvne O O 0 stage: 1.0,nv . O 0 9.0.9. n.131. O O . n.nenv 051 C O . 888 see 888 16L 0 e e 9901 10. 12. 11. 19 6.5 6.8 5.1 5.5 6.5 11.6 11.7 11.h 11.7 6.7 8.1 8.0 11.3 8.0 8.0 11.3 8.2 11.5 11.3 8.h 9.2 11.1 11.3 11.1 26 28 25 Infected lean Control Mean 28 he 38 ’|-I-I-l-I—.-l-l-I- 314 I-I-u-g-.-'_.‘.-I.."p‘ 5:1 30 S A g 26 a o 22 Q E 18 ES Cholera pigs— 1’4 Control pigs -I-I- 10 - 3 «2 -1 O l 2 3 h S 6 7 8 9 10 11 12 DAYS POST- INOCUIA'I‘ION i- i, *— L— _ __ _—' Figure 7. Mean Hematocrit Values for Infected and Control Pigs. 800 700 "‘ ’. 6 ‘—I-I-l-l-I-|" 00 7 ." 13 500 3 [mo E 5‘. 300 200 Anticipated II-I- 100 Actual _ 8 7 6 5 h 3 '2 1 L_ IMYSIEFOEEIHMIH Figure8. Mean Food Consumption of Infected Pigs Prior to Death. 29 TABLE VI. Food Consumption of Infected Pigs (ml.) Days Prior to Death P18 No. 7 6 5 4 3 2 1 W548 mm 286 4 555 630 561 400 200 5 ' 555 630 615 450 575 265 155 6 630 650 620 395 575 445 185 7 555' 650 496 455 380 380 8 555 630 500 495 465 200 ' 9 650 525 520 555 545 470 250 10 455 530 630 630 630 500 230 11 ‘ 630 630 650 580 540 450 210 14 555 555 445 ' 500 260 15 #30' 540 510 #70 430 440 210 16 630 510 510 550 490 440 300 17 630 630 465 440 410 400 195 18 495 465 425 ' 350 , 500 210 150 19 465 550 600 410 400 550 210 21 530 510 480 480 550 490 310 22 630 550 540 400 350 540 210 23 480 540 540 540 570 630 330 25 540 540 540 540 . 570 610 '555 26 410 400 385 350 390 200 150 28 570 650 65c _ 475 £525 550 551 549 492 484 457 249 Ant 8 M an 555 630 630 630 650 645 720 3O Inactivity, depression, and weakness increased as the disease progressed. Signs of central nervous system involve- ment, starting 1 to 3 days prior to death, were observed in 5 pigs. These signs consisted of muscular spasms with twitching of the head and limbs, paddling movements of the legs, and hyperexcitability or depression. Posterior paresis was pre- sent in nearly all of those pigs surviving 9 days or longer. Initial and final body weights are summarized in Table VII. Mean birth.weights were 2.76 pounds for control pigs and 2.71 pounds for cholera-infected pigs. Mean death weights were 5.83 pounds and 4.53 pounds, respectively. This indicated a net weight gain of 40% less for the cholera-infected pigs. Splenic extracts from 3 infected pigs, inoculated into 6 farm-raised, hog cholera-susceptible pigs, produced death with temperatures, blood counts, and gross lesions typical of those seen in hog cholera. Six hog cholera-susceptible littermates, given commercial anti-hog cholera serum simultaneously with the splenic extract, developed a transitory leukopenia but otherwise remained normal. Bo .LLGOSW All 21 pigs died within 4 to 15 days after virus inocula- tion (Fig. 9), with 76% of the deaths occurring from 6 to 12 days after inoculation. Necropsies were performed as soon after death as possible. Consistent gross necropsy findings (Table VIII) included petechial and ecchymotic hemorrhages in the cortex and medulla of the kidney (Fig. 10). Hemorrhages were 31 Body Weights of Infected and Control Pigs (1b.) TABLE VII. Death Birth Pig No. Control Gain Weight Wei ht 889 o o o 555 878 O O O 222 1 2 12 200 o o 960 o o o 556 760 o o o 223 20 24 27 Infe ted 981 4:414 199 322 3 4 5 861:4 .0. 141:4 856 000 222 990 113 250 8:4,: 36nvo 223 10 11 9:826 O C O 1:414 hwh;o 222 14 15 16 1.8 1.7 1.3 .4 52 8:414 ’00nt 222 17 18 19 1.9 1.3 1.9 02h. 5:41: 21 22 23 1.8 1.9 2.1 195 0.. 144:4 3nvol4 232 25 26 28 Control Mean 3.06 5.83 2.76 Infected Mean 1.82 4.53 2.71 32 no'. DEATHS 1 3 5 7 9 11 13 15 17 DAYS POST-INOCULATION —_.'— 7 Figure 9. Time of Death of Infected Pigs Following Inoculation with Virus. ., J . (1 m (D 2 q 1,, v“ 4 - ‘ f f m I. \ 5" N f" "‘r ~t § I f F: F d’ S: 5;.vgv: P 1 _— — _....— ___.7 - __.__._ _ ___‘_._ ..__—__——.H__u Figure 10. Variations in Kidney Hemorrhages Observed in Infected Pigs. 33 TABLE VIII. Summary of Gross Necropsy Findings in 21 Infected Pigs Lesion Number Showing Lesion Kidney Hemorrhages Cortex Medulla Hemorrhagic Lymph Nodes External Inguinal Prefemoral Prescapular Parotid Mandibular Suprapharyngeal Mediastinal Gastric Splenic Hepatic Mesenteric Renal Cecal Colic Internal Inguinal Petechial Hemorrhages of Urinary Bladder (Musoca) Body Neck Petechial Hemorrhages of Small Intestine (Mucosa) Duodenum Jejunum Ileum Petechial Hemorrhages of Small Intestine (Serosa) Duodenum Jejunum Ileum Petechial Hemorrhages of Colon (Mucosa) Petechial Hemorrhages of Colon (Serosa) 21 21 .17 21 21 12 15 21 21 18 8 9 15 10 18 21 15 19 21 15 15 O\ (I) (INDCDCD 34 TABLE VII. (Continued) Lesion Number Showing Lesion Hemorrhages on Lungs Apical Lobe Cardiac Lobe Diaphragmatic Lobe Intermediate Lobe Hemorrhages on Meninges Hemorrhages on Diaphragm Hemorrhages on Heart Ventricle Atrium Infarcts in Spleen Petechial Hemorrhages on Parietal Pleura Petechial Hemorrhages in Thymus I-" H N) U NUTU'I U1 0\ QNHQQ Gastric Serosal Hemorrhage 35 consistently found in the lymph nodes, either at the peri- phery or throughout the tissue. Petechiae were found less commonly in the mucosa of the urinary bladder and the mucosa and serosa of the intestine. Gross hemorrhages were also seen in the lungs, meninges, diaphragm, heart, parietal pleura, thymus, and stomach; but the appearance of these lesions was" inconsistent from one animal to another. Gross hemorrhages on the epiglottis and larynx, ulcers of the intestine, hyperemia and discoloration of the skin, and prepucial ulcers were not observed. The severity of the lesions was not dependent on the length of survival time. Renal hemorrhages amounting almost to suffusions were present in l pig dying 5 days after inoculation while 2 pigs dying 11 and 12 days, respectively, after inoculation had only a few petechial hemorrhages in the kidneys. C. MicroscOpic Lesions Histologically, the lesions found in hog cholera in- fected pigs were congestion, edema, and hemorrhage, ac- companied by thrombosis and necrosis, and degenerative changes in blood vessel walls. 1- 9.1181129122822292 MicroscOpic lesions were not observed in the skin, lip, cheek, or ear of hog cholera-infected pigs. A few areas of congestion were present in the skin of 3 infected pigs. 2. Tonsils Though the lymphatic tissue of germfree pigs proved to be considerably less well deve10ped than that in 36 conventionally reared pigs (4l,55,54), the tonsils were ap- parently better developed than other lymphatic structures. Various degrees of hemorrhage and thrombosis of under- lying vessels were found in the tonsillar areas in 15 of 2l infected pigs. These lesions varied from slight, focal hemorrhage within the lymphatic tissue to extensive hemor- rhage, necrosis, and thrombosis of vessels with total to partial occlusion (Figs. ll and 12). Hemorrhages were ob- served also in the musculature underlying the tonsillar areas. Suggestions of endothelial proliferation, endothelial pyknosis, and degenerative alterations in blood vessel walls were observed, but comparison with tonsils from control pigs yielded no definitive conclusions. 3. Maxim Numerous changes in the blood vessels of the tongue were observed in all infected pigs. The muscular elements and other structures were apparently normal. Endothelial proliferation (Figs. 13 and 14) and hyaline changes (Fig. 15) were seen in blood vessels. Pyknosis of endothelial cells (Fig. 16) was found. Conges- tion and hemorrhage were associated with these changes (Fig. 17). Most of these degenerative vascular changes and the accompanying hemorrhage were in the mid portion or base of the tongue. Lesions were not observed in the anterior third of the tongue. Hemorrhages and edema of the diaphragm were found in 12 of 21 infected pigs (Fig. 18). The hemorrhages were sub- 37 Figure 11. Hemorrhage in Tonsillar Area. Hematoxylin and Eosin. x 50 J. Epithelium K. Erythrocytes L. Lymphocytes Figure 12. Hemorrhage, Edema and Thrombi Underlying Tonsillar Area. Hematoxylin and Eosin. x 105 M. Thrombus N. Erythrocytes O. Edema Figure 15. Endothelial Proliferation in Blood Vessel of Tongue. Hematoxylin and Eosin. x 540 Figure 14. Proliferation of Endothelial Cells in Blood Vessel of Tongue. Hematoxylin and Eosin. x 540 38 Figure-ll Figure 14 Figure 13 39 Figure 15. Hyalinization of Blood Vessel Wall in Tongue. Hematoxylin and Eosin. x 540 P. Hyalin Material Q. Tongue Muscle Figure 16. Pyknosis of Endothelial Cells of Blood Vessel in Tongue. Hematoxylin and Eosin. x 540 Figure 17. Hemorrhage in Papilla of Tongue. Hematoxylin and Eosin. x 196 R. Stratified Squamous Epithelium S. Erythrocytes T. Propria Mucosae Figure 18. Hemorrhage and Edema of the Diaphragm. Hematoxylin and Eosin. x 28 U. Serosa V. Erythrocytes W. Muscle 41 serosal and generally on one side only. It could not be determined if this was the thoracic or abdominal sides, or both. Occasionally hemorrhages were found within the mus- culature. Suggestions of blood vessel degeneration were observed. 4. Epiglottis, Trgghea gngflggngg No lesions were observed in or on the epiglottis or. trachea of any infected pig. The surrounding structures were commonly congested, but no other lesions were noted. Congestion of blood vessels was common in most lungs from infected pigs. Frequent and extensive hemorrhages were found in 10 of 21 lungs; smaller hemorrhages were found in lungs from another 5 infected pigs. The apical and inter- mediate lobes appeared to be more commonly involved. Hemorrhages in the lungs were commonly found associat- ed with blood vessels and infiltrating the interlobular con- nective tissue (Fig. 19). Free blood in the alveoli was not uncommon (Fig. 20). Pleural and subpleural hemorrhages were also observed (Fig. 21). Edema of the lung with numerous alveoli filled with proteinaceous exudate was found in the lungs of 12 of 21 infected pigs (Fig. 22). Occasionally interlobular edema was found (Fig. 23). 5. Saliyary Glands 3nd Pancreas Comparison of salivary glands of hog cholera infected and control pigs did not reveal essential differences. It appeared that more glands were collapsed (especially mucous glands) in infected pigs than in non-infected control animals, but it could not be determined if this was 42 Figure 19. Perivascular and Interlobular Hemorrhage in Lung. Hematoxylin and Eosin. x 32 X. Artery Y. Erythrocytes Z. Interlobular Area . A. Alveoli Figure 20. Hemorrhage in Alveoli of Lungs. Hematoxylin and Eosin. x 224 B. Erythrocytes C. Alveolus Figure 21. Hemorrhage and Edema of Visceral Pleura. Hematoxylin and Eosin.. x 105 D. Erythrocytes 8., Edema F. Alveoli Figure 22. Proteinaceous Fluid in Alveoli of Lung. Hematoxylin and Eosin._ x 140 1 .N ‘ U'J' ‘ Y 7 -’ ‘ If I" -. a E , 4:! \"Q'. . , .. -. 3". " - 3' I"? "K " '. it"? °“ "1 (I I y . ’ J"5 b o ‘\ “ v - 3.9" v “. (7 -. "x V' 3 ‘1 .. A}? r. .1 '5 .4 ' 4‘ u .' '1 \‘ . 1.. ‘ ‘ I , f [q . 'v ' "I," .‘5 ', (0 ‘ 51.1.“. e ' V 4 F A- '0 Ju w. t . z. “‘2‘ 3m. ‘ J_ ‘ .._. 'l'r ‘-. ‘ ‘\ ' ,1, 9., _ V H ‘ uh: ‘3. \ ' . d'.‘ ‘_ - 1 ' 3 a) '- .a -‘ C e‘\' g T’.‘ ; C" ’ ‘ -Y -‘ a ‘f - l’.- . a a \ 1"“ - v- 1 '. ‘- J‘ '7‘ .1 ' ‘ I , - '- ' s. '5 h It 7.". x .7 .. ‘ I , .- “ e' 31's.: 7 \ 1" ~tz>}- w" 7‘53" 8 a . *’ , ' ' ‘ W‘- h ‘ v ..'i ‘\ . k, . I‘ ‘ . 4 A - W: f" ‘ " -.‘ , )» Figure 21 “5"“ ( Figure 22 44 Figure 23. Interstitial Edema of Lung. Hematoxylin and Eosin. x 105 G. Alveolus ' H. Interstitial Edema Figure 24. Hemorrhage in Salivary Gland. Hematoxylin and Eosin. x 140 I. Mucous Gland J. Erythrocytes K. Duct Figure 25. Hemorrhage in Myocardium. Hematoxylin and Eosin. x 140 Figure 26. Epicardial Hemorrhage. Hematoxylin and Eosin. x 140 L. Erythrocytes M. Myocardium. Figure 25 - 81.88.26 46 absolute. An occasional small hemorrhage was observed in the salivary glands of 6 infected pigs (Fig. 24), and con- gestion was not uncommon.‘ ‘ Suggestions of hyaline changes in blood vessels of the pancreas were present; no other microscopic lesions were observed in the pancreas of infected pigs. 6. MMM . Histological alterations could not be detected in the aorta of infected pigs. Thrombi were seen in smaller ves- sels near the aorta. Hemorrhages were common both.within the musculature of the heart and subepioardially in 16 of 21 infected pigs (Figs. 25 and 26). Subendocardial hemorrhages were found in only 3 pigs. These were commonly in the same areas as Purkinje fibers (Fig. 27). The hemorrhages were more common and more extensive in the ventricles; attempts to trace hemorrhages to blood vessel lesions were unsuccess- ful. Endothelial proliferation in blood vessels or degen- erative changes in blood vessel walls were not observed in the hearts of infected pigs. An outstanding feature of the histopathologic lesions in the heart, in addition to hemorrhages, was the great in- crease in numbers of Anitschkow myocytes (Fig. 28). Not only were their numbers increased, but the size of the individual nuclei was considerably greater. Increased amounts of nucleoplasm with very distinct prominent central bars of chromatin and accompanying strands radiating to the 47 Figure 27. Subendocardial Hemorrhage Associated with Purkinje Fibers. Hematoxylin and Eosin. x 140 .N. Myocardium O. Purkinje Fibers P. Erythrocytes. Q. Endocardium Figure 28. Anitschkow Myocytes Associated With Myocardial Hemorrhage. ' Hematoxylin and Eosin. x 315 R. Anitschkow Myocyte Nuclei s. Myocardial Cell Nucleus T. Erythrocytes. Figure 29. Anitschkow Myocytes in Myocardium. Hematoxyli. and Eosin. x 1200 Figure 30. Anitschkow Myocytes in Myocardium. Hematoxylin and Eosin. x 1200 U. Anitschkow Myocyte Nucleus V. Myocardial Cell Nucleus :9 k \ I. \ . C ‘ \ \ (. o C " \ a ‘ \ ‘. I. ‘ '0 ‘ . . 0.. . q. I" e- ’1. .3 ‘ '. \ . .“ I ~ ‘s ‘ '- o \ “s . ‘ . \ R C- r A\ a | e \‘ \ $ \. . ‘ \ \ \.. a .3 A Q .\ \ Figure 29 48 , '9. ‘E Figure 28 Figure 30 ‘___ _ ___ 49 nuclear membrane were found in numerous Anitschkow myocytes of infected pigs (Figs. 29 and 30). These cells were found in the hearts of control pigs, but they were not nearly so numerous or so prominent nor did they show the large amounts of nucleoplasm and the distinct central bars (Fig. 31).’ The Anitschkow myocytes were found most commonly in the ventricles, especially in the basal and middle portions. Few were found in the apex. The atria were nearly devoid of these cells. .Frequently the AnitschkOw myocytes were assoc- iated with the intramuscular focal hemorrhages. Myocytes were commonly found without adjacent hemorrhage, but few hemorrhages were found without adjacent Anitschkow myocytes. 7. mmmm Hemopoietic areas were commonly observed in the spleens 'of control pigs. These foci were absent or their numbers greatly reduced in infected pigs. Increased numbers of (erythrocytes were found in the cortex of the spleens of in- fected pigs; uniform lymphocytic depletion was common in in- fected pigs (Figs. 32 and 33); splenic corpuscles were not distinct in all spleens. Hemorrhages were present on the surface of the spleens, of infected pigs with accumulations of erythrocytes fre- quently distending the capsules (Fig. 34). Some of these foci appeared to be intracapsular. Definite infarctions were not found histologically, but were observed in 3 of 21 in- fected pigs on gross examination. 50 Figure 31. Anitschkow Myocyte in Heart of Control Pig. Hematoxylin and Eosin. x 1200 W. Myocardial Cell Nucleus X. Anitschkow Myocyte Nucleus Figure 32. Spleen of Control Pig. Hematoxylin and Eosin. x 42 Y. Trabecula Z. White Pulp Figure 33. Spleen of Infected Pig. Hematoxylin and Eosin. x 34 A. Trabecula B. White Pulp C. Capsule Figure 34. Hemorrhages on Surface of Spleen. Hematoxylin and Eosin. x 140 D. Erythrocytes E. Parenchyma “ 51 in n a " ‘. 52 The white pulp of the spleen of infected pigs had numerous pyknotic and degenerating lymphocytes (Fig. 35). These were quite evident when compared with spleens of control pigs (Fig. 36). Changes in blood vessels were observed in spleens of all infected pigs. These consisted of endothelial pro- liferation and swelling (Fig. 37). hyaline changes (Fig. 38% and vacuolar degeneration of the subendothelial and medial layers of blood vessel walls (Fig. 39). A summary of lesions found in lymph nodes of in- fected pigs is presented in Table IX. Edema was consistent in lymph nodes of infected pigs (Fig. 40). Congestion was common but not consistent. Hemorrhages were present in nearly all lymph nodes of all infected pigs. For conven- ience, these hemorrhages were arbitrarily classified as (1) moderate peripheral, (2) extensive peripheral, (3) moderate diffuse, and (4) extensive diffuse. Examples of these are shown in Figs. 41, 42, 43, 44 and 45. Congestion was of similar occurrence in both internal and external lymph nodes of infected pigs. Edema tended to be more common in external nodes; hemorrhages were more extensive in internal nodes. Lesions were found in the blood vessels of most lymph nodes of all infected pigs. These consisted of endothelial proliferation, endothelial swelling (Fig. 46), hyaliniza- tion of blood vessel walls (Figs. 47 and 48), and thrombosis of some smaller blood vessels. All blood vessel lesions Figure 35. IWhite Pulp of Spleen of Infected Pig. Hematoxylin and Eosin. x 540 . F. Central Artery G. Pyknotic Lymphocytes Figure 36. White Pulp of Spleen of Control Pig. Hematoxylin and Eosin. x 540 H. Central Artery I. Lymphocytes Figure 37. Blood Vessel in Spleen Occluded with Endothelial Cells. Hematoxylin and Eosin. x 540 J. Endothelial Cells K. Lymphocytic Cells Figure 38. Hyalinization of Wall of Blood Vessel in Spleen. Hematoxylin and Eosin. x 540 L. Hyaline Material M. Immature Lymphocytes ; v» -' fl )‘ l.’ t ' - ’ I _ -,_ __.__‘. U '5‘ _-‘,M . L . . 55 Figure 39. Vacuolar Degeneration of Blood ‘Vessel in Spleen. Hematoxylin and Eosin. x 315 N. Lumen 0. Vacuoles P. Smooth Muscle Figure 40. Edema in Cortex of Lymph Node. Hematoxylin and Eosin. x 140 Q0 Stroma . R. Macrophage with Engulfed Erythrocytes S. Edema Figure 41. Lymph Node of Control Pig. Hematoxylin and Eosin. x 32 Figure 42. Lymph Node with Moderate Peripheral Hemorrhage. Hematoxylin and Eosin. x 32 T. Erythrocytes _ U. Lymphocytic Cells Figure 4C Figure_39 L 2 .4 o e .. 5...? 8 s”; mT.- w. Us“... .s... Figure 41 ATS: in... .. . 1 Lr\b\.(yn\ m9... . d .3... 57 TABLE IX. Summary of Microscopic Lymph Node Lesions in 21 Infected Pigs. (Number of pigs showing each lesion.) wExtent of * Hemorrha e Node Edema Co estion l 3 4 Egternal ‘ External Inguinal 16 6 l2 4 l 4 Prefemoral l6 4 8 4 l 2 Prescapular 12 2 6 6 6 Parotid 14 10 9 7 4 l Mandibular 18 l2 l7 4 Suprapharyngeal‘ 18 ll 12 4 l 1 Internal ‘ Mediastinal 7 4 l l 2 4 Gastric 4 3 2 2 5 Splenic 6 4 l 2 7 5 Hepatic 7 5 l 2 4 5 Mesenteric 9 6 4 3 4 7 Renal 10 1 4 4 6 7 Cecal 5 3 3 3 4 5 Colic 4 l 2 7 4 6 Internal Inguinal 6 4 5 2 7 7 *Extent of Hemorrhage l - Moderate Peripheral 2 - Extensive Peripheral 3 - Moderate Diffuse 4 - Extensive Diffuse 58 Figure 43. Lymph Node with Extensive Peri- pheral Hemorrhage. I Hematoxylin and Eosin. x 32 V. Erythrocytes W. Lymphocytic Cells Figure 44. Lymph Node with Moderate Diffuse Hemorrhage. Hematoxylin and Eosin. x 32 X. Erythrocytes Y. Lymphocytic Cells 'Figure 45. Lymph Node with Extensive Diffuse Hemorrhage. Hematoxylin and Eosin. x 32 Z. Erythrocytes A. Lymphocytic Cells Figure 46. Swollen Endothelial Cells in Blood Vessel of Lymph Node. Hematoxylin and Eosin. x 1200 B. Lumen c. Endothelial Cells 59 . 'T'" ‘mj’ '- ‘ _' '.v"“ " ‘ - .‘ «Jivm‘;‘ ';" 5’ ,2: him-Y . use? ' 7" f.’1~“'~- . . 4 . ‘ -‘ .. a." ‘ \QLF'ff*?¥m»w!£fl.fiu IRmenvetr~ e l - -' -' « . ‘ ' $5.3 ‘ 31.11531“ 3")”- B r‘ 'l' 3“ * ‘ ' ‘0 $5 "‘9‘; .O'D‘ ‘ 3% \. .. . ‘ 1 I .l -‘ 0 I N - l‘ i _. «A N .O _| . I r v I .f 'h, ‘ L ’3' ~ - ' .‘A . . c ‘ I _ ' : .iu _.‘ A I. 1‘ “ J T“. . , o ' a ’\ \ a e - 'a a V V 2"" I.) '4‘. t e'_ x '7‘ .5 - '2‘ ‘1 l (a I 7 v 9‘. A \ tn v I5 ‘ e .\ ,\ :1 OJ. .' ' 30‘ 4":‘0‘ -I , 2 a p' e r " b: ”i a- " ‘\ V .0 ' l’ ._: I P I e (A .-.'. \— r '.'. ‘ .|-’ "Q \ ‘ l‘. c . .‘ _ ' ~ I _ - C ‘ . -. .4 .4”; ”tr" .3, an. y ':. ~.' V . . ‘ »- . ‘ . '-' ' :\'\' h“ 4‘." 'I ' r ‘ ._ " e I. ' n. = . . ' 1" 1 " ,.‘ .nl . . 1 ..‘ J er‘.‘ no .1" . 'ex 1" ' . . - ' . ' .- ~ e x . ' '. ‘U \ I A._ 'Kt'll, ‘_“ “a: ' . ,_ '0. ‘- .. , ‘t‘t‘f‘ ' .n e \u'h“~‘--~‘ _ . , , Q a f ‘ . ‘ I ‘\‘. . :50 ‘ "‘ ‘ v ‘Q . Figure 44- -I '. M a O I‘r an“ e /'. .Vx «an i Figure 46 60 Figure 47. Hyalinization of Wall of Blood Vessel in Cortex of Lymph Node. Hematoxylin and Eosin. x 540 D. Hyaline Material . E. Stroma Figure 48. Hyalinization of Wall of Blood Vessel in Medulla of Lymph Node. Hematoxylin and Eosin. x 540 F. Hyaline Material G. Degenerate Lymphocytes Figure 49. Macrophages in Lymph Node Laden with Erythrocytes Hematoxylin and Eosin. x 540 H. Erythrocytes I. Nucleus of Macrophage Figure 50. Subcapsular and Intratrabecular Hemorrhage in Thymus. Hematoxylin and Eosin. x 32 J. Erythrocytes K. Thymocytes L. Capsule Q - “a ‘.\‘fi' Figure 47 Figure 48 62 were similar to those previously described in other*organs. These were not always distinct nor specific. Large numbers of pyknotic cells were observed within the lymph nodes of infected pigs, especially in the cortex of internal lymph nodes. These were thought to be degen- erating lymphocytes. ' Macrophages were prominent within the cortex of each lymph node. They were commonly engorged with erythrocytes (Fig. 49). 8. Eggogrine Oggans The thymus of all infected pigs had areas of hemor- rhage, varying from focal accumulations of erythrocytes to large subcapsular suffusions (Fig. 50). Hemorrhage fre- quently extended into the interlobular connective tissue. Edema of the subcapsular tissue and congestion of blood vessels was a common observation. The adrenals, thyroids and pituitary glands of hog cholera-infected pigs had no observable microscopic lesions. 9. Mgninges Extensive submesothelial hemorrhage was present in the pachymeninges of 6 infected pigs (Fig. 51). Five other pigs had less, but nevertheless distinct, submesothelial hemorrhages. Sufficient lymphocytic and neutrophilic infiltration was present to justify a diagnosis of hemorrhagic pachymeningitis. Con- gestion and edema were consistently found in the pachymen- inges of infected pigs. Some indications of endothelial 63 Figure 51. Hemorrhage and Edema of Pachymeninges. Hematoxylin and Eosin. x l40 M. Mesothelium N. Erythrocytes .O’ Edema Figure 52. Hemorrhage and Edema in Propria Mucosae of Urinary Bladder. _ Hematoxylin and Eosin. x 140 P. Surface Epithelium Q. Erythrocytes R. Edema Figure 53. Hemorrhages and Dilated Tubules in Cortex of Kidney. Hematoxylin and Eosin. x 38 S. Erythrocytes T. Dilated Tubule Figure 54. Hemorrhage in Medulla of Kidney. Hematoxylin and Eosin. x 38 U. Hemorrhagic Area V. Normal Tubules 64 . ....l.l.) '))’Iu|’|rl’|l.ll * Figure 52 Figure 51 . a e . .4 a... D I .1 rel .. 5. 4 ti . - Q a O . .‘o ... J . . .VJ... 1f... , . .4I\.l.. .. r \ I a . a . I l _ s ls l \o .. v. . u. - . a. . . s a /- .e . J a o . .. . . 4 4 . . 0.. a I I- f . o L .9 a .J. “O." u. - .. n . .2 . . f .1 .u . o .0 v.4 .-,1 ‘n... 5' Q t I ' . I u I .v. 0' 'I I . .e . , ..1. . . l ‘3... \ . .1“. ' ..\ t . . . 2. . n a. c . I? Q . . Q ‘ Q n‘ K s a \ O \ . \‘I v . e ;. aw . e I. I. — I. ‘ b . I; ~ . 0 I‘ a . .1 i a. a Q . . w y u c ..k.‘- ‘ r... . O .1! e s A! . v I :3! . fl." ._ . . .3 . . .9... .44. e s, . i, . 4. . .. ‘0. n c .- unfin“ ((1)1 ..tb‘l. II' \. ..lI-l'u. «)illl'lllll I C a... . . . . . I. I a A ...fi.. .. . . I r t 3.: e V e w : . . _ . v o . , .1 v a l A b - . . . . l . _ v . . . a I A . l o . . . ..a. . l I I d to Figure 54 Figure 53 65 Proliferation were present, as well as paravascular* ac- cumulations of lymphocytes. 10. Urinary Dggtg,ggg Bladder The urinary bladder of infected pigs consistently had microscopic submucosal hemorrhages with considerable varia- tion in sine (Fig. 52). Vessels of the lamina propria were consistently congested. Edema frequently accompanied this hemorrhage and congestion. A few subserosal hemorrhages were also observed. Subtle changes were noted in blood vessel walls, but endothelial changes and degenerative alterations in blood vessel walls were indistinct. Changes were not observed in the ureters of hog cholera-infected pigs. The_corpus cavernosum of the ab- dominal urethra in the male was consistently congested, but no evidence of degenerative changes in blood vessel walls or hemorrhages were observed. 11. Kigggy Hemorrhages were observed in the cortex of all kidneys of all infected pigs and in the medulla of 15 of 21 in- fected pigs. The hemorrhages were quite variable in size and location (Figs. 53 and 54). Many subcapsular hemor- rhages were found as well as extensive accumulations of erythrocytes between tubules in both medulla and cortex. *Paravascular, meaning beside the blood vessel. Perivascular, accumulations of lymphocytes (meaning around the blood vessel) were seen only occasionally in the various organs of infected pigs. 66 Those kidneys with extensive accumulations of erythrocytes between collecting tubules frequently had dilated or cystic tubules in the cortex of the kidney (Fig. 53). Proteinaceous casts (Fig. 55) were present in many tubules, together with hyaline changes in the blood ves- sels (Fig. 56). The glomeruli did not seem to be affected; inflammatory infiltration was not present; degenerative changes in the tubules were not observed. 12. ELEEE‘EEQ Gallbladder The lobular appearance, commonly seen in livers of con- ventionally reared pigs, was indistinct in all animals; the interlobular connective tissue was not well developed. Hepatic cells of all non-infected pigs were heavily laden with what appeared to be glycogen, in many cells to such an extent as to render morphologic detail unrecogniz- able (Fig. 57). Hemopoietic areas were numerous throughout the tissues (Fig. 58). The livers of hog cholera-infected pigs contained little glycogen (Fig. 59) and few active hemopoietic foci. Occasionally, near a central vein of an infected pig liver, a limited fatty change was noted (Fig. 60). Focal areas of coagulation necrosis with characteristic pyknosis and accompanied by hemorrhage were observed through. out all livers (Figs. 61 and 62). These areas were not limit- ed to any specific location, and varied in size from a few liver cells to areas covering nearly all a high power field (250 microns). Infected pigs had similar lesions except the accompanying hemorrhage was of considerably less volume. 67 , Figure 55. Hemorrhage and Proteinaceous ,Casts in Cortex of Kidney. - Hematoxylin and Eosin. x 105 W. Cast . X. .Erythrocytes Figure 56. Hyaline Changes in Blood Vessel Wall and Degeneration of Endothelial Cells. Cortex of Kidney. I Hematoxylin and Eosin. x 540 Y. Hyaline Material Z. Tubule Cells Figure 57. Large Amounts of Glycogen in Liver of Control Pig. Hematoxylin and Eosin. x 315 Figure 58.. Hemopoietic Foci in Liver of Control Pig. . Hematoxylin and Eosin. x 315 A. Hemopoietic Focus B. Glycogen-laden Hepatic Cells Figure 57 .«-——‘—-—-r‘ e -‘ ‘ . ‘I 'Q“ . " m’ . \ 3* , .. -r l "\ "q- ' . e ' _, ‘, ,rs C ' k . I ~ ' .. . , a i “v 'c D‘ -‘ \ O A e . I, ' I . ‘ O! I Z . . n .r \’ l l ' ' . . .10 c . . , ‘X ' . .. -‘ Q. 0"- I a I r ’ ‘ . . g, . .. o ' 0 a . °' - .7. " .- ' , {a ~ " ‘ \ c u ' . . . ( _.' ‘ " ' . . e\ O . ) s. O" t _ 4, x ' 7- fl \ ‘ . V . :5). — .; 0 r‘ -e "I . a I .2: ’n \ .' ’ \ . \ 68 ./V,C' ‘ind .r‘_.lu Figure 56 ,3 ‘D 7. IL- 3"“ ‘5, '..o o" w ,_, ‘ C l i ! b (Figure 58 69 Figure 59. Absence of Glycogen in Hepatic Cells of Infected Pig. ' 4 Hematoxylin and Eosin. x 140' Figure 60. Fatty Changes Near Central Vein of Liver. Hematoxylin and Eosin. x 315° C. Vein D. 'Fat Droplet in Hepatic Cell Figure 61. Coagulation Necrosis Near Central Vein of Liver of Control Pig. Hematoxylin and Eosin. x 140 E. Hepatic Cells F. Necrotic Area C. 'Lumen of Vein Figure 62. Coagulation Necrosis in Liver of Control Pig. Hematoxylin and Eosin. x 140' H. Hepatic Cells I. Necrotic Area a... t. .. . o. \ a c e ., ..0... D e I .. O OI . Us 0 «.Q a 1 e O O J. _ I .0 Cl C s t a 1 . A . . . e MNW . .. . . e 0 . S Q 0 .‘Q .0 e. D m, b .- . ..n. . .. v0. ex: . e. .. 1. is I t . . 9 . .. “x .-Jr A . o. 1.‘ . . e. x. 1 0*. O \ O .. t. a .~ . .~' .0 c . . . . . . . . O 4 0 . as e‘ \ r I. 0 ' o O I o 1 e. I. o O . 0 p. a . a... 9 e. .. . o 0.. a v - . . . o ‘ M‘ . no . '0 "O ..l\ 1‘. up no.0 4 .' O. 0 e .I e 0 o o... O o O 39:913.. .u . n a a. 0 O a. . . . O I I ' u .0 v ' . e0. 3. D. Y. .e . o .v .. I .0 . J10. .. O Q. a. .. 0.1. . 0 (not. a, . ”.0 .. . ; . .. o. o c . e 4‘ \04 “v00 a Q ‘ a . O 7 ‘ , e . 0 . O 0 . I Vb .s 40 a . I , ti .. . .1 . ..3. . . . O: I , e) .‘ ' ..l... a. . .. ..3. .91 . ... . .. . _ ' .e 00 .Oe 0" ’.O 0% l . o e n e (- 00 .u r e , a o .1... e . . .1. e .3 f .o I I ... o.h ‘ I. ' o 6 .0 D I~ " 0 ‘0 o! O ‘ e I. 0’ . o A ‘1 r” I... . 9 l _ .’ 0 .4 (J 0.. o a 7' e o .. e a 0’0 . . It Q. I u o 0 ‘ co . .l o. .0 a... O '0 'V 941 e . :o e...u...e an... a . ,.u . .. Mfl'nflac 0.. H e0 an. 9.. one-u. a o t. e 0‘ . . x! q. v.1... . 0 ”red. . o . :)u,|l..ll)L Figure 60 Figure 59 Figure 62 Figure 61 71 Interlobular veins in livers of infected pigs Fre- quently had paravascular accumulations of cells, pre- dominately lymphocytes (Figs. 63,64,65 and 66). Endo- thelioid cells, eosinophils and an occasional basophil were also observed. In several instances, the paravascular in- filtration was such that it caused a projection into the lumen of the vessel (Figs. 67 and 68). Some lymphocytic infiltrations were found in the connective tissue accompany- ing large blood vessels and occasionally near bile ducts (Fig. 69). A tendency toward actual "cuffing" of blood vessels was noted only in the liver of one infected pig. The paravascular infiltration had little effect on sur- rounding hepatic cells; an occasional area was noted with some pressure degeneration of liver cells. Vascular changes in infected pigs were indistinct and non-specific. Some suggestion of endothelial swelling and proliferation and degenerative changes in blood vessel walls was present, but not sufficient for positive conclusions. Some subcapsular hemorrhages were noted (Fig. 70), as well as hemorrhages in the connective tissue near large vessels. Moderate congestion of the hepatic sinusoids was a consist- ent finding in infected pigs. Vessels in the lamina propria of the gallbladder were consistently congested. Fifteen of 21 infected pigs had subepithelial edema of the gallbladder with accompanying (congestion, some paravascular infiltration with lympho- cytes, and early degenerative changes (Fig. 71). Distinct 72 Figure 63. Focus of Lymphocytes in Inter- lobular Area of Liver. Hematoxylin and Eosin. x 140 Figure 64. Focus of Cells, Predominantly Lymphocytes, in Interlobular Area of Liver. Hematoxylin and Eosin. 1'140 J. Hepatic Cells K. Blood Vessel L. “Cellular Infiltration Figure 65. Paravascular Infiltration of Lymphocytes Near Interlobular Vein. Hematoxylin and Eosin. x 140’ M. Vein N. “Lymphocytes 0. Hepatic Cells Figure 66. Paravascular Infiltration of Lymphocytes in Liver. Hematoxylin and Eosin. x 168 P. Hepatic Cells Q. Vein R. 'Lymphocytes \\ . o O. .‘ v . H . _ .v. e u o .o . q u A o a \ * h a. .. s 0‘ e o 0 6 in Q . . 0 . e . o . \ . . or .0. M 1‘"! o . . . . o g t . . . ... ...: .. _ . .V . . ... ”o‘od I . u _ U .‘ O- ..l‘.‘ I ' e ... O I! e .4 a o . _ ‘ ‘9 ,I‘ a.l' 1'. ‘ . w . \ o .. v... r . r .\. o k \ V ..- . h o .. r 0.. u ’f e n I . 0.1.01 ; a. 9‘ o u v . . . P ._ 1 . .. . t n G.- ..lutdl‘.'. ‘ . £ 0 0 g .o \ I \ O a . I o f. C V I . I - .0. . fl 1. o . e. O o a! O a . o ‘c a O U u a... O S O c c I Q. Ct .I n O ' I .. . . . u -. -. . .. a . . c .. . r _ . . . on. n o a. h an a . .0 O A. I . .0 . e . . . o a . . .0 w ' _.' .0 |.¢ .1 .4 o 0 c .o I O . .. O — \O . o C . 10.. . o. a o . I a. 0 .~ . . . . 9w o s. . . .r 0 ’ cf ’ . . . .a Q C .0. . . . on “fig . . . .c. . Q . e \ a .pfl ‘0‘. a. i .. 1. f... e . 3 l. O 0‘. 'Q. 1. C. . O ’ . I o O . e o . U 0 . \ 0.. o a a I s 7 .a... . tame... . n ..V . . a Q ' v'JJ ~ 0 a ”I I .0 . ‘ O H 4 \u . . o . Q a fie I... O . . . . . ad on On e .1 ‘1‘ . 1‘ or' .UO% 1 0' 0L 0 C . o . a u... 9. 017‘. a 0 o .o \ \8 § L .t . ov 0 O . Figure 64 Figure 63 Figure 66 Figure 65 74 Figure'67. Mass of Lymphocytes Protruding into an Interlobular Vein of the Liver. Hematoxylin and Eosin. x 140 S. Hepatic Cells T. Bile Duct H. Lymphocytes. V. Lumen of Vein_ Figure 68. Paravascular Infiltration of Lymphocytes in Liver. Hematoxylin and Eosin. x 140 W. Hepatic Cells X. Lymphocytes Y. Lumen of Vein Figure 69. Lymphocytic Infiltration Around Interlobular Bile Ducts in Liver. Hematoxylin and Eosin. x 140 Z. Vein A. Bile Duct B. Lymphocytes Figure 70. Capsular Hemorrhage of Liver. Hematoxylin and Eosin. x.140 C. Erythrocytes D. Hepatic Cells ._ . .r'e... .— ..--—~.._ Figure 68 Figure 67 Figure 70 Figure 69 76 Figure 71. Edema of Gallbladder Mucosa. Hematoxylin and Eosin. x 70 E. Edema F. Congestion C. Leukocytic Infiltration Figure 72. Focal Necrosis of Gallbladder .mucosa. Hematoxylin and Eosin. x 70 H. Necrotic Area I. Leukocytes J. Edema Figure 73. Necrosis of Mucosa of Esophagus. Hematoxylin and Eosin. x 98 K. Normal Epithelium L. Necrotic Areas Figure 74. Degenerative Changes in Wall of Blood Vessel. Submucosa of Esophagus. Hematoxylin and Eosin. x 504 77 72 Figure Figure 71 Figure 74 78 focal necrosis of the gallbladder mucosa was observed in 5 infected pigs (Fig. 72). The base of the necrotic areas con- sisted of degenerated fibroblasts and epithelial cells, with neutrophilic infiltration. Degenerative changes in blood - vessels of the lamina propria were not distinct. l3. Esophagus and Stomach The thoracic portions of the esophagus from 8 of 21 infected pigs had focal areas of necrosis in the epithelium with slight extension into the lamina propria (Fig. 73). Moderate lymphocytic and neutrophilic infiltration accom- panied these degenerative changes. Congestion was present in the esophagus of nearly all infected pigs. Hyaline and vacuolar changes were present in some blood vessels (Fig. 74). Subserosal hemorrhages were found in several areas near the thoracic inlet. These were frequently accompanied by hemor- rhage in the surrounding musculature. Freduently, blood vessels within the musculature of the stomach were surrounded by lymphocytes. These accumula- . tions were present in both control and infected pigs. Rather extensive areas of necrosis were found in the mucosa of the stomach in 12 of 21 infected pigs (Fig. 75). These were accompanied by underlying infiltrations of neutro- phils and lymphocytes (Fig. 76). Areas of hemorrhagic gastritis with erosion of surface epithelium and only a slight leukocytic infiltration were found in the stomach of 3 of 21 infected pigs (Fig. 77). Lymphocytic foci were found commonly in the submucosal areas of the stomach of infected 79 Figure 75. Necrosis of Mucosa of Stomach. Hematoxylin and Eosin. x 38 .M. Normal N. Necrotic Figure 76. Necrotic Area of Stomach Mucosa with Leukocytic Infiltration. Hematoxylin and Eosin. x 140 O. Necrotic Epithelium F. Neutrophils and Lymphocytes Q. Submucosa Figure 77. Necrosis and Hemorrhage of Stomach Mucosa. Hematoxylin and Eosin. x 98 R. mucosa S. Erythrocytes ' T. Necrotic Epithelium Figure 78. Hemorrhage in Propria Mucosae of Small Intestine. U. Epithelium V. Erythrocytes 80 Figure 76 Figure 75 81 pigs. Generally congestion of blood vessels was not a common finding. 14. gmall Intestine, gggum and gglgn The epithelium of portions of the small intestine (especially the terminal portions) of control pigs con- tained many vacuoles. These vacuoles were much less numer- ous and smaller in size in infected pigs. The submucosa and adjacent mesentery of the small intestine were congested in nearly all infected pigs. Mucosal, submucosal, and serosal hemorrhages varying great- ly in size and location were found in 15 of 21 infected pigs (Fig. 78). Hemorrhages were common near Peyer's patches and other lymphocytic aggregations (Fig. 79). Areas quite suggestive of thrombosis were found (Fig. 80). Necrosis and sloughing of the mucosa were observed in 10 of 21 infected pigs (Fig. 81). The degenerative changes were limited chiefly to the epithelium with only slight ac- cumulations of inflammatory cells. Some suggestion of increased lymphocytes was present- ed in the lamina propria of the mucosa of infected pigs, but interpretation was difficult because of normally occurring lymphocytes in this area. Edema, congestion and possible thrombosis (Fig. 82) were found in the cecum of some infected pigs. Necrosis was not observed within the mucosa. Hemorrhages of various size and in various locations were found in 12 of 21 infected pigs (Figs. 83 and 84). 82 Figure 79. aHemorrhage in Peyer's Batch of Small Intestine. Hematoxylin and Eosin. x 140 W. Erythrocytes X. 'Lymphocytio Focus Figure 80. Thrombus in Blood VeSsel of Propria Mucosae of Small Intestine. Hematoxylin and Eosin. x 140' Y. Thrombus Z. 'Propria Mucosae Figure 81. Necrosis, Hemorrhage, and Congestion of mucosa of Small Intestine. - t . Hematoxylin and Eosin. x 140' A. Necrosis of Epithelium B. 'Erythrocytes Figure 82. Hemorrhage, Edema and Thrombi in Submucosa of Cecum. Hematoxylin and Eosin. x 91 ' C. Erythrocytes D. Edema E. 'ThrOmbus 83 _.'-- — 80 Figure Figure 79 Figure 82 Figure 81 84 Figure 83. Hemorrhage in Submucosa of Cecum. Hematoxylin and Eosin. 1'91 F. Erythrocytes G. Lymphocytic Focus Figure 84. Hemorrhage in Propria Mucosae of Cecum. Hematoxylin and Eosin. x 91 H. Erythrocytes I. Globlet Cells Figure 85. Hemorrhage in Fold of Submucosa of Colon. Hematoxylin and Eosin. x 116 J. Epithelium. .K. Submucosa .L.' Erythrocytes Figure 86. Thrombi in Submucosa of Colon. Hematoxylin and Eosin. 1.140 -N. Submucosa .0. Thrombi -P. Mucosa \ Figure 86 86 Hemorrhage and congestion were present in the large intestine of 14 of 21 infected pigs (Fig. 85). Necrosis was not seen. Suggestions of thrombosis were found (Fig. 86), and possible degenerative changes in blood vessel walls with some endothelial proliferation were observed. 15. Reproductixg Organg ' Histopathologic examination of male and female reproductive organs failed to reveal any abnormalities, except for occasional moderate congestion of the male accessory glands and the female uterus. V. DISCUSSION The clinical signs and lesions in the hog cholera-in- fected pigs were attributed to the hog cholera virus alone. The findings from this experiment discount the role of Salmgnglla species and other bacteria in contributing to the clinical syndrome, mortality rate, and gross lesions of hog cholera as seen under field conditions. Bagillus guppilig is a common laboratory contaminant (3). Its mode of entrance into the isolators was not determined but the possibility exists that the organism was not destroyed during autoclaving of the diet. .B. gubtilig was apparently of no importance as a complicating factor in the cholera- infected pigs because clinical and gross pathologic differ- ences could not be detected between germfree and monocon- taminated animals. The diphasic temperature curve was quite distinct for each cholera infected pig in this experiment. No deaths oc- curred until after the first peak was passed. The 3 pigs which died at 4, 4.5, and 5 days, respectively, after inocula- tion had all passed the first temperature peak and were at the "valley" stage or moving upward toward the second peak. The remaining pigs died during or after the second peak tempera- ture. An anemia existed from the second day after inoculation. This anemia increased in severity as the disease progressed, with a noticeable drop in both hemoglobin and hematocrit values after the sixth day. This suggested a deleterious effect of the hog cholera virus on erythrocytes. While 87 88 determining the differential leukocyte counts, there was re- vealed large numbers of nucleated red blood cells. A close relationship was found to exist between the unadjusted total leukocyte count (Fig. 4) and the numbers of nucleated eryth- rocytes (Fig. 5). The apparent recovery in numbers of leuko- cytes originally observed was actually due to these nucleated cells; the leukopenia itself persisted. Comparison of the hemoglobin and hematocrit values (Figs. 6 and 7) with the appearance of nucleated erythrocytes (Fig. 5) revealed a delay of approximately 5 days between the decline in hemo- globin-hematocrit values and the appearance of large numbers of circulating nucleated erythrocytes. These nucleated cells suggested hyperplasia of the bone marrow or premature release of these cells from the bone marrow as a result of the exist- ing anemia. Differential leukocyte counts were not notice- ably different from preinoculation levels or from values ob- tained from control pigs, suggesting that the leukopenia was absolute and the virus had similar effects on all types of leukocytes. The most noticeable effect of the virus on food con- sumption was the loss of interest and enthusiasm in eating. The actual decrease in the quantity of food consumed was not marked until the day before death, but the time required to consume the milk was greatly extended. The exact onset of diarrhea was difficult to detect. The feces of infected pigs living beyond 7 days were fluid to semisolid in consistency; pigs surviving the longest had 89 the most severe diarrhea. This undoubtedly caused some de— hydration and may have accounted for a portion of the lower body weights of infected pigs. Blood findings did not reveal, nor did they exclude, the possibility of hemoconcentration. Gross pathologic findings were hemorrhages of various sizes, shapes, and in numerous locations. They were consist- ently found only in the kidneys and lymph nodes. The cortex of the kidney of infected pigs was always involved, the medulla less often and less severely affected. Of the 15 groups of lymph nodes examined in this study, the minimum num- ber showing gross lesions in one pig was 10. Most infected pigs had 12 to 15 of these nodes grossly involved. In paired nodes, bilateral involvement was commonly, but not always, found. The external and internal inguinal nodes, renal, paro- tid, and mandibular nodes always had some degree of hemorrhage. The mediastinal, gastric, and hepatic nodes were the least fre- quently involved, with the incidence of hemorrhages in other nodes being between these two levels. Correlation could not be made between the number of organs showing hemorrhage and/or the severity of these hemor- rhages, with the elevated temperatures, leukopenia, survival time, or other clinical signs. Apparently the lesions were somewhat independent of the clinical signs; death, in turn, was probably not dependent on severity of either clinical signs or gross lesions. Microscopic lesions of hog cholera in gnotobiotic pigs consisted of congestion, edema, and hemorrhage accompanied by necrosis and degenerative vascular alterations (endothelial 90 swelling and proliferation, hyalinization, vacuolar changes, and thrombosis). Lymphocytic elements were uniformly deplet- ed in lymph nodes and in the spleen of infected pigs; the tonsils appeared to maintain their lymphocytes. Varying degrees of edema and hemorrhage were common in these 3 structures, as well as degenerative blood vessel changes. Pyknotic cells, thought to be degenerate lymphocytes, were found in the cortex of lymph nodes and in the white pulp of the spleen of infected pigs. Lymphatic tissue within the intestinal tract appeared to be neither stimulated nor de- pleted. The respiratory and urinary systems were characterized by hemorrhages while the digestive system had, in addition, focal areas of necrosis with cellular infiltration. Para- vascular infiltration of the liver was mostly with lympho- cytes, although large numbers of paravascular endothelioid cells were found. The exact nature of these endothelioid cells was not determined. Necrosis and edema found in the gallbladder and tubular portions of the digestive tract of infected pigs were ac- companied by infiltrations of neutrophils, with a small number of lymphocytes. Dilatation of the renal tubules ap- peared to be dependent on the amount of hemorrhage in the medulla. This dilatation was, perhaps, merely a mechanical condition. The presence of focal necrosis of the liver in both infected and control pigs was uniform and persistent. The 91 cause of this condition was not determined. Similar lesions have not been observed in other germfree pigs (41,53,54). A dietary exclusion could be considered in view of the high temperatures and pressure used to assure sterility of the diet. The significance of the Anitschkow myocytes in the heart was not ascertained. They were, however, a consistent finding, commonly associated with myocardial hemorrhage, and were mostly ventricular in location. They were considered by some (5,17) to be histiocytes, part of the reticulo-endo- thelial system, and evidence of inflammatory processes with- in the heart. Attempts to trace hemorrhages to blood vessel lesions were unsuccessful. Frequently the blood vessel alterations were subtle, and comparisons with similar structures in con- trol pigs often permitted no definitive conclusions. Apparent glycogen depletion of the liver was observed in infected pigs. Unfortunately material was not preserved in fixative suitable for glycogen stains. Fat stains showed that the material in the liver was not fat. Previous work (41,54) showed similar material to be present in livers of gnotobiotic pigs, and this was proven to be glycogen. It was thought, therefore, that it could be rather safely as- sumed that this material in control pig livers was actually glycogen. Though control pigs were fed a similar quantity of food (reduced as anorexia developed in infected pigs), gly- cogen depletion was not evident. Indeed, much of the archi- tecture of the liver cells was obscured by the large amounts 92 of glycogen in the livers of control pigs. Apparently the metabolic rate of hog cholera infected pigs was altered so that the glycogen was needed and the liver depleted early in the course of the disease. ‘It was thought the associated fever may have required the utilization of this glycogen. Pigs dying 5 days or earlier after inoculation with virulent virus had glycogen depletion similar to that found in ani- mals surviving 15 days. Erythropoietic foci were consistently absent in the livers and spleens of infected pigs. When present, they were very small and contained only a few nucleated cells. Control pigs, on the other hand, had numerous large focal areas of namepoiesis, especially in the liver. Megakaryocytes were not uncommon. It would appear that the virus is capable of either depleting or depressing (or both) the extramedullary hemo- 5 poietic centers in the young pig. The large numbers of nucleated erythrocytes found in the circulating blood may have partially arisen from depletion of the extramedullary centers. It seems unlikely, however, that the great numbers of nucleated erythrocytes found in the blood could have been present without bone marrow hyperplasia, in addition to the depletion of the extramedullary centers. Certainly no hyper- plasia was evident in these extramedullary hemopoietic centers, but it could not be determined if the virus actually "de- pressed" these areas. Bone marrow studies have not been included. 93 Comparison of gross and microscopic lesions of lymph nodes indicated that congestion, edema and moderate hemor- rhage were not always detected grossly. Microscopically, the external lymph nodes had hemorrhages of less extent and severity than the internal lymph nodes. . 7 The results of this experiment emphasized the marked variation in the response of individual pigs to hog cholera virus. An attempt was made to hold the number of variables in this work to an absolute minimum by using littermate pigs whenever possible, by maintaining the pigs in a bacteria-free or monocontaminated environment, and by giving a standardized dose of virus to each pig. In spite of these rigidly con- trolled conditions, the gross lesions varied from one pig to another, as did the survival time. It is not surprising, therefore, that epizootics of hog cholera are characterized by such marked variability of clinical signs, lesions and mortality rate. The assumption was made that changes were due to the hog cholera virus since bacteria were absent and similar lesions were not found in control pigs. While birth weights were within normal limits, it was evident that.the final weights, even in control pigs, were considerably less than expected for pigs reared to this age under conventional conditions. This undoubtedly reflected the need for a more adequate ration for pigs raised from birth under artificial conditions. The net weight gain of the infected pigs was no doubt influenced by the fact that 94 some of these pigs died early in the experiment. If the control pigs had been killed at comparable times, the de- ficiency in weight gain of the infected pigs might not have been as noticeable.‘ Both control and infected pigs were kept in plastic isolators within the same room, and no precautions other than those routinely employed in rearing germfree pigs were used. Cross infection between isolators did not occur. This, in itself, was significant and suggested that the technique used may be a useful tool in the study of infectious disease of domestic animals. v1. SUMMARY AND CONCLUSIONS Germfree techniques were employed to study the clinical signs and lesions caused by the hog cholera virus. Ten germ- free and 11 monocontaminated pigs were inoculated at 14 days of age with hog cholera virus. Six uninoculated pigs (4 germfree and 2 monocontaminated) were raised under similar conditions and served as controls. Rectal temperature curves were diphasic following virus administration, reaching 2 mean peaks of 106 F. and 106.3 F. Total circulating leuko- cytes decreased to approximately one-third their preinocula- tion levels within 24 hours. There was a reduction in hemo- globin and hematocrit levels, followed by a rise in numbers of circulating nucleated erythrocytes. Death occurred in all 21 inoculated pigs, with the time of death ranging from 4 to 15 days after inoculation. Necropsies consistently revealed hemorrhagic lymph nodes and petechial and ecchymotic hemor- rhages in the kidneys. Microscopically, hemorrhages were found in the tonsils, tongue, diaphragm, lungs, heart, lymph nodes, meninges, urinary bladder, kidneys, liver and tubular digestive tract. Hemorrhages were most extensive in the internal lymph nodes. Necrosis was found in the gallbladder, spleen, lymph nodes, and tubular digestive tract. Leukocytic infiltrations were found in the liver and in association with necrotic foci of the digestive system. Anitschkow myocytes were found in large numbers in the heart. Lymphocytic depletion was 95 96 common in the lymph nodes and spleen. Degenerative changes were present in the smaller blood vessels of the major tissues. Livers of infected pigs were characterized by glycogen deple- tion and absence of extramedullary hemopoietic areas. These signs and lesions are attributed to the effects of the virus itself, and serve to emphasize the marked variation in the response of individual pigs to the hog cholera virus. 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