gag. .Efififig $2.... ._ . _ , ‘3; . : .EIU.§_..._ir x35... hmma_m..w§..§§ «7...: . . .3 . _ :21: . 3%. . .mmawmm. a. fix“... ”3;. . «wk v .r. ._ a... y. a m u... a. 23w . .a k. .2". a- gwmmm M . . . .. .31”... .u ..._m..w an. . . 31;? my... E v . . u: "m R. £376 wk “9‘ . k? a. ._1..T.9.._.- . u. .. e. . a. 93?. firm. a? . m. r. ~ 2.5.: aw. , .m. trauma . . . if?) «52?. . .113” 3.2% ‘7’... 1? ‘v a? at“ 3 33. g 5.... a... . . . and c: As; . . ...\mm‘__...“. 4. 449 _. U. a . . 4...? $1 , .. .flmfiw _.. _. . m. . . . .35 "x . . r: .a. . .3. 3a.. , ..a..~...%._. L. 11 . . $01.3 . m 5.: , . k. w. L... I- A“. a ‘1 .4 1.4. .a- 51.31 a. .1 5.9: 7 .1 . .- 55x. I? V. . 3* H! J. . 4k..2$fi~v fiiu:..r1:.:«. . . z... x ‘22:. {:5 1M. 3.2.5.:33 . .E {.9 . .1“! 7.3:. .4331“ 5. . «is. “.232, . . n. £3.53 a . . . , _ . . . : qwadah5nu§ gt, . in .. .34... x b.3213... . . 27!: .1 .P .slww. ’ Ipyflatfiru.i . Jazz. . . . f . .1 . 4 .. 1.: v . u v , ‘- ‘11-’u2. Q‘ i . ~ a). I-\ D; 44.», 1.? 9.3%»... 7.. 41.6 4‘ _ 62.1 .x. 251 I’VE” 9) .. inn! :1 :K . Let: .3. 5, 1.5L): _ . .......:1 a. 3.53. )iniufiflKt 733.1: 3!... a. $92.59.: a . E ..fl...aa!a , 35...): ghfi). . . a. .5 :: riie I“... . €11¥~5R1 ,1}: 11.177: :3) . 3:1.) I. «1:. 1.3.: [.35 . 2.315.“. ’.1.u14;...12 he“ 2.: 74 1.1.4 , . “I 757:» . , ‘7V\<)"V\t’ ; 3...: 2.2% . :51: Z. . 3.31 . V; ‘1 .N.n.>. ....2 . 7‘ .q a a :55 #1:: 3.. 41!! Ignxasl—ci 1 . tar . 3 ”fr 3 3...? 121...}...1: as: .31? , y ‘ €71“ in... x I 1.5 .s t 113...... its» ‘2 {IQ-‘51. . J 1y;...._>\.z. 3.1%. 3..) 35:12! . iii}: 3:113:91 . .193r35VXs. 7. PS». . L .3: I 1.431. .3 .A I 7. 3:11 11.: A... ..V...v1...5?..3.u. {12:53 . 2.1:. . i. .1; :qufiurnfliafis ”v... 1ky’chl)‘. ita‘ . , . . .. r. 13.3.10... 306.313. 73.1.33: .17) . . :72}£.£§Y35..¢:i11 . s... z 1.41.; ‘5‘: 513:2... c): . 1...; 531.; 5r. . > . a...:.........3 1!. .2 , 1.)... > {15 :3: {Pg . 4 7.1 v duuycuxiilh. s. ‘1. . 9 .\ .t:........ is w} 1.3).)...2 3...... . . AIVZ . .3 2.1.3.4: all}, ,4 . 3.). ~yss )7 .17.; 9!... b... a) w 77 s. .6. 2.3%? If“ 1 7 5.! II. .. \ . I .2 .3 . .1 .24.. i €2.52 THESlS This is to certify that the thesis entitled Pathology of Protein Malnutrition and Infection presented by Kenneth K. Keahey has been accepted towards fulfillment of the requirements for C. K. Whitehair Major professor Date August 21, 1963 0-169 LIBRARY Michigan State University ABSTRACT PATHOLOG‘.‘ O? r”? .IEEJE MAUHJTRLTIOS #3er ZEFECT’I'IJ'W ‘1‘; Kenneth E. Keahey Three experiments were conducted to evaluate and study the pathology of pretein nalnutritien and infection. The baby rtg was used as the experimental.aninals The pigs'were divided into two groups. one t which was fed a lewaprotein {six oer cent casein‘ ration and the other fed a high-protein (32 or 2% per cent casein} ration. Control animals were selected from each group and the remaining nine were infected at approximately seven weeks of age with transmissible gastro~ enteritis t'fifiz rirus. The effect of infection an the nutritional state of the host was sraluated by means of :rswth rate, symptomatolng:. hematology, nitrogen balance. sodium balanse. potassium balance. total serum protein, electrophoretic distribution of serum protein. gross pathology and histopathology. Growth responses of the pigs fed the low-protein ration were poor. Pigs fed either high— or loubprotein rations gained lees weight following inoculation with the virus. The most outstanding gross and microscopic lesions were observed in the livers of pigs fed the low-protein ration. and these lesions “12- swan more severe in tasse animals infected with TGE. Grossly, they appeared as dark red. depressed. irregular areas on the surface of the liver and were distributed throughaat the parenchyna. Some livers were yellowish in color. Hieroscopieally, these lesions uDre characterized by cirrhosis and fatty metamorphosis, and massive 1ver necrosis. Kenneth K. Keahqy There was some reduction in nitrogen retention for a period of at least nine days after infection with the T68 virus. The animals fed a lowbprotein ration regressed into a negative balance. while those fed the high-protein ration remained in positive balance. All pigs infected with the virus had a reduction in potassium and sodium retention for three days after inoculation but then returned to normal levels. No differences were noted for control animals on the high- or the lowbprotein rations. The hemOgrams revealed a leukopenia and a slight anemia following inoculation of the virus. Total serum protein was slightly low'for animals fed the loweprotein ration. In those pigs fed the low- protein ration, the electrOphoretic distribution of serum proteins indicated that there was a relative decrease in serum albumin and a relative increase in serum gamma globulin. The cirrhosis and fatty metamorphosis observed in the liver were possibly associated with low-protein intake. Ihe massive liver necrosis was probably due either to a deficiency of FactorIIl (Schwarz, 1960) or insufficient vitamin E in the ration. The dramatic changes in the metabolism of nitrogen, sodium and potassium were probably primarily due to infection. One of the great contributing factors in metabolic changes with onset of symptoms of infection was a decreased intake of feed. The interrelationship of nutrition and infection resulted in a more adverse effect in pigs fed the low-protein ration. The lowe protein control pigs made less weight gains. consumed less feed. ha! Kenneth E. Koshey more marked lesions and changes in electrolytes than the infected aniaals in the high-protein ration. The results help to explain the need of additionsl nutrients during stresses such as infection. PATHOLOGY OF PROTEIN MALNUTfilTION AND INFECTION By Kenneth K. Keahey A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTJR 3F.PHILOSOPHY Department of Veterinary Pathology 1963 ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. C. K. Whitehair and Dr. G. L. waxler for their invaluable guidance and assistance throughout this study, and for their critical reading of this manuscript. Sincere thanks is expressed to Dr. E. J. Benne for generous cooperation in the use of his laboratory and facilities for nitrogen determinations. The author wishes to express his appreciation to all members of the Department of Veterinary Path010gy for their generosities and considerations. and for technical and professional aid and guidance. Thanks is expressed to the National Institutes of Health for providing to him a Postdoctoral Fellowship in the Division of General Medical Sciences. The writer especially wishes to acknowledge his gratitude and appreciation to his wife. Norlyne. whose sacrifices and encouragement made this study possible. ii I. 11. III. TABLE OF CONTENTS mmODUCTION O O O O O O O 0 O 0 O O O O OBJECTIVES O O O O Q 0 O O O O O O O O O me 0? 151mm 0 O O C O O O O O O A. B. C. D. F. Early EXperimentation with Proteins Protein Requirement in the Pig . . . Protein Malnutrition in Children 1. Terminology ........ 2. History' . . . . . . . . . . 3. Geographical Distribution . “. Predisposition . . . . . . . S.Symptoms........... 6. Pathology . . . . . . . . . a. Gross pathology . . . . b. Clinical pathology . . . c. Microscopic pathology . 7e Traltmant o e e e e e e o e EXperimental Protein Malnutrition in Animals Protein Malnutrition in Animals Resulting in Livar NOCt0513 e e e e o e e o o e e e o e e 1. P18 0 e e e e e e e e e e e e e o e e e 2. Rat e e e e e e o e e e e e e e e e e e interactions of Nutrition and Infections . . iii Page (r ‘3 “Q r. IL!) EV. V. “-3. HaTEElALS AND METHODS :7 l'el :1 A. 3 3?..ISU'LT S A. Transmiseible Gastroenteritis in Terms Q- . O O O C Virus threation e Bacterial Infections Other Infections . Emperimental Animals . QPO Of ADiMLlS e e e mtion O O O O O O O O ?ch°g0n e e o e e e e Facilities and General Womation O Pigs U63), 0 O O O O O An‘lyses e e e e e e e e e e e e e e e e e e l. HematOIOgy e e e e e e e e e e e e e e e Nitrogen Determination . . . . . . . . . Sodium and Potassium Determinations . . Serum-Protein Fractions 0 0 O O O O O O BiStOpathOlogy e e e e e e e e e e e e 0 Specific- Procedures for Each Experiment . . I i v 9 ~e 3. marimfin‘t 1 o e e e 0 Experiment 11 . . EXPBriment III e e mperiment I . . . . . 1. 2. q .1. Grcuth e e e e e e Analyses e e e e e mp toms I O O O 0 iv 0 O O O O O O O 2 .5 26 3o 3o 30 30' VI. VII. B. C. h. Gross Lesions . . . 5. Microscopic Lesions Experiment 11 . . . . . 1. Growth . . . . . . . 2. Analyses . . . . . . 3. Symptoms . . . . . . u. Gross Lesions . . . 5. Microsoapic lesions Experiment III 1. 2. 3. a. 5. O O O O GrOWtheeeeee-e AnalyQOSeeeeee Symptoms...... Gross Lesions . .Microsconic Lesions DISCUSSION . . . . . . . A. B. l. 2. 3. h. C. D. E. SUMMARY Growth....... Mdysaseeeeee Symptoms an? Cress Hematology . . . Serum Proteins . Nitrogen Balance 0 Sodium and Pbtassium Balance Microscopic Lesions ’ 4 4:95 tsnls General Observations . . . . ' O O O O O O O 0 O O O O LJST OF REFERENCES . . . . . . . 6 122 LIST OF TABLES as: £9.32 Megment I 2. Composition of swine ration. no. 1 . . . . . . . . . 33 LI. Composition of swine ration. no. 2 . . . . . . . . . 38 is;. Composition of swine ration. no. 3 . . . . . . . . . 33 IV. Heights of infected and control pigs on 32 percentcasomrationeeeeeeeeeeeeeeo #8 V. 'Weights of infected and control pigs on 6 per cent casein ration . . . . . . . . . . . . . . . #8 VI. Hemocytometer leukocyte counts of infected and control pigs on 32 per cent casein ration . . . . . #9 VII. Hemocytometer leukocyte counts of infected and ' control pigs on 6 per cent casein ration . . . . . . 4% VIII. Hemoglobin levels of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . . $0 IX. Hemoglobin levels of infected and control pigs on 6 per cent casein ration. . . . . . . . . . . . . 50 X. Hematocrit values of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . . Kn 5.: XI. Hematocrit values of infected and control pigs on 6 per cent casein ration. . . . . . . . . . . . . ‘1‘ b) \n I“) XIIe Mean differential lankocyte counts 0 e e e e e e e e XIII. Paper electrophoresis of serum protein of infected and control pigs on 32 per cent casein ration . . . Sb XIV. Paper electrophoresis of serum proteins of infected and control pigs on 6 per cent casein ration . . . 5“ XV. Serum nitrOgen of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . . . 55 vi 35919 XVII. XVIII. XIX. XXII. XXIII. XXIV. XXVI. XXVII. XXVIII. XXIX. XXX. XXXI. Serum nitrogen of infected and control pigs on 6 per cent casein ration. e e e e e e e e e o e e Nitrogen balance of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . . Nitrogen balance of infected and control pigs on 6 per cent casein ration. . . . . . . . . . . . . Sodium balance of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . . Sodium balance of infected and control pigs on 6 per cent casein ration. . . . . . . . . . . . . Potassium bal*.ce of infected and control pigs on 32 per cent casein ration . . . . . . O O O O O 0 Potassium balance of infected and control pigs on 6 per cent casein ration. . . . . . . . . . . . . Wiesel; Weights of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . . . . . ‘Weights of infected and control pigs on 6 per cent casein ration. . . . . . . . . . . . . . . . Hemocytometer leukocyte counts of infected and control pigs on 32 per cent casein ration . . . . Hemocytometer leukocyte counts of infected and control pigs on 6 per cent casein ration. . . . . Hemoglobin levels of infected and control pigs on 32 per cent casein ration . . . . . . . . . . Hemoglobin levels of infected and control pigs on 6 per cent ca59in ration e e e e e e e e e a e Hematocrit values of infected and control pigs on 32 per cent casein ration . . . . . . . . . . Hematocrit values of infected and control pigs on 6 per cent casein ration . . . . . . . . . . . Mean differential leukocyte counts vii 55 56 56 \J‘ 00 (.D \_"| ?2 7? XXXIII. XXXIV. XXXV. XXXVI. XXXVII. XXXVIII. XXXIX. XLV. XLVI. Paper electrophoresis of serum proteins of infected and control pigs on 32 per cent casein ratian o e o o e o e e e e e o e e e e o o a Paper electrophoresis of serum proteins of infected and control pigs on 6 per cent casein ration . . . Total serum protein of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . Total serum protein of infected and control pigs on 6 per cent casein ration . . . . . . . . . . . . Nitrogen balance of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . . . Nitrogen F“1n:e of infected and control pigs on 6 per cent C3381“ ration o e e e e e o e o o e O 0 Sodium balance of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . . . Sodium balance of infected and control pigs on 6 per cent casein ration . . . . . . . . . . . . . Potassium balance of infected and control pigs on 32 per cent casein ration . . . . . . . . . . . Potassium balance of infected and control pigs on 6 per Cfint casein ration e o e i e o e e e o o e §§periment LI; Weights of infected and control pigs on 2h per cent casein ration o e o e e e e o e e e e e e e e Weights of infected and control pigs on 6 per cent casein ration e o e o e o e e o o o o e e e e Hemocytometer leukocyte counts of infected and control pigs on 2% per cent casein ration . . . . . Hemocytometer leukocyte counts of infected and control pigs on 6 per cent casein ration. . . . . . Hemoglobin levels of infected and control pigs on 2# per cent casein ration . . . . . . . . . . . viii 78 79 83 85- 123 124 p; h) \I‘ XLVII. XLVIII. XLIX. LII. LIII. LIV. Hemoglobin levels of infected and control pigs on6percentcaseinration........... Hematocrit values for infected and control pigs on 2% per cent casein ration . . . . . . . . . z Hematocrit values for infected and control pigs on 6 per cent casein ration . . . . . . . . . . . Mean of differential leukocyte counts . . . . . . Paper electrOphoresis of serum proteins of infected and control pigs on 2h per cent casein ration . . Paper electrophoresis of serum proteins of infected and control pigs on 6 per cent casein ration. . . Total serum protein of infected and control pigs on 2“ per cent casein ration . . . . . . . . . . Total serum protein of infected and control pigs on 6 per cent casein ration . . . . . . . . . . . ix 0 126 127 128 129 U (”VON-MI? 10 ll 12 13 14 15 l6 IST OF FIGURES Metabolic balances of (A) control pigs fed 32 per cent. (B) infected pigs fed 32 per cent. (C) control pigs fed six per cent. and {D} infected pigs fed six per cent casein rations. . . . . . . . . . . . . . . Hyperkeratosis of the skin . . . . . . . . . . . . . Increased numbers of lymphocvtes and plasma cells in the lamina prOprla Of tha inadcnum e o o e e e o o e Edema in the submncosa of the cecum . . . . . . . . Hydropic degeneration of the liver . . . . . . . . . Hepatic necrosis . . . . . . . . . . . . . . . . . . Hepatic necrosis . . . . . . . . . . . . . . . . . . Control pigsfed 32 per cent and six per cent casein ration e o o e o o e e e o e o e o e o o e O O C O O ?igsfed 32 per cent and six per cent casein ration and iflffiCtOd With TGE e e e e e e e e e o e o o e e Nitrogen balances . . . . . . . . . . . . . . . . . A control pig fed six per cent casein ration . . . . Skin lesions of a pig fed six per cent casein ration and infOCted With TGE e e o e o o o e o o o e e e e Abdominal organs of a pig fed six per cent casein ration and posted ten days after inoculation with TGE . . . . . Abdominal organs of a pig fed milk and posted two days after inoculation with TGE . . . . . . . . . . . . . . . The liver of a control pig fed six per cent casein ration e e o o e e o e o e o e e e e e e e e o e e e o e The liver of a pig infected with TGE and fed six per cent casein ration e e e e e o e o e o e e o e e e e e o X 5? 62 38 Active sweat glands of a control pig fed six per cent i'aseinrationooeooeoeeeeeoooeeoee Pustule of the skin of a pig infected with TGE and fed six per cent casein ration . . . . . . . . . . . . . . ?ustule of the skin . . . . . . . . e . . . . . . . . in organized thrombus in a blood vessel of the heart . Focal myocardial necrosis . . . . . . . . . . . . . . Focal myocardial necrosis . . . . . . . . . . . . . . Myocardial edema . . . . . . . . o o . . o . . o . . . Anitschkow myocytes in the edematous area illustrated in .igure 2} . . . . . . . . . e . . . . . . . . . . . Intra-epitheliel focal abscess of the esophagus . . . Intra-epithelial focal abscess of the esophagus . . . Parakeratosis of the cardia of the stomach . . . . . . Edema in the subnucosa of the glandular cardia cf the stomach . . . . . . . . . . . . . . . . . . . . . . . Murinous degeneration of the pylorus of the stomach . Catarrhal gastritis . . . . . . . . . . . . . . . . o Atrophy c: zancreatic acini . . . . . . . . . . . . . Cortex of kidney . . . . . . . . . . . . . . . . . . . Eafly'fibrosis of the kidney . . . . . . . . . . . . . Hyalnsvdegeneration around a small blood vessel of the kidney..o..........o..o.o.o.o. Clobules of fat in the cytOplasm of cells lining the proximal and collecting tubules of the kidney . . . . Fatty metamorphosis of cells lining the proximal conWluted :Ubujl.“ Of the kidney 0 e e o o o o o o o 0 An isolated basophilic hepatic cell . . . . . . . . . Basophilic stippling in the cytoplasm of necrotic ilVez‘cells'...-............o.... xi Page 96 98 98 99 van Kossa positiVe substances in cytoplasm of necrotic liver 99113 e o o o a o o o o o o o o o o o o o o o o o 110 Von Kossa positive sibstances in the cytOplasm of recrOtiC livar 30115 e o o e o o o o o o o o o o o o o 110 Hepatic nficrOSiS o o o o o o o o o o o o o o o o o o o 111 Hepatic necrOSis o o o o o o o o e o o o o o o o o o o 111 Hydropic degeneration of hepatic cells . . . . . . . . 112 Liver changes of a control pig fed six per cent casein ration o o o o o o o o o o o o o o o o o o o o o o o o 1L2 Liver changes of a control pig fed six per cent casein ration o o o o o o o o o o o o o o o o o o o o o o o o 113 47 as 1+9 50 51 53 55 56 57 58 fiydrOpic degeneration and perinuclesr basophilio condBHSQtion 0f the cytOplasm o o o o o o o o o o o o 113 Lobular necrosis and cirrhosis of the liver . . . . . 115 Cirrh°513 Of the liver 0 o o o o o o o o o o o o o o 11: Cirrhosis Of the liver 0 o o o o o o o o e o o o o o 116 Indentation of the Capsule of Glisson by hepatic necr°815 o o o o o o o o o o o o o o o o o o o o o o 116 i more normal hepatic lobule. surrounded by necrotic liver tifisnfis o e o o o o o o o o o o o o o o o o o 0 ll? LiVQr regeneration o o o o o o o o o o o e o o o o 0 ll? Liver regeneration . . . . . . . . . . . . . . . . . 118 Fatty metamorphosis of a hepatic lobule . . . . . . . 118 Intra-epitheiial abscess and parakeratosis of the Esophagus o o o o o o o o o o o o e o o o o o o o o o 138 Intra-epithelial abscess and parakeratosis of the 950phagus o o o o o o o o o o o o o o o o o o o o o o 138 Intro-epithelial abscessesvin the nonglandular portion ofthecardiaofthestomach............. 1‘60 Hepatic necrosis 0 o o o o o o o o o o o o o o o o o o 140 xii H'Vircpic degeneration 0f the liver 0 o e o o o o o o c e LinrOpi: degeneration Of thO liver 0 o o o o o o o e o o xiii am like I‘d I. INTRODUCTION A voluminous amount of research has been conducted on protein metabolism in the normal animal. Likewise much research has been reported on the pathogenesis of specific experimental infections. However. only a very limited amount of experimental data is available on the influence of an infection on protein metabolism. The primary purpose of this research was to obtain information on the influence of infection on protein metabolism using the growing pig as the experimental animal. This information is of value in arriving at protein requirements of both man and animals under natural conditions where infectious agents may have an influence on protein requirements, and also is of value in determining resistance to and recovery from specific infections. II. OBJECTIVES The specific objectives of this research were as follows: A. To determine the influence of an infective agent on protein metabolism. as measured by (l) nitrogen balance, (2) electrolyte balances. (3) serum protein distribution, (4) total serum protein. (5) clinical symptoms, (6) gross and (7) histOpathological lesions. B. To observe and record gross and microsopic lesions associated with protein malnutrition as influenced by an infection. C. To obtain information concerning the effects of specific lesions on absorption and metabolism of protein, potassium and sodium. D. To evaluate the effects of good and poor protein intake on susceptibility to and recovery from an experimental viral infection. E. To characterize the effects of transmissible gastroenteritis infection in normal and protein-deficient pigs. III. REVIEW OF LITERATURE A great volume of literature has accumulated on various aspects of protein malnutrition and upon various aspects of infection. A lesser amount of work has. however. been reported.on the interactions of these two entities. A complete literature review on these topics is beyond the scape of this dissertation. An.attempt has been made. however. to review those publications most pertinent to this study. This review will.be concerned principally with protein malnutrition and infection of the growing animal. A. mwmnnmam- Levenson and watkin (1959) reviewed the literature on protein requirements in injury and in acute and chronic diseases and stated that Hippocrates. some 2.500 years ago. admonished all to pay close attention to the connection between nutrition and disease. He indicated that the interactions of malnutrition and infection have been a problem from the beginning of time. McCollum (19h8) traced the development of our knowledge of protein in nutrition. According to his survey of the subject. F. Magendie (1816) appears to have been the first to perceive that there exist several kinds of nutrient principles. among them proteins. In 18h8. G. J. Mulder coined the term protein. C. Voit. in 186h. showed by experiment that when a dog was given gelatin only. or gelatin with fat. the animal excreted more nitrogen than it ingested in its diet. When. however. a small increment of good quality protein was added to 3 the gelatin. nitrogen equilibrium was brought about. Voit and associates fostered the hypothesis that the protein molecule undergoes metabolism as a whole. There'was a steady but slow'advance of knowledge through experiments designed to reveal the significance of the amino acids rather than "protein" in nutrition. K. A. H. Mbrner. in 1899. announced the-discovery of the amino acid. cystine. This was the beginning of the discovery of more than 20 individual amino acids. According to Maynard and Loosli (1962). J. B. Boussingault carried out the first basic balance experiment in 1839. He measured the carbon. hydrogen. oxygen. nitrogen and ash in the food of a dairy cow fed a ration that maintained her weight and the outgo of the nutrients in the feces. urine. and milk. Later he made similar studies with a horse and other species. B. Ergtgin Baguigggent in,thg_§ig. Wintrobe (1939) determined that at birth pigs require daily from 100 to #00 calories and at four weeks of age 35 to 200 calories per kilogram )f body weight. Suckling pigs consume 20 to 3“ grams of protein per kilogram of body weight and at three weeks of age 7 to 11 grams. The most satisfactory experimental diet consisted of casein 9.5 grams. lard b.0 grams. codpliver oil 0.5 grams. sucrose 21.0 grams. salt mixture 2.2 grams. giving a total of 37.2 grams. He further determined that sucrose was satisfactory as the source of carbohydrates. In fact. it was as good as lactose. dextrin or whey powder. Reber. Whitehair. and MacVicar (1953) found that rations containing #1 per cent protein produced maximum weight gains and feed efficiency for the very young pig. As the pig approached eight weeks of age. a level of 20 per cent protein appeared to be used as efficiently as higher levels. .mawtsn _t,gl. (1953) raised pigs that had not received colostrum on dried skim milk. vitamins. minerals. and antibiotics and lard. Pigs did not survive on diets without lard. Carmpton and Mass (195“) develOped a meal mixture suitable as the entire ration to be fed to pigs weaned at ten days of age and observed that 30 per cent protein was preferable to 26 per cent. They also determined that 13 per cent fat gave a better feed efficiency than eight per cent. Peo 23.21. (1957) reported that the protein requirement in baby pigs is high and essentially agreed with Babe: and coaworkers (1953). The pigs weaned at seven pounds body weight and kept on a twoaweek test period made maximum gains on a 30 per cent protein ration which was the highest protein ration fed in this experiment. Pigs on a four—week test period from the above average body weight made maximum gains on a 20 per cent protein ration. Jensen gtflgl. (1957) concluded that the minimum protein requirement in pigs weaned at from two to eight weeks of age was approximately 17.5 per cent protein. Rutledge gthgl. (1961) suggested that since cows' milk contains 33 per cent protein on a dry weight basis that pigs weaned at three weeks of age have at least 20 per cent of dietary protein of high quality to promote maximum nitrogen retention. Lloyd and Crampton (1961) suggested that pigs weaned at two weeks of age be fed a 24 per cent protein ration for a foureweek period and at that time changed to an 18 per cent starter ration. Manners and McCrea (1962) reported that pigs from two days of age to 28 days of age should be fed a ration containing approximately 25 per cent protein. The National Research Council (1959) in the publication Nutrient W 2.1“. his: rewmanded that piss weighing ten 0 pounds be fed a ration containing 2“ per cent crude protein. Mertz _e_t_ al. (1952) reported that the aminc acids which must be present in the diet to promote good growth of the weanling pigs are arginine. histidine, isoleucine, lysine. methionine. pherwlalanine. threonine, tryptophan. and valine. Dudley gt 3;. (1962) measured the composition of essential amino acid mixtures as the sole source of nitrogen for the baby pig and their results were somewhat higher than those suggested by the National Research Council. Using the rat as the experiment animal. Harper (1959) deter- mined the essential amino acids in a diet containing a low level of casein (six per cent) as the only source of protein and determined the limiting amino acids for the growth of the rat in the following order: First, sulphur-containing amino acids: second. threonine: third. tryptophan. isoleucine, leucine. histidine. valine and phemrlalanine: fourth. lysine; and finally. arginins. In addition he reported that when sucrose is the source of carbohydrate the total food intake is low while when dextrin is the source of carbohydrate. the intake is high. A mixture of all of these amino acids. together with glutamic acid. was required with six per cent casein to support a rate of gain of young rats equivalent to that obtained with compa. a}. L-‘- rats fed an adequate purified diet. Rama Rao (1961), in an experiment similar to that of Harper in the rat. determined the pattern of amino acids required by the growing rat. Basically. the figures that he determined were slightly lower than those of Harper. Longenecker (1959) concluded that the limiting amino acids in the dog on a.ration of casein as the protein source are: arginine. methionine. phenylalanine, histidine. threonine and lysine. respectively. .c..mmwsenmmi 1cm Platt (1958), Director of the British Medical Research Council. stated that two main types of protein malnutrition can be distinguished: marasmus and kwashiorkor.. Marasmus can.be defined as a protein deficiency accompanied.by a usually severe caloric deficiency. Kwashiorkor may be described as a protein deficiency unaccompanied by a.caloric deficiency. Protein deficiency in children in most areas of the world occurs in association with some degree of caloric deficiency. When the latter is marked. many authors prefer to designate the corres- ponding clinical syndrome as marasmic kwashiorkor. 2. gistogy Kwashiorkor was recognized by many African tribes who gave it a variety of names, most of which indicated certain features such as edema. changes in the color of the hair and skin. or the displacement of the child from the breast by the new pregnancy. Trowell and Davies (1952) reported that modern knowledge dates from the studies in the 1920's and 1930's of Normet in French Indo-China. Procter in East Africa, and Payne in Haiti. ‘Williams (1933) insisted that the condition. as she saw it in children. was a new clinical entity, and two years later‘Williams (1935) called this syndrome ”kwashiorkor”. The term kwashiorkor was from the Ga language of the Gold Coast and literally translated means ured boy”. However. she indicated that this term was in use on the Gold Coast to refer to "the disease that the deposed baby gets when the next one is born". Mere than it names have been given the various syndromes. the main and common element of which is an absolute or relative insufficiency of protein in the body. and usually in the diet. of the affected subjeCL (Flatt. 19 8). The term kwashiorkor has been accepted by most investi- gators only within tne past few'years. 3. Geogzgphical Distribution The clinical syndrome commonly known as kwashiorkor has been described in most countries of the world. Trowell g;_;;. (195b). Today it is. however. primarily found in techr‘sally underdeveloped countries where there is a deficit of animal protein and where prejudice and poverty limit the child's diet largely to starches or cereal gruels and thin soups. Since kwashiorkor is found in so many areas. the clinical and pathological manifestations vary somewhat from place to place. this being due primarily to differences in diets and feeding customs. There are several well documented studies of the condition in Jamaica (waterlow and‘Wills. 1960a. 1960b). India (Bagghi. 196G; Chanda. 1958; Rao, 1959). Haiti (Jelliffe and Jelliffe. 1960}, Africa (Mayer. 1959; Senecal. 1958: Suckling and Campbell, 1957: Trowell §t_gl.. 1952), Central America (Ascoli and Brush. 1960; Echar. 1958; Fernandez. 19633, and certain other tropical and subtrOpical areas (Baptist g£_§l.. 1959). There has only been one case reported in the literature of the United States. and that one was a report of three cases in Navajo children (wolf. 1961). hwy-lea Mest investigators agree that this condition occurs after 'weaning and most generally occurs before five years of age. The child is usually weaned and is suddenly changed to a full adult diet which may contain little protein, little fat and much carbohydrate and roughage. Rao (1959) stated that malnutrition strikes the child at a time when he is being eXposed.to increasing dangers of infestation and infection. and when his protein requirements are higher than in any subsequent period. Trowell and Davies (1952) explained that after weaning there is a gradual weight lag and in the second year of life the weight drifts still further. There are frequent occurrences of diarrhea. sow Follis (1958). in his book, described a variety of symptoms. and he stated that it is generally agreed that the syndrome usually consists of: retardation of growth, weight loss with muscular wasting, apathy and other psychic changes. dermatitis, changes in hair. edema, diarrhea. and enlargement of the liver. Jones and Dean (1959) found a high degree of correlation between the degree of retardation in skeletal development and.measure- ments of height or subischial length. Heard g§,§l, (1958) stated that subcutaneous fat and fluids are increased as a result of high carbohy- drate intake. Edema is a constant feature of all advanced cases. It usually starts in the feet but the face is soonlinvolxed. Eventually it becomes severe and involves all the dependent parts. Trowell gt_£l, (1952) wrote that mental apathy is profound and consistent and it is most unusual for a child to retain any semblance 10 of alertness. Skin lesions most frequently appear first in the inguinal region and later may be scattered over the body. Their dis- tribution appears to be determined largely by local.trauma or sites of pressure. The dermatitis begins as dusty patches and becomes more darkly pigmented with time. The skin.may remain.dry with some tendency to scaling, but in severe cases it tends toward exudation or suppuration. Diarrhea is a constant symptom. Ascoli and Brush (1960) reported in Central American communities 391 episodes of diarrheal diseases per 100 children, ages 6 to 11 months. with a gradual decrease accompanvng LiLLZ‘fi59 mytga‘ R30 gt_§;, (1959) in a survey in India reported that one quarter of the children examined had had recurrent diarrhea. and enlargement of the liver was common. 6. Eathology a. Gross pathology Post-mortem examination reveals commonly a pale. fatty. almost diffluent liver. Terminal pneumonia may be noted and occasionally atrOphy of the pancreas. b. 9 inical pathology A moderate normocytic. normochromic anemia is usually present. and MacDougall (1960) implied that it is not primarily associ- ated either with a deficiency of protein or of vitamin 812. MacDougall (1960) and Walt 22.51. (1956) have reported a megaloblastic anemia but they think that this condition is secondary. Baptist gtflal. (1959). Kinnear and Pretorius (1956), and MacDonald (1960) have established that hypoproteinemia. particularly hypoalbuminemia, is characteristic of kwashiorkor. MacDonald suggested ll that the protein metabolism in kwashiorkor is similar to that in marasmus. and that the clinical differences found between the two conditions are due to some other factor. possibly the relative excess of dietary carbohydrate in kwashiorkor. Fasting blood-sugar levels in patients with kwashiorkor were shown to be substantially lower than those of normal controls. Slone ; (1961) suggested that the occurrence of fatal hypoglycemic attacks in protein malnutrition may in part be due to the presence of a defect, in gluconeogenesis. Schendell g; 3;. (1959? measured the amino acids in the urine in four infants with kwashiorkor before treatment and obtained an average value of 33.85 mg. of amino acids per kilogram per day: normal mean value should be 14.23 mg. per kilogram per day. The increased amino aciduria was due to a slight increase in all of the 17 amino acids measured except histidine, alanine, and cystine. Following a short period of treatment with milk, the mean excretion had fallen to 23.66 mg. per kilogram per day. In another report. Edozien gt 2;. (1960) proposed that the aciduria could be the result of a defect in the reabsorption mechanism of amino acids by the renal tubules. Mentgomery (1960} indicated that infants with kwashiorkor have a magnesium deficiency though no correlation was noted between the clinical state and the magnitude of the magnesium depletion. Whterlow and Wills (1960a and 1960b) have observed a phosphorus depletion in.this syndrome and they suggest that the greater amount retained upon repletion may have been taken up by bone. c. Microscopic pathology Williams (1933) described fatty infiltration of the L 12 liver as a characteristic lesion found in kwashiorkor. It begins in the peripheral cells of the hepatic lobule. The fat globules increase in size and gradually encroach on the central areas. until in severe cases the whole lobule may be filled with fat. Davies (l9h8) and Chanda {1958) report that perilobular fibrosis develops and increases to the point that the fibrosis eventually strangulates the peripheral liver cells. MacDonald (1959) explained that the increase in hepatic lipid is not associated with an increase in the phospholipid or non- saponifiable portions, but with an increase in fatty acids which tend to become more saturated. MacDonald (1960) suggested that this fat is the result of the excess dietary carbohydrate and not the protein deficiency itself. Thompson and Trowell (1952) observed that the pancreas had. first, a reduction in the zymogen granules and, later. total atrophy of the acinar cell cytoplasm. The islands of Langerhans appear to remain intact. Parallel with the above changes. there is a decrease in enzyme production which may help to explain the passage of large quantities of undigested food in the stool. 7. Treatment Most investigators stress the role of infections in precipi- tating cases of kwashiorkor. especially those causing diarrhea. Behar (1958) recommended the use of antibiotics during the first eight to ten days of hospitalization because of the prevalence of infection. The standard method of avoiding protein deficiency is the use of cows' milk as a supplement to the remaining portion of the diet. Behar studied a vegetable mixture containing adequate propor- tions of most of the essential amino acids. These trials in Central America have given good results; no complications have developed during a loo-day interval. The average retention was the same as that per equivalent weight of milk protein. Another mixture was devaloped by Clegg and Dean (1960) for use in Central Africa. This diet produced nitrogen retentions that ‘were approximately the same as those produced by a milk diet. This mixture has given excellent results in the treatment of acute kwashiorkor. 1:. wmwmm Knowles (1957) and McCance (1960) produced lesions resembling kwashiorkor in the young pig by feeding diets with lowaprotein content. The pigs lived longer than six months if they were not started on the IOWAprotein rations (four and one-half to six and one-half per cent) until they were at least two weeks of age. Knowles stated that there appeared to be a somewhat critical age before which it is not desirable to start feeding pigs the lowbprotein ration. Heard.§t_3;, (1958) noted that striking differences have been obtained in the reSponse of young pigs by supplementing a lowbprotein ration with additional carbohydrate. The results indicated that pigs fed on a lOWbprotein ration developed a syndrome resembling marasmus in children: the assiduous feeding of extra calories as carbohydrate. precipitated a form of protein malnutrition resembling kwashiorkor. MacDonald and Charavi (1960) used a similar ration in the rabbit and concluded that the presence of an excess of sucrose. in a diet that was lb low in protein. seemed to aggravate the protein deficiency and the extent of the changes in serum proteins and liver lipids were related to the amount of sucrose consumed. Lowrey and associates (1962) at Cornell University. placed five groups of threedweek-old pigs on the following regimens: (1) control group. (2) five per cent protein (casein) with three per cent fat, £3) five per cent protein {caseinl with 23 per cent fat, (4) five per cent protein (wheat gluten) and three per cent fat. and (52 five per cent protein (wheat gluten) with 23 .er cent fat. The pigs were given the above feeds 3g,libitgg for lb weeks. Deficiency symptoms noted were similar to those reported in kwashiorkor in humans. Symptoms were most severe in pigs fed wheat gluten with 23 per cent fat and least severe in those fed casein with three per cent fat. The pigs in the former group were emaciated and feeble during the last two weeks. had reduced rectal temperature. and had lower hematocrit values than the other groups. Hemoglobin was depressed with wheat gluten as compared with casein. but total serum protein declined to about four grams per 100 m1. after six weeks in all groups and then remained constant. Total globulins remained constant in all groups throughout the experiment. weight gains were similar during the first eight weeks when casein was fed in the presence of 23 per cent fat, but weights reached a plateau and remained constant during the remainder of the trial. Wheat gluten failed to support body weight gain at either level of fat. Pellett (1957‘ related that when protein content of the diet of the pig was raised from 4.5 per cent to 7.5 per cent, digestibility immediately increased from 56 per cent to 80 per cent. He suggested that the period on the high level of protein allowed the development of increased amounts of digestive enzymes to occur. Sidransky (1960) described lesions in the rat that resembled some of the alterations described in kwashiorkor. This was produced by force-feeding them from three to seven days with a purified diet devoid of certain essential amino acids or with diets in which the sole source of protein was natural plant foodstuffs known to be deficient in certain amino acids. Animals fed the same diet 9g ligitgg_for seven days. in contrast to those force-fed. consumed less food and fewer pathologic changes were found at necropsy. It was concluded that the difference in results between the force-feeding and the 5g libitum feeding was thus related to the quantity of the diet consumed. Williams {1961) reported that the amour' of nitrogen per liver cell of protein deficient animals dropped to 68 to 74 per cent of that of pairedpfed control rats. The nitrogen per cell of pairedpfed CORP trols fell to 68 to 8b per cent of the normal 2g iggiagmlcontrols. The ratio of liver weight to body weight increased markedly in the protein- deficient rat. French g;_5l, £l9hl) demonstrated that rats on low- protein or nonprotein regimens. overcompensate for loss of body protein in periods of realimentation. As compared with controls. such animals require a longer time on a low-protein diet before their urinary nitro- gen falls to an acceptable endogenous level. Sidransky and Baba (1960) force-fed rats a purified diet devoid of the amino acids. valine and lysine. and in earlier experi- ments fed diets deficient in threonine. methionine and histidine. The changes that occurred in all of these trials were very similar and included a fatty liver, excess hepatic glycogen” and atrophy of the pancreas, parotid salivary gland. thymus and spleen. This suggests , .3 CF. 1shat deficiencies of many of the essential amino acids may produce changes which reflect an overall protein deficiency rather than a deficiency of a specific amino acid. Kumta gt_g;, (1958) noted that nitrogen intake decreased and the percentage of absorbed nitrogen retained was reduced when rats were fed an amino-acid-imbalanced diet. Ousterhout (1960) published the survival time of chicks fed diets lacking the following amino acids: isoleucine, 18 days: valine. 19: both phenylalanine and tyrosine, 23; methionine, 26; arginine, 27: threonine 27; leucine. 28: both methionine and cystine. 28; all essential amino acids, 28; phenylalanine, 3%: the combination of valine, isoleucine and leucine. 3“: all amine acids. 35; tryptophan, 36; lysine. 53: and histidine. 60 days. The survival time was longer in case of diets devoid of all essential amino acids and all amino acids. than for most single.amino-acid deficiencies. Heard.g;_§l, {1958) and Knowles (1957) reported substantial loss of serum albumin in the pig placed on a lowbprotein.diet with additional carbohydrate. MacDonald {1960) found a similar decrease of serum albumin in the rabbit. Yuile gt 3;. (1959) demonstrated that the rate of plasma-protein turnover is more rapid in dogs fed adequate dietary protein than when a diet devoid of protein is fed. The difference in turnover was reflected in a total protein half-life of 4.8 days with protein feeding versus 7.8 days without protein in the diet. Additions of dietary protein from 10 to 30 per cent caused no further increase in the rate of plasma-protein turnover. Friend g§_a;. (1960) commented that signs of vitamin A deficiency with protein malnutritionaee due to IOV'serum albumin. which reduces the transport of vitamin A from the liver, where stores may be adequate, by restricting production of the water-soluble albumin-vitamin A complex of plasma. McLaren and Bagghi {1958‘ believed that protein deficiency neither hastens the onset nor enhances the severity of xerophthalmia. MacDonald an Charavi {1960? reported that. as determined by serum electrophoresis. there is a marked decrease in the albumin, alpha-globulin and beta-globulin fractions in rabbits on a high dietary sucrose intake. Periportal lipid accumulation in the liver has been produced in all species placed on a lowaprotein diet with added carbohydrate. Wilgram 2§_g;, {1958‘ produced similar pathological changes in primates on a choline-deficient diet. burhin {19603 obserVed that excess glyco- gen in the liver is as characteristic a feature of protein deficiency as is the deposition of fat. Through a series 0: ex eriments. they demonstrated that glucose-é-phosphatase fails to d Velop in pigs o. low-protein plus carbohydrate diet. The addition of five per cent casein to these diets allows normal enzyme synthesis to proceed and also improves the pig's ability to mobilize liver giycogen. Flatt {1960}, Stewart and Heard {19593. and Volk and Lazarus (1960) observed that there are alterations in the islands of Langerhans and in the exocrine portion of the pancreas. Volk and Lazarus suggested that the acinar atrophy is probably due to lack of methionine. 7he changes in the pancreas support the concept of a precariously balanced endocrine system resulting from prolonged deficiency of dietary protein. (I) P. JI Platt and Stewart (1960) studied the nerve cells of the spinal cords of pigs on low-protein diets and reported that dust-like Nissl granulea.increased number of glial nuclei and satellitosis were present. Godwin (1960) noted that pigs on a lowbprotein diet with additional carbohydrate had a skin which appeared dry, scaly. wrinkled and cracked. and the hair was long, fine, dry and had an unkempt appearance. MicroscOpic examination showed a progressive thinning of the epidermis. which was often only one cell thick. The dermal papillae were almost absent. These changes are complicated by the possibility that in protein malnutrition other dietary factors may not be available to the skin. Cabak 2;,;;. (1962) explained that under-nutrition. by preventing growth, prevented the normal eXpansion in the animal‘s surface area and maintained a high hair and gland count per cubic millimeter. They further stated that, in spite of the under-nutrition, the hair grew slowly in length and to some extent, in thickness. The result was a long silky coat. Under-nutrition did not lead to follicular or glandular atrophy. E. Erotgin Malnutrition inLAnimals figsultigg in Liver Necrosis Lag Obel (1953) reviewed the early literature on liver necrosis in swine and stated that Kleinpaul, in 1907, was probably the first to observe and report on this syndrome. Others on the continent of Europe periodically reported on liver necrosis in swine. Some of the German authors believed they found some correlation between the appearance of toxic liver dystrOphy and cod-liver-oil feeding. 1? Quin and Shoeman (1933) described cases of idiopathic hemorr- hagic hepatitis in swine in the United States. Graham gt_§;, (19h0). at the Illinois Experiment Station. reported on liver degeneratiOn in swine that had consumed pulverized clay pigeons and coal tar. Naftalin and Howie (l9h9l,in Scotland. observed a similar necrotic and fibrotic liver lesion in swine raised under poor hygienic conditions. Obel (1953). in an extensive paper. suggested that the disease be called hepatosis diaetetica. She mentioned that Hjarre in 1952 stated there are two meanings of the term toxic liver dystrophy. In the one case it may refer to some etiologically uncertain. frequently acute and fatal disease with characteristic liver changes occurring in swine and other animals. In the other case it may refer to processes in the liver arising from various causes and, most frequently. characterized by centrolobular. degenerative or necrotic changes. She reported that hepatosis diaetetica was one of the most common swine diseases occurring in the autopsy material at the State Veterinary Medical Institute. Stockholm. Sweden. ranging from 6.7 per cent to lb.5 per cent of all pigs posted in the years 19“? through 1951. The greatest number of cases occurred during the months of October and November and in pigs between 5 and 1h weeks of age. Gross examination revealed anemia, waxy degeneration of skeletal muscles. and pericardial. pleural and peritoneal effusions. The most outstanding lesions were in the liver. The liver tissue presented a mottled. mosaic-like pattern because of regressive changes and hemorrhages in groups of lobules. The undamaged liver tissue had a lighter grayish-brown color. The changes were distributed throughout the lobes. The older lesions were 2? depressed and there was an increase in perilobular connective tissue. Twenty-two per cent of the cases exhibited ulcers in the fundus of the stomach. There were occasionally edema in the submucosa and hemorrhages in the fundus. Histologically. the liver changes involved single ldbules or groups of lobules while others close by remained entirely intact. The hepatic cells undergoing slight change exhibited an eosinophilic cytOplasm and the cells were somewhat separated from one another. In many cases water-clear. Scharlach R-negative. vacuoles were found in peripheral cells of the lobule. Fatty degeneration of the liver cells was infrequent and did not appear to belong to the typical picture. There was either hyperchromatosis of the nuclear wall or no change in the initial stages. In advanced regressive pro- cesses, the hepatic cords were often not distinguishable. Necrosis was indicated by eosinophilic cytOplasm and pyknosis. karyorrhexis and karyolysis of the nuclei. Necrotic liver cells were often affected by dystrophic calcification. In lobules with more pronounced regressive processes. numerous erythrocytes had migrated to the spaces of Desse. and the sinusoids were compressed. Necrotic liver cells underwent autolysis. The empty space left by the liver cells was graduallv filled by mononuclear cells and fibroblasts. The necrotic parts of the lobules were gradually replaced by fine fibrillar granulation tissue. Finally, there was left a small scarred lobule. greatly compressed by pressure of the surrounding liver tissue and enveloped by thickened perilobular connective tissue. From intact liver cells remaining in the periphery of the lobule. a regeneration could take place. with the formation of small islands of hepatic cells. The new liver cells possessed strongly basophilic cytoplasm and enlarged nucleoli. Some nuclei were double normal size. Cbmmonly there was proliferation of bile ducts in the interlobular spaces. It was characteristic for the lobular necrosis occurring in hepatosis diaetetica to appear in patches. and a hemorrhagic tendency was pronounced. These liver lesions closely resembled those occurring in dietary liver necrosis in rats. Hove and Seibold (1955l in a limited report, observed a fatal liver necrosis in growing pigs fed a diet deficient in vitamin E. and containing six per cent protein as furnished by soybean meal. and two per cent cod-liver oil. Of six pigs fed this diet from weaning. three died suddenly with massive acute hemorrhagic liver necrosis. Two of the survivors. when slaughtered. had necrotic cirrhosis of the liver. but little liver fat. Five of the six animals in control lots on the same basal diet but supplemented with alpha-tocopherol acetate survived to slaughter and failed to have appreciable liver damage. Eggert 33‘3l. {1957) conducted an experiment with three groups of six pigs each. Four of six pigs. receiving a basal diet devoid of vitamin E, died within 53 days after the start of the test. Deaths occurred suddenly without previous outward manifestation of ill health. Each pig that died had a marked necrosis of the liver. A yellowishébrown discoloration of the body fat and hemorrhages in the lymph nodes and gastrointestinal tract were evident in two of the four pigs. No deaths occurred in the other two groups, one receiving vitamin E and the other selenium. Average daily gains and feed efficiencies 'were good for all animals and no marked differences in growth rate were noted for any of the treatments. One-half of the animals in the vitamin E- and seleni'm-supplemente-d groups were illed, and there was no gross evidence of liver necrosis. Dodd and Howling (1960) published a paper on a natural ort- break of liver necrosis in New Zealand. In addition to liver necrosis. there were severe degenerative lesions of skeletal and cardiac muscle. Forty animals in a group of 200 had clinical signs of muscular damage. Lear. (1961'? reported on pigs reared on diets low in tocopherol and essential fattv acids and found no differences in body weights or carcasses at slaughter. These animals were placed on emerimental rations at ten pounds live weight and remained on experiment until they reached 200' pounds live weight. Plasma taco-pherol levels were two to seven times higher in pigs on rations supplemented with alpha- tocOpherol. In a following experiment. plasma levels were seven to 13 times greater. ‘ ~ ~ - c. . 1 Elias 9; 11.. (1951+) described what is comonty called a normal liver of the pig and they stated that the pig liver, characterized by Sharply defined lobules separated from each other by connective tissue SI.” . represents a form of portal cirrhosi of unknown etiology. 2‘. .At -.~Jeichselbaum =;.L9’3_Sr wrote that rats fed a diet deficient in rho Wstine and methionine became ill and died in about six weeks. c. .‘ o 1 a ‘- post-mortem examination, the livers of mouse animals contained definite hemorrhages throughor'. t . Schwarz {1951+} stated that it is quite certain that. in exPerimental animals, two completely different deficiency syndromes 23 can be distinguished. These are fatty liver and cirrhosis on the one hand and acute (massive) liver necrosis, preferably called necrotic liver degeneration. on the other hand. These two disease entities can be produced separately and even occupy a mutually antagonistic position in response to various dietary factors which are protective. Gyorgy and Goldblatt {1939) gave the first clear description of liver necrosis. They fed rats a diet designed to produce cirrhosis of the liver and found that some of the animals died of acute liver necrosis. The distinction between the pathogenesis of liver cirrhowzs and acute massive liver necrosis was made a few years later by Daft. Sebrell and Lillie (l9h2‘. They observed that choline prevented fatty infiltration and cirrhosis and cystine prevented hemorrhagic necrosis. and methionine prevented both cirrhosis and hemorrhagic necrosis. McLean and Beveridge {1952) observed no liver necrosis in groups of rats fed rations devoid of protein or containing casein at levels of one to two per cent. At concentrations of three and four per cent casein there was a low inciience of liver damage, and none at levels of greater than four per cent. Their results indicated that some factor other than cystine. methionine, and alpha-tecOpherol had a profound effect on the development of acute massive hepatic necrosis. Schwarz {lSfih} oXplained that three different nutritional factors have been recognized to be specifically connected with dietary necrotic liver degeneration: cystine. vitamin E. and Factor III. Duets for the production of the deficiency in rats must be low in all three of these factors. f Bonetti and Stirpe (1962) found that adequate dietary protein 2h level provided by 20 per cent casein as well as dietary addition of sulphur-containing amino acids did not afford complete protection against liver necrosis in the rat. Vitamin E or selenite protected completely when added in sufficient amount to either ration. It was concluded that the mortality observed in rats fed the 20 per cent casein diet was due to lack of a Factor III-active selenium complex in the casein used. The regeneration of the liver was slightly impaired in rats fed five per cent casein diets when compared with those fed 20 per cent casein; it was not affected by dietary addition of methionine. qystine or vitam1n E. Schwarz g§,§l. {1959) concluded that the protective effect of L-cystine against dietary liver necrosis is primarily the result of contamination with traces of a Factor III-active selenium compound. Necrotic liver degeneration is. therefore, attributable to the simul- taneous lack of only two essential nutrients: namely. vitamin E and Factor III. rather than the three. Schwarz advanced the hypothesis that vitamin E and Factor III participate in alternate pathways of intermediary metabolism; for instance. of electron transfer. Schwarz (1960) noted that one atom of selenium. in the form of Factor Ill. affords as much protection as 700 to 1.000 molecule; of vitamin E in preventing dietary liver necrosis in the rat. L—cystine. in turn. possesses only a very small fraction of the potency of tocOpherol. Methionine and qystine. when clearly free from traces of selenium. do not prevent liver necrosis. Regardless of the interrelationship, selenium neither spares vitamin E, nor does it substitute for it. It is an essential dietary constituent in its own right. 25 Fite (195b) characterized the pathogenesis and histological liver changes of more than “00 rats on diets which precipitate this syndrome. The most significant finding was the essentially normal histoIOgic appearance of the liver almost up to the point of appearance of necrosis. Liver glchgen content was normal in all animals examined except for those undergoing necrosis. in which it was deficient. There were no definite abnormal fat deposits except in animals with Mrly necrosis, in which droplets of moderate size were distributed in moderate numbers throughout the liver. Two distinctive types of changes in the liver cells were observed. The first of these was diffuse karyolysis and karyorrhexis cf the liver cell nuclei with relatively little change in the cytoplasm of the liver cells. The second type of degenerative change Wes observed in many animals. Fine eosinophilic granules, one to two microns in diameter, were seen dis- tributed in the cytoplasm. These granules increased in size and finally underwent calcification. Initially. the nucleus was unaltered but later it faded out of existence without undergoing karyorrhectic changes. The so-called hemorrhage was an accumulation-cf blood within the sinusoids at the margins of necrotic areas rather than extravasaticn outside normal channels. F3 Intgzagtign§,g§_Nutrition and Lnfection Llama ScrimshaW'gt,al, (1959) published a detailed review on this subject and used the term antagonism as that situation in which a nutritional deficiency results in decreased frequency or severity of an infection.and synergism as that situation wherein a deficiency results in an increase in frequency or severity. In general, synergism was the usual result when the dominant action n” fie nutritional deficiency was on the host, and antagonism when the main impact was on the infectious agent. Scrimshaw further noted that there are two forms of malnu- trition: in the first, a reduction in intake of all essential nutrients leads to a state of undernutrition or inanition called marasmus in children; in the second, there is a relative or absolute deficiency of one or more specific nutrients. The distinction is important because the former may not be associated with detectable physiological or biochemical changes, while signs of specific nutritional deficiencies develop as a result of disproportion among nutrients. The many bio- chemical changes in kwashiorkor and their absence in infantile marasmus furnish an example. 2. Kings Lnfectiog Cannon (1949), at the Pathology Department, University of Chicago, was of the opinion that on the basis of research. practical experience and assessment of available data, dietary protein constitutes a major immunologic contribution to antimicrobic defense. If dietary protein is to be effectively utilized, it should be accompanied by an adequate intake of other dietary essentials, including calories, vitamins, and salts. In the field of experimental virus infection, Rous (1911) undo one of the earliest observations concerning the effect of nutrition on the transmissible chicken sarcoma. He reported that intercurrent illness of the host checked the development of the tumor and that L¥_ young, healthy, well-nourished fowl proved more susceptible than did the thin and the ill bird. Olitsky gt‘gl. (1928) noticed a similar response in guinea pigs inoculated with the virus of foot-and-mouth disease. Frequently, the animal suffering from malnutrition or intercurrent infection had a delayed appearance of the primary and secondary vesicles. Rivers (1939) made the observation that unhealthy and mal- nourished rabbits had less reaction to vaccinia virus and a lower titer to the active agent than did healthy animals. Sprunt (1942) described an experiment using rabbits as follows: In group A, they received no food but had free access to water; group B received neither food nor water: and group C received no food but had access to water and received in addition intraperitoneal injections of 50 ml. of physiological-saline solution twice daily. These regimens were maintained for ten days‘before virus inoculation and two days thereafter. The animals were ther fed amounts sufficient to maintain weight, and group B was given.free access to water. The animals were killed seven days after vaccination and the following observations were made: 1. Starvation but with access to water (group A) was associated with fewer or smaller vaccinia lesions than in controls. 2. Starvation plus dehydration (group B} was associated with even more marked decrease in vaccinia lesions. 3. Starvation with increased interstitial fluid (group C) was associated with increased number and size of lesions. Sprunt suggested that the greater inhibition in group B may be due to the restriction of the spread of virus particles by dehydration so that fewer host cells were exposed. while increased interstitial fluid as in group C had the Opposite effect. Clark (19“9) suggested that some of the violent changes that occur during alterations in water balance are responsible for changes in cell permeability and therefore. susceptibility. Olitsky and Schlesinger {l9fll} stressed the factor of edema when they observed that hypertonic salt solution, injected into the base of the tail of albino nice three and one-half to 2n hours before the cutaneous injection of herpes virus, increased infection both on the basis of success with loo-fold greater dilution of the virus and also shortening of the incubation period by one-third. Mixtures of hypertonic solution plus virus. injection of the hypertonic solution after the virus. and application of virus after the edema had subsided. had no enhancing effect. They suggested that the factors in edema which dilated the lymph vessels. rendered their walls more permeable and increased the rate of flow. bringing the virus into contact with a greater number of fibers in the cerium and therefore aiding infection. MacCallum and Miles (19h6) reported that attempts to transmit the three types of human hepatitis to the usual laboratory animals have been unsuccessful. However. the virus was successfully transmitted by inoculation of blood and feces from patients with infective hepatitis into rats on a proteinpdeficient diet. Kearney gt_gl. (1948) found that low-protein (nine per cent casein} and high-protein (36 per cent casein) diets exerted no influence on infection of mice with Theiler's GD VII virus. A marked effect of this disease has. however. been produced with diets deficient in tryptophan. with a lack of the characteristic signs of infection in 29 the majority of deficient mice. Pond 2£.éi- (1952) noted that tryptophan. isoleucine. methionine and valine deficiencies were most effective in reducing the incidence of paralysis and prolongation of the incubation period in Theiler's G. D. VII encephalomyelitis in mice. but none of the essential amino acids prevented eventual death of mice after infection. Davies gt 2;. (1952) observed that tryptophan and isoleucine deficiencies were most effective in prolongation of incuba- tion period. a decrease in total number of animals paralyzed. and an increase in death without typical symptoms of Lansing poliomyelitis in mice. Sprunt and Flanigan (1956) preposed that in mice and chickens there is an initial phase of increased susceptibility. a secondary phase of increased resistance. and a final phase of increased suscepti- bility of the host to viral infection. The relative resistance of the host on a low-protein intake is a function of the duration on incomplete diet administration before virus inoculation. and consequently. a function of the host's state of depletion. Therefore. they proposed that these animals on a low-protein diet show a cyclic susceptibility to virus infection. Squibb (1961) related that Newcastle disease virus infection in the immature White Leghorn cockerel. resulted in a greater growth depression when complete rations. rather than those imbalanced by deficiencies of lysine. were supplied. Trypsin or amylase supplementa- tion of a low—lysine diet resulted in increased growth of Newcastle disease virus-infected chicks. This phenomenon was observed. however. only when the experimental rations were deficient in lysine. 30 Scrimshaw 55,5l. (1959) stated that antagonism is the common and well-defined reaction in virus infections associated with nutritional disorders. Synergism is less frequent. Protein deficiencies ordinarily have little or no effect on viral infections. 3. figctegial Lnfections Clausen (1934). Howie (1948). McClung (1949) and Scrimshaw gt,gl. (1959) have very carefully reviewed the literature on the effect of nutritional deficiencies on bacterial infections. All of these studies indicated a more or less preportional loss of resistance with severe protein depletion. therefore were almost uniformly synergistic. b. chgr lnfections Scrimshaw'gtflal. (1959} in reviewing the available literature stated that all studies of the interaction of malnutrition or protein deficiency and rickettsial infections showed synergism. With few exceptions. the consequences of parasitic infection are generally more serious in malnourished and protein-deficient hosts-- a synergistic reaction. Synergism and antagonism were observed with almost equal frequency in protozoan infections. G. nggmissiblg gastroenteritis in Pigs ”3E! Doyle and Hutchings (1946). at Purdue University. first described and named this disease in pigs. The symptoms were diarrhea. vomiting in some cases. rapid loss of weight. and a high death rate in baby pigs. The most nearly constant post-mortem finding was a large amount of highly fluid intestinal content having a whitish. yellowish. or greenish color. Feenstra gt‘al. (1948). at Michigan State 31 University. noted similar findings and confirmed the results of Doyle and Hutchings. Whitehair 35,31. (1948) observed a morbidity and mortality rate from 70 to 100 per cent in pigs less than one week of age. They suggested the presence of an infectious agent other than a bacterial microorganism. In additional research at Purdue University. Bay gt_gl. (1949) reported that the disease was readily transmitted by a ground suspension of intestine. kidney. spleen. liver. brain or lung. They further reported that intracranial. intravenous. intramuscular. intra- peritoneal. subcutaneous or oral administration of filtrates produced the disease. Heat at 56° C. for 30 minutes inactivated the causative agent. as did 0.5 per cent phenol in filtrates incubated for 30 minutes at 37° C. It was therefore assumed. since filtrates were effective in reproducing the disease. that the causative organism was a virus. Doyle (1951. 1958). Bay (1952) and Young gt_gl. (1955) further reported on general features. symptoms and treatment of TGE. At the Nebraska Experiment Station Young and associates (1955) found a particle size of approximately 200 millimicrons and considered it a virus. The TGE virus multiplied in the lungs of pigs at two hours of age but not at six days of age. It did not multiply in the nasal mucosa at two hours of age but multiplied in the nasal mucosa at six days of age. The virus multiplied in all sections of the gastrointestinal tract and viremia was common 48 hours or more after inoculation. One hundred per cent mortality occurred in pigs less than one week of age: infections occurred at 11 to 17 days of age with low mortality; and at 24 days of age TGE infection occurred but was inapparent. Lee §;__1. (1954a) published a report on the mortality in young pigs as follows: 100 per cent in pigs inoculated when less than five days old. 67 per cent in pigs inoculated from six to ten days of age. 30 per cent in pig: iroculated from 11 to 15 days of age and four per cent in pigs inoculated after 16 days of age. Viable TGE virus could be isolated from the feces of 46 per cent of the pigs five weeks after inoculation and eight per cent eight weeks after inoculation. Lee _£_g;. (1954b) observed that oral administration of either filtered or unfiltered intestinal suspension from infected animals readily and regularly produced characteristic signs of TGE illness. and that parenteral inoculation did not produce the disease. It was therefore concluded that the natural route of infection was oral. In the acute phase of illness. the virus was found in low concentration in the blood. liver. spleen. brain and lung. and in tigh concentration in the intestines and kidneys. The virus was recovered from feces for two to eight weeks following inoculation. recovered from the lungs and kidneys for only a short time after inoculation. recovered from the blood only during clinical illness. and was never recovered from the urine. The mortality rate in a total of 111 pigs infected with TGE was 100 per cent in animals under five days of age; 67 per cent between six to ten days of age: and 30 per cent between 11 and 15 days of age. Pigs over 16 days of age when infected usually recovered. Gibbons (1957) found that the incubation period was from 12 to 96 hours in older pigs. Immunity can be transferred to pigs through the sows' milk by exposing the sow to natural infection or '33 feeding her infective materials 20 to 40 days before farrowing time. Attempts to innnunize sows in other ways have failed. Reber and Whitehair (1955) studied the effect of transmissible gastroenteritis on the metabolism of baby pigs. The pigs were fed a 35 per cent casein ration and were inoculated at 26 days of age. The average amounts of water retained by the pigs following infection was about one-half that retained during the preliminary period. The fecal water excreted by the pigs during the preliminary period was found. to average 1.5 gm. per 24 hours. while following infection there was a forty-fold increase. The nitrogen retained by the infected pigs was about one-third that which they retained during the preliminary period. There was a considerable decrease in sodium and potassium retained by the infected pigs with a negative potassium balance for the period of 21+ to 48 hours after inoculation. TGE infection did not influe-..:e the RVerage body temperature. plasma-protein level. or the serum-scdium lGVel. The infection did cause a decrease in the average blood glucose and a slight decrease in serum potassium. while the hemoglobin value "‘8 increased. It. was concluded that the mechanisms for absorbing and re‘llaining sodium by the pig were not disturbed but for potassium they “ere disturbed. initehair 32 g. (1950) reported that in one-day-cld pigs. bl00d urea values increased from 25 to 215 mg. per 100 m1.; in five- day-old pigs from 6.? to 41 mg. per 100 m1.; and in Zi—day-old pigs from 12.8 to 23.1 mg. per 100 ml. on the seventh day after exposure. All pigs dying from exposure to the infectious agent had high urea ”Id nonprotein nitrogen (NPN) values shortly before death. Clinical ; recovery from this infectious agent was associated with the return of urea and NPN values to normal. It was also observed that there was a decrease in bloodpsugar values from the time of exposure until the seventh day. Reber (1956) disclosed that TGE could be airborne for distances of 23 to 42 inches at air velocities of 15 and 52 cubic feet per minute. The infectious material apparently became airborne at the time of initial appearance of vomition or diarrhea. Incubation for four- to sixrday-old Hampshire pigs varied from 18 to 24 hours. Yusken 93,51. (1959) noted that total blood-protein and hemo- globin values were higher in infected pigs due to dehydration. Blood- glucose values were the same in normal and infected animals. but liver glycogen decreased to almost zero in the infected animals. They further reported that there was an increase in blood urea and nonprotein nitro- gen due to kidney damage. From zero to 54 hours after inoculation. total body weights and organ weights (lung, gastrointestinal tract. kidney, spleen and liver) were equal in normal.and infected pigs but from 54 to 90 hours they were lower in the infected piss. Reber and Yusken (1956) found that the per cent of nitrogen intake excreted in the urine was about the same for infected and non- infected pigs; however. the percentage of nitrogen intake excreted in the feces was about nine times greater in the infected group. The percentage of sodium intake excreted in the feces was about two times and that of potassium about six.times greater in the infected group.' They had increased the amount of sodium in the ration by about five- fbld over previous rations fed to pigs and concluded that a high level or sodium was of value in retaining sodium and potassium. Bay 23.51. (1953) observed that 52 of 62 pigs farrowed by sows exposed orally with TGE material lived following exposure. while only three of 36 pigs farrowed by nonexposed sows survived. Their results strongly suggest that a degree of immunity results when sows recover from either naturally or experimentally induced TGE and that there is a transfer of immune bodies through the sows' milk to nursing pigs. Nelson (1954) reported that in a large herd. losses from TGE ranged between 80 and 90 per cent of pigs. He infected 156 sows in this herd before farrowing and lost only 35 per cent of the pigs in the first 30 sows to farrow. Subsequent farrowings showed diminished clinical evidence of infection for about 25 days and the death loss fell pro- gressively until it was no more than would normally be anticipated. Other methods of producing immunity have been tried by Hurst and Doyle (1956) and Reber and Beamer (1956). but they have been unsuccessful. IV. MATERIALS AND METHODS A. Egpezimgntgl gnimals The baby pig was used as the experimental animal in these studies. This experimental animal was chosen because it (1) was of a size that permitted collection of tissue and excreta in quantities for analytical work. (2) was large enough to make clinical observations. (3) was readily available at a minimum of expense. and (4) was uniformly susceptible to an infectious virus that produced diarrhea. B. anaemia; The pigs of Experiment I and II were maintained in individual metabolism cages designed for quantitative collection of urine. feces and refused feed.- In Experimentjfil each experimental group of pigs was maintained in an individual pen. ‘ The pigs that were infected were maintained in separate isolation rooms and were cared for by different caretakers. The pigs were given supplemental iron either by swabbing the sows' udders once a day with a solution containing FeSOu. CuSOu. and sucrose in water or by injecting two milliliters of iron dextran intramuscularly. To maintain uniform consumption of feed. the paired-feeding technique was used in all experiments. The pigs selected for this work were usually two to four weeks of age. and placed on a good and poor protein ration for two to five weeks. At this age they were susceptible to an infection which produced the desired type of pathology. 36 37 Accurate records as to food consumption. urine and fecal excretions and general observations. such as temperature. weight changes and symptoms. were maintained during Experiments I and II. Urine and fecal excretions were not collected in Experiment III. C-Bstisa The pigs in Experiments I and II were placed in metabolism cages and were fed a mixture of vitamin D fortified cows‘ milk and a semi- purified dry ration {32 per cent casein). as a four-day period of con- ditioning the animals to the semipurified diet. The animals of experi- ment III were weaned and placed directly on semipurified rations. A semipurified ration containing 32 per cent casein was fed in EXperiments I and II (TABIE i). a ration containing 2k per cent casein was fed in Experiment III (TABLE 11). while a ration containing six per cent casein was fed in all three experiments {TABLE 117}. The only differences in the three rations fed were increases or decreases in casein content with corresponding decreases or increases of cerelose. The rations were prepared dry in a Hebert mixer and stored under refrigeration at 36° F. Quantities were prepared and stored in the refrigerator for a maximum of three days and were removed immediately before feeding. Feed intake was regulated daily with all pigs receiving the amount that the lesser consumed. 'Water was offered ég,libitgm, D. Pathogen The transmissible gastroenteritis (TEE) virus was used as the pathogenic agent. The virus of known pathogenicity was obtained from «I CI) TABLE I. Composition of swine ration. no. 1. W M’s“ Crude casein 32.0 Cerelose 53.0 Phi1lips and Hart Salt Mix (Ivy- 5.0 Lard 10.0 W“ i *Phillips and Hart719333. "Vitamin A 7.00. vitamin Dz 0.8. thiamine hydrochloride 1.32. riboflavin 3.3. nicotinic acid 22.0. calcium pantothenate 13.2. pyridoxine hydrochloride 1.1. choline chloride 1000.0. vitamin 312 0.022. ascorbic acid 300.0. biotin 0.02. inositol 1000.0. p-aminobenzoic acid 100.0. folic acid 0.5. DL alpha tocopherol 11.7. menadione “.0. mg. per kilogram of feed. TABLE II. Composition of swine ration. no. 2. lam-£5.92: _ _ i r _ PeLfiLnt Crude casein 2 .0 Cerelose 61.0 Phillips and Hart Salt Mix (IV)‘ 5.0 Lard 10.0 Vitamin supplement** *and "same as-in composition of swine ration. no. 1. TABLE I. TABLE III. Composition of swine ration. no. 3 W _ fiat—gm! Crude casein .0 Cerelose 79.0 Phillips and Hart Salt Mix (IV}‘ 5.0 Lard W" ‘and **same as in composition of swine ration. no. 1. TABLE I. 39 the Pathology Department', Purdue University. To detect the presence of the transmissible gastroenteritis virus in the inoculated pigs. the follwing prOcedures were followed: Iwo pigs. three days of age. weaned from the saw and placed on vitamin D- fortified cows' milk. were each incculated with two milliliters of a suspension of TGE virus pg; pg. Vomiting was noted in both pigs c.t approximately 40 hours after inoculation and a very watery yellowish diarrhea was noted at approximately 32 hours. The pigs were killed #8 hours after inoculation and the following changes were noted: the stomachs were distended with ingesta and some was. and the SMJll intestine: were filled and somewhat distended with a fluid material that contained some undigested ingesta. The small intestines. with their contents, were removed from the carcass and were then out up with scissors into apsroXJ- mately two-centimeter lengths. Tue cut-up intestine plus ontents measured 0 200 ml.. and this was diluted to 500 ml. with distilled water. The mixture was placed in a Waring Blender and further minced for approximately three minutes. A portion of the resultant mixture was placed in test tubes and centrifuged in an EEC International Centrifuge, EiZe a. Model V. at ZSQO r.p.m. for one hour. The fluid material was removed. placed in screwcae tubes and quick-frozen to a minus 79° F. Another portion was filtered three times through a double layer of cheesecloth.filtered through coarse filter paper. filtered through a coarse Seitz filter. and finall . to 9 remove bacteria. the filtrate was passed through a Sales 03 filter. This material was then placed in sterile tubes and quick- r zen to a minus ?”° ?. The two types of prepared inoculum. as just described. are referred to as the infective crude material and the infective filtered *Obtained through the courtesy of Dr. E. 0. Haelterman. Purdue University. Lafayette. Indiana. 2.0 material. respectively. Eight crossbred Yorkshire pigs. seven days of age. were treated as follows: Three pigs were each inoculated orally with two milliliters of the infective crude material and three pigs were each inoculated orally with two milliliters of the infective filtered material. and the remaining two pigs were isolated as controls. Animals in both infected groups began vomiting approximately 24 hours following inoculation and both groups had severe watery diarrhea beginning at approximately 36 hours after inoculation. Temperatures remained near normal for both groups. controls had no diarrhea and/or other symptoms of infection with TGE virus. The eight animals were killed with an overdose of sodium pentobarbital and necrOpsies were performed. Gross changes were as follows: The stomachs of all infected animals were filled and distended with a whitish-yellow. curdled milk. Figure la, page 93. The small and large intestines and coca were filled with a very clear watery fluid in which undigested curds of milk could be found. The mucosal surface of the stomach. small and large intestine. and ceca appeared to be covered.with a slight excess of mucus. The mesonteric blood vessels were hyperemic in about one-half of the infected animals. No lesions were evident in the organs of the thoracic cavity. None of the above changes were noted in the control animals. which appeared normal in all respects. The above symptoms and gross changes are consistent with those found in transmissible gastroenteritis of swine. 3- mmmw The pigs were maintained in Barn Number Five on the Veterinary Research Firm. Michigan State University. Research was initiated in June 1961 and was terminated in June 1963. #1 The information reported in this thesis was the result of data accumulated from a total of 68 pigs; 22 pigs were discontinued and data not reported because of complications from a different pathogen. Separate experimental groups were initially designated as follows: Experiment I consisted of 12 pigs: Experiment II, 11; EmperimentIII, ll: Experiment IV. 1h: Experiment V. 8: Experiment VI, 8; and Experiment VII. 26. Experiment I was reported in this paper as Experiment I. Experiment II and III were discarded; for convenience. Experiments IV. V, and VI were grouped tagether and reported as Experi- ment II: and Experiment VII reported as Experiment III. Selections were made by using random numbers (Goulden , 1952). F- M. l. fiemgtolggy Blood samples were drawn from the anterior vena cava. using ammonium and potassium oxalate as the anticoagulant. The samples were obtained Just prior to inoculation with the virus and. depending on the experiment, several collections were made at selected intervals following inoculation. Hemoglobin was determined by the cyanmethemo- globin method. Hematocrit values were determined by the micro method (capillary tube). White-blood-cell counts were made using Turk's diluting fluid. and the hemocytometer. Blood smears weretmade at the time of bleeding, stained with Wright's stain and a differential white- blood-cell count wee determined. ’42 2- WW Samples of urine. feces, feed and serum were analyzed for nitrogen content by the macro-Kjeldahl (Osborn n g. . L935). Con- tainers of urine were transferred from the freezer to the refrigerator the day previous to Kjeldahl determination and allowed to stand at room temperature for approximately one-half hour before running the sample. The specimen was completaly melted before measuring. the three- milliliter aliquot. All specimens were determined. in duplicate and titrations‘ with standard 0.10 N NaOH agreed within 0.30 milliliter. All results were reported in milligrams of KJeIdahl nitrogen per milli- liter of urine. Several methods of sampling fecal specimens were attempted. and uniform results were. obtained by grinding frozen feces with mortar and pestle. Approximately one-gram aliquots were taken using the mettler balance. run in duplicate. and duplicates agreed within 0.15 milligrams per cent. Que-gram samples were employed for assaying nitrogen content of feed by the procedure used in fecal de- termination. Serum samples were determined similarly to that of urine. except the aliquot was only one milliliter. 3- WMWW Sodium and potassium were determined in urine, feces and feed. using the Model 21. Colman Flame Photometer (Operating Directions for the Colman Model 21 Flame Photometer D-ZIISA. 1957). Lab-trol (Dede Reagents) as controls and Harleco (Hartman-Leddon Company) standards. g 4. mm The serum-protein fractions were separated on a Spinco. Model R. paper electrOphoresis system (Spinco Model R Paper Electrophoresis System Operating Instructions. 656824} at room temperature. Spinco- prepared buffer. dye and fixatives were used and the relative inten- sities of the separated proteins determined by scanning with a Spinco Model RB Analytrol. Routine sections were collected. preserved in Zenker's fixative. and were stained with hematoxylin and eosin. Sections of the liver. kidney and pancreas were preserved in acetate-buffered. ten per cent formalin. stained with; Sudan IV for determination of fat content. Sections of the liver. kidney and pancreas were preserved in Carnoy's fixative. stained with Best's carmine for determination of glycogen content. Other special stains were auployed when necessary. The histological procedures followed were according to the Armed Forces Institute of Pathology Manual of Histologic and Special Staining Techniques (1957). G. Specific ‘Progedggeg {9; Each W 1- W 1. Twelve Yorkshire crossbred baby pigs were taken from their dams at seven days of age and placed on a mixture of cows' milk and 32 p9” cent casein ration as a four-day conditioning period. When they H. were 11 days of age. six pigs were placed on a ration containing 32 per cent casein and six were placed on a ration containing six per cent casein. They remained on the respective diets for 32 days. at which time three animals on the 32 per cent casein ration and three animals on the six per cent casein ration were transferred to an isolation room and inoculated orally with two milliliters of a suspension of transmissible gastroenteritis virus. t the time of inoculation. daily food consumption was 195 grams of dry ration. Records were kept of feed intake and collections were made of focal and urinary excretions for three consecutive Zhehour periods. following inoculation with the Virus. Excreta were collected accordi.g to the method described by Lassiter g; 3;. £1956}. firine was collected in wide-mouthed. $.000-milliliter bottles containing 100 milliliters of seven per cent acetic acid. The volume of urine was determined. and a loo-milliliter aliquot was taken and frozen at 0° F. for later determinations. Fecal material was acidif ed with seven per cent acetic acid, dried in a hot-air oven. removed from the oven, allowed to come to equilibrium with air moisture. weighed and frCZen at GC F. for later determinations. If excess fecal material was collected. an aliquot of approximately 30 grams was taken, discarding the excess. The infection wnth TGE virus produced vomiting with onset of symptoms. and it was impossible. with the aparatus used, to separate the vomitus from urinary and fecal collections. The pigs were observed regularly for signs of illness. fleets; temperatures were taken twice daily. both prior to and following administra- tion of the virus. :iJ- 1 All animals were killed with a mixture of amobarbital sodium and butabarbital sodium. administered intravenously, at #6 days of age. and were examined for gross changes. Sections from the following organs were preserved for routine and special histological examination: stomach. duodenum, jejunum. ileum. cecum. spiral colon, descending colon. liver. kidney. pancreas. spleen and skin. 2. Experiment 1; Thirty Yorkshire crossbred baby pigs were taken from their dams at approximately 21 days of age and fed a mixture of cows' milk and 32 per cent casein ration for four days. When they were 25 days of age. 15 pigs were placed on a ration containing 32 per cent casein. and 15 were placed on a ration containing six per cent casein. All animals remained on the respective diets for a period of 35 days. at which time ten animals on the 32 per cent casein diet and ten animals on the six per cent casein diet were transferred to an isolation room and inoculated orally with two milliliters of a suspension of TGE virus. There were four collection periods of 72 hours each for urinary and fecal excretions. One three-day collection consisted of the three days prior to inoculation with the virus. The other three collection periods, each being three-day collection periods, extended .from the time of inoculation through the ninth poetninoculation day. Fbur samples of blood were drawn. with a sample taken at the end of Bach urine and fecal collection period. Total serum protein was determined by the biuret method. German 9; 5;. (1918). 36 All animals were killed with an overdose of barbiturates at approximately 65 days of age. after which the necrOpsies were performed. The number of tissue sections was increased. as well as the number of organs sampled. Sections. in addition to those described under Empori- ment 1. included the esophagus. adrenal gland. mesenteric lymph nodes, heart, and lung. 3- Willi Twenty-six.Yorkshire crossbred baby pigs were taken from their dams at 28 days of age. and ten pigs were placed on a ration containing 24 per cent casein while 16 were placed on a ration con- taining six per cent casein. The pigs remained on the respective diets fer a period of 16 days, at which time one animal was killed in each of the respective ration groups and necropsies performed. Six pigs on 2# per cent and ten pigs on six per cent casein rations were isolated and each inoculated orally with two milliliters of a suspension of TGE virus. Five days after inoculation, animals were killed and necropsies performed from the following groups: one control pig fed 24 per cent casein. one control pig fed six per cent casein, two pigs infected with TGE and fed 2“ per cent casein ration, and two infected pigs fed six per cent casein ration. Six days after inoculation. two six per cent control pigs were transferred to the 2k per cent control pig ration, and four six per cent infected pigs were transferred to the 24 per cent infected group. The pigs remained in these respective groups for a period of 16 days, at which time they were killed with an overdose of barbiturates and necropsies performed. V. RESULTS A. menses; Lane. The data on weight gains for pigs infected with transmissible gastroenteritis (T83) virus and control pigs on 32 per cent and six per cent casein rations are presented in TABLES IV and V. The control pigs on each of the two rations made gradual gains from the tins of weaning to #5 days of age. The animals on the low-protein ration gained weight much more slowly than those on the high-protein ration. The animals that were infected had made gradual weight gains up to the time of infection. After this date. however. their body weights decreased. 2cm». Data on total white-blood- cell counts are presented in TABLES VI and VII. 'lhe leukocytes decreased in number three days following inoculation with the virus. This leukopenia was greater in pigs infected with TOE and fed 32 per cent casein ration. Hemoglobin levels (TABLES VIII and II). hematocrit values hams x and x1). and lean differential leukocyte counts (new x11) were not significantly different between the experimental groups . a? L8 TABLE IV. Weights of infected and control pigs on 32 per cent casein ration." Experiment I. (lbs.) Fig A e a s no. _7 In 2; 28 35 4g__ 53 Central 1 9.8 10.0 10.2 11.2 13.0 1b.5 15.h 2 9.7 9.7 10.7 11.7 13.6 15.2 1h.6 6 6.2 7.0 7.5 8.5 10.7 12.2 12.8 9 5.8 6.1 7.2 7.9 10.3 11.1 12.9 me 3 10.0 8.7 9.3 10.9 12.6 1h.l' 7 6.6 6.6 7.8 8.0 12.6 lO.2* 10.6 12 h.2 4.6 5.5 6.0 7.9 9.2‘ Contggl Mega F‘fi 7.9 8.2 8.9 9.8 11.9 13.3 13.9 Igfected Mean .9 6.6 7.5 8.3 11.0 11.2 10.6 ‘Infected on h2nd day. “Infected refers to infection with transmissible gastroenteritis virus. TABLE V. Wei ts of infected and control pigs on per cent casein ration. Experiment 1. (lbs.) Pig A e D s §g£_ 2 15 21 28 35‘ ‘7132 hfi Contgol 5.h 5.5 5.1+ 5.7 7.0 7.2 7.8 10 6.3 6.3 6.0 6.1 7.0 7.2 7.9 Info ted 5.5 6.5 6.3 6.5 7.0 7.0’ 5 6e8 6e8 6e3 700 705 795‘ 11 70“ 7e“ 7e5 7e8 8e6 8e1' Contgol Mega F" 5.9 5.9 5.7 5.9 7.0 7.2 7.9 e e 09 6e9 6e? 701 707 7e5 —viflilnfected on 52nd day. 1. 4'1 TABDE VI. Hemocytometer leukocyte counts of infected and control pigs on 32 per cent casein ration. Experiment I. (cells/cmm.) Pig Da s Aft 0 tie ‘ me a A _ gentrgl 1 11.200 2 22.050 19.650 6 16.700 8.900 9 17.550 6.800 Infested 3 16.300 6.050 7 29.500 9.350 12 12.550 7.550 W 18.767 11.638 ect an “Days After Inoculation refers to oral inoculation with trans- missible gastroenteritis virus. TABLE VII. Hemocytometer leukocyte counts of infected and control pigs on 6 per cent casein ration. Experiment I. (cells/emu.) ==IE=I===I=II=I===IE=EE=EE===El=====Ii==IIIIIIIIIIIISII=I=Il==l=l======t Pig Dafs After Inoculation __ 0e - 3 Cent 01 V‘— 5 13.650 10.050 10 13.750 7.800 e ted 13.050 13.850 5 13.650 10.850 11 14.100 8.000 W 13.700 8.925 ed Mean 13.600 10.900 {.0 I TABLE VIII. Homeglobin levels of infected and control pigs on 32 per cent casein ration. Experiment I. (Gm./100 m1.) Pig De s to o _.i £2.11. :1_ :1 Qantas; l 13.h 2 15.3 13.7 6 15.7 12.b 9 14.? 12.7 Infected 3 13.4 12.0 7 13.4 11.7 12 12.7 10.1 Eontggl Mann “ 15-2 13.1 Infected flenn 13.2 11.4 TABLE IX. Hemoglobin levels of infected and control pigs on 6 per cent casein ration. Experiment I. (Gm./lOC m1.) a 11.7 11.u 10 10.u 10.u Lnfgcted 12.“ 12.u 5 11.u 9.8 11 12.0 12.1 Santzal.naan. 11.1 10.9 Iafsaiaé_hssa " ‘ _J l TABLE X. Hematccrit values of infected and control pigs on 32 per cent casein ration. Experiment I. (packed cell volume per cent) 1 g P: 8 Aft-r 90.1, JtOo ------- “fl“ #“fl ~ . Rant 1 w ~12 3 gnu :2 1 38.0 2 “300 3705 6 10.5 37.5 9 u2.5 38.5 e ted 3 141.0 3700 7 38.0 36.0 12 37.0 33.0 W "— 1‘3-0 37.9 W 38.7 35.3 TABLE XI. Hematocrit values of infected and control pigs on a 6 per cent casein ration. Experiment I. Qpacked cell volume per cent) Pig 5 t 0 tie Np - JV 5 32.0 32.0 10 31.5 28.5 i 30.0 38.0 5 38.5 30.0 11 37.0 36.0 2112;211:2111 ‘— 31.8 30.3 Wheat 35.2 39.7 52 TABLE XII. Mean differential leukocyte counts.’ Experiment I. (per cent} Let s Aft nocul tio XEIKEEIEflirs 3;: 3—7 A. Neutrophils e_, Segmented 51.0 39.5 _ Nonsegmented 1.2 5.8 lymphocytes “6.3 50.8 Mbnocytes 1.2 2.5 Eosinophils 0.5 0.8 Basophils 0.0 0.8 B. Neutrophils Segmented 59.7 51.7 Nonsegmented 1.0 7.7 lymphocytes 38.3 39.7 Mbnocytes 0.7 .2 Eosinophils 0.3 0.7 Basophils 0.0 0.3 C. Neutrophils Segmented 66.0 49.0 Nonsegmented 2.5 3.5 Lymphocytes 30.5 “9.5 Monocytes 1.0 2.5 Eosinophils 0.0 0.0 Basephils 0.0 0.5 D. Neutrophils Segmented 51.7 56.7 Nonsegmented “.7 1h.3 layuphocytes 41 . 0 27 . 0 Mbnocytes 1.7 2.; Eosinophils 1.0 0.0 Basophils 0.0 0.0 ‘Pi s fed (I) Control 32?. (B) Infected 323. (0) Control 6i. and (D Infected 6% casein ration. 53 The data for paper electrophoresis of serum proteins (TABLES XIII and XIV) indicated that control and infected pigs fed 32 per cent casein ration had a distribution of serum proteins that approached normal values. Gamma globulin was slightly but not significantly high: while albumin was slightly but not significantly low. The control and infected animals fed six per cent casein ration had higher than normal gamma globulin values and lower than normal albumin levels. Significant differences were not detected for serum nitrogen levels of control animals or animals infected with TGE fed 32 per cent or six per cent casein rations (TABIES XV and XVI)- Nitrogen balance data (TABLES XVII and XVIII and Figure 1.) indicated that control animals fed six per cent casein ration had lower than normal nitrogen values but they remained at a rather constant level. Animals infected with TGE and fed 32 per cent casein ration had greatly decreased values but remained in positive nitrOgen equilibrium. Animals infected with TGE and fed six per cent casein ration had decreased nitrogen retention and were in negative nitrogen equilibrium through the first. second and third days after inoculation with the virus. The sodium balance data (meme xxx and xx) revealed that control pigs fed 32 per cent and six per cent casein rations maintained strong positive sodium balance. Pigs infected with TGE and fed 32 per cent casein ration significantly decreased in sodium retention but remained 151 positive balance. while the pigs infected with ms and fed six per cent casein ration recessed into a strong negative balance the first day. returned to a slight positive balance on the second day. and declined to a strong negative balance on the third day after inoculation. an TABLE XIII. Paper electrophoresis of serum proteins of infected and control pigs on 32 per cent casein ration. Experiment I. (per cent) We _ :12 ji:_ Pig gamma Bite If:§Ipha gamma beta alpha . H ' t ‘1‘ ' ' .’ P ‘ _. 9 ' '1‘ 0 § ° 2" ‘. ° ' _Q _' ’4’ 9 _ ' ,‘1 -_ ' 1 -‘ L 1 14.4 24.7 30.8 30.1 17.3 26.0 27.2 29.6 2 12.9 11.5 43.9 31.7 11.2 17.8 30.8 40.2 6 14.1 22.3 33.0 30.6 16.7 18.0 25.3 40.1 9 24.0 20.9 32.2 22.9 21.2 14.3 28.2 36.3 Jaguars 14.6 25.9 25.6 33.9 17.6 21.9 27.2 33.2 7 17.4 22.8 30.8 29.0 20.7 14.7 28.9 35.8 12 16.0 17.6 29.6 36.8 11.6 16.3 26.9 45.2 o i_i 18.4 19.9 35.0 28.8 16.6 19.0 27.9 36.6 Inflected Mean 1 .0 22.1 28.7 33.2 16.6 17.6 27.7 38.1 TABLE XIV. Paper electrophoresis of serum proteins of infected and control pigs on 6 per cent casein ration. Experiment I. (per cent) ‘3. s Aft oc at o __I _ —*7 :17. i3_ Pig gamma beta alpha gamma beta alpha \. _..__, .. a. 1... . Albid. ..J_. .. pg. _.. 1. . L" M 8 15.8 24.8 32.6 26.8 21.1 17.6 29.4 31.9 10 33.4 19.0 30.8 16.8 23.5 17.7 31.1 27.7 mated 11 23.1 20.1 32.8 24.0 18.5 17.8 33.3 30.5 4 18.3 20.1 37.8 23.8 15.8 22.0 40.1 22.1 5 25.7 22.4 33.6 18.3 18.7 19.7 38.8 22.8 24.6 21.9 31.? 21.8 22.3 17.? 30.3 29.8 W 22.4 20.9 34.7 22.0 17.7 19.8 37.4 25.1 *5 . TABLE XV. Serum nitrogen of infected and control pigs on 32 per cent casein ration. Experiment 1. (mg. nitrogen/m1.) Pig 5.1} e o ati L '; l Emmi 11.5 2 11.8 10.4 6 11.2 9 11.0 10.1 Inflected 3 11.9 11.1 7 9.4 10.5 12 10.1. 9.9 W * 11.5 10.6 Infaaiss.naaa. 10.6 10.5 TABLE XVI. Serum nitrogen of infected and control pigs on 6 per cent casein ration. Experiment 1. (mg. nitrOgen/ml.) 553 Da 5 r o ' tio N2._ 1 -1 3 g 9.9 8.1 10 9.1 8.2 £ 9.1 9.8 5 9.1 8.7 11 9.2 11.2 921121.121; fit 9.5 8.2 W 9.1 9.9 <4 I 5 TABLE XVII. Nitrogen balance of infected and control pigs on 32 per cent casein ration.‘l Experiment I. (Gm./day) - P18 —_ 4_::fiv —- s I o t o ’— ::‘~7 - E21- 1 _, _. 3L;..... l 5.09 4.38 7.16 2 6.39 5.06 7.30 6 3.01 3.39 5.35 9 3.56 4.13 6.61 10:22122. 3 2.74 1.72 2.59 7 2.16 .0003 -0081 12 0.92 -o.96 0.58 mm W I I 4.51 4.25 6.61 d me 1.94 0.24 0.79 ‘Intake Gm. nitrogen - (Erinary Gm. nitrogen -0-'fecal_¥(1m:ww nitrogen) = balance. TABLE XVIII. Nitrogen balance of infected and control pigs on 6 per cent casein ration.‘ Experiment I. (Gm./day) lllzlll====::: fi—— Pig 5 ts o o m .— _.,a_ 3 "f _ Control 8 1.38 1.35 1.62 10 1.07 1.29 1.63 111229221 4 -1.16 0.36 -1.20 5 -1001 .0067 -O.34 11 -1.27 -0.36 -2.66 gontggl £953 —' '— 1.23 1.32 1.63 f ed -l.15 -0.22 -1.57 *Intake Gm."nitr5gen - (urinary Gm. nitrogen + fecal Gm. nitrogen) = balance. 57 ...... . . . . .36.,” . 1 2 3 1 2 3 1 2 3 1 2 3 Days after inoculation with [he TGE virus. Figure 1. Metabolic balances of (A) control pigs fed 32 per cent, (B) infected pigs fed 32 per cent. (C) control pigs fed six per cent and (D) infected pigs fed six per cent casein rations. Intake is plotted downward from the zero line; fecal and urine outputs are then plotted upward from the point of intake. When total output touches the zero line. equilibrium is represented; when output fails to reach the zero line. positive balance is represented; when output extends beyond the zero line, negative balance is represented (Reifenstein e_t_ £00 1945). TABLE XIX. Sodium balance of infected and control pigs on 32 per cent casein ration.‘ Experiment I. (mg./day) Pig gays Aftg; Igogglation _i, Contzol 1 459.13 244.82 641.52 2 587.24 287.28 656.70 6 294.91 260.30 451.24 9 151.85 433.15 623.66 new 3 312.95 - 16.74 7 350.21 262.85 - 58.88 12 125.07 151.60 313.00 gont§217u§§§" '77 “'- 373.28 306.39 593.28 Infected Mean 262.74 132.57 127.06 'Intake mg. sodium - (uriniry mg. sodium + fecal mg. sodium) =‘7 balances TABLE XX. Sodium balance of infected and control pigs on 6 per cent casein ration.‘ Experiment I. (mg./day) Kg A w V ww11m: gte; Indggetign W' T r 112.: 1 _ _fie— —3 a t oi 469.12 539.04 566.79 10 405.36 528.48 601.90 E t '53203" 197082 'Me62 5 -343.45 126.03 -871.23 11 -591.54 - 11.61 ~323.69 M 489011 104.08 -513018 balance. *Intake mg. sodium - (urinary mg. sodium + fecal mg. sodium) = 59 The data on potassium balance (TABLES XXI and XXII ) disclosed that control pigs fed 32 per cent and six per cent casein rations remained in strong positive potassium balance. Animals infected with T08 and fed 32 per cent casein ration had a negative balance the day following inoculation with the virus and steadily increased retention on the second and third days to positive potassium equilibrium. Pigs infected with TGE and fed six.per cent casein ration had a negative balance one day after inoculation. a slight positive balance on the second. and a negative balance on the third day following inoculation with the virus. S-M All pigs fed six per cent casein ration had synptoxs of vomition and severe watery diarrhea from 12 to 16 hours following inoculation. Two pigs fed 32 per cent casein ration began vomiting within 40 to 48 hours and had severe diarrhea within 60 to 72 hours after inoculation with the virus. The other pig fed 32 per cent casein ration and inoculated with the virus did not have observable symptoms of transmissible gastroenteritis. 4. ngss Lesigns The most obvious gross change was the difference in growth rate between pigs on the low and high protein rations. The animals developed a dry and scaly appearing skin and the hair became thin and long after approximately four weeks on the lowbprotein ration. 60 TABLE XXI. Potassium balance of infected and control pigs on 32 per cent casein ration.’ ' Experiment 1. (mg./day) Pig ___ 05x5 Aftg: I92::l:tigg __f .__ . Ho. 1 2 3_ Qantggl 1 517.37 577.98 1027.13 2 902.69 696.25 1052.u5 6 “50.68 5&8.82 753.h1 9 330.17 562.22 820.92 liliuflas: . ‘ 3 205.79 370.62 h82.09 7 -32u.67 262.52 155.53 12 -306.05 3.12 b53.98 W ‘" “ ’ 550.23 506.32 913.h8 infssisé.naan. -141.6b 212.09 360.53 .- '—"Intake mg. potassium ~1urinzty “80433t333inm * fecal “8:?“ potassium) = balance. TABLE XIII. Potassium balance of infected and control pigs on 6 per cent casein ration.‘ Experiment I. (mg./day} 12 __ W _ .2 -: N°~ _:lr EL, 3+ 2252221, “’— u96.oo 796.30 923.76 10 370.02 583.h3 985.21 lafiasiad -170.77 380.75 -229.82 5 .212.74 57.19 -265.16 11 -715033 "' 709“ 400.83 2225221.!220. 933.21 689.87 959.99 12:22229.Haan. -367.61 143.33 .298.60 *Intake mg. potassium - (urinary mg. potassium + fecal mg. potassium) =‘ba1ance. 6.1 All the animals that had symptoms of TGE. at necropsy. had greatly distended stomachs. and they uniformly contained large quantities of curdled casein but only small quantities of gas. The small intestines. ceca and large intestines were distended with very watery contents in which were found curds of undigested food. The mucosa of the gastro- intestinal tract appeared hyperemic with a slight excess of mucus adhering to the surface epithelium. thus indicating catarrhal enteritis. The mesenteric blood vessels were congested in approximately one-half of the infected animals. 5. Microscopic Lesions Hyperkeratosis was distributed on the surface of the skin of pigs fed six per cent casein ration. but was more extensive in animals that were in addition infected with TGE (Figure 2 _‘. Hyperkeratosis was more commonly located in the skin of the back and shoulder. There was a general increase in reticular cells of the red pulp and in the area of the venous sinuses of spleens of pigs infected ‘With TGE and fed six per cent casein. The reticular cells had enlarged :nuclei and appeared active. Malpighian bodies were small. HistopathoIOgical changes were similar in the stomach. duodenum. .5ejunum. ileum. cecum. spiral colon and descending colon. Control aumimals fed 32 and six per cent casein rations had few histological <11Lfferences. Animals infected with TGE on the respective rations had iiticreased connective tissue and an increase of neutrOphils. lymphocytes fluid plasma cells in the lamina propria throughout the gastrointestinal ttract (Figure 3 l The infected animals also had an increase of mucus ':=r-~a ' Qvnarksratc513 02 inc skin. Fi; “ .-.-.. . b «an! casein ratinn and infected ' in .“L. nemaLOXVl n and . n- Fi§"re 1. Increased numb r cell: Lfi the l=mfna crop: a G? the duodenum. ?ig .ed six per can? casein rattan and infected with TOE. Hematcxyiin and Ensin. x Ld7. H-au 63 0‘ adhering to the mucosa. Approzdmately one-half of the infected animals on the respective rations had submucosal edema Figure 4 ), The pancreas of control and infected animals fed six per cent casein ration contained acinar cells that were smaller and contained a lesser number of secretory granules. Sections of the pancreas from animals of all treatments in the experiment were negative for fat when stained with Sudan 1‘}. and negative for glycogen when stained with Best's carmine. The epithelium lining the proximal and collecting tubules of the kidney of animals infected with TGE and fed six per cent casein ration had increased numbers of fat-containing vacuoles. This was evident in sections stained with hematoxylin and eosin and of sections stained with Sudan IV. All kidney sections were negative for glycogen when stained with Best's camine. The livers of control animals fed 32 and six per cent casein rations were strongly positive for glycogen. while liver sections from animals that were infected with TGE contained lesser amounts. Liver sections of pigs from all dietary regimens were negative for excess fat content. The parenchymatous cells. around the periphery of the hepatic lobules of control pigs fed six per cent casein ration. had many poorly defined cytoplasmic spaces that were too large to be spaces containing gylcogen (Figure 5 ). Since these cells were negative for eaccess fat the spaces represented hydropic degeneration. Liver sections from infected animals on six per cent casein rations had widely separated a~nd disorganized hepatic cords leading from the central vein toward the periphery of the lobule. Isolated liver lobules had occasional cells 65 near the periphery that stained intensively eosinophilic. were smaller than normal cells and contained pyknotic nuclei Figures 6 and 7 ), Bo WE. 1.9222521. The data on weight gains for pigs infected with TGE and control pigs on 32 per cent and six per cent casein ration are presented in TABLES XXIII and mm. These tables disclosed that pigs in all four experimental treatments continued to gain weight from 19 days of age to 61 days of age. Weights were recorded on surviving control and infected pigs on six per cent casein ration at 68 and 70 days of age. and. during this period of time. both groups lost weight. Figures 8 and 9 illustrate comparative sizes of pigs in the four treatments of this experiment. 2. Analyses leukocyte count data (rams xxv and mm;- denoted that there was a distinct leukopenia in animals in both dietary regimens three days following inoculation with transmissible gastroenteritis virus: however. on the sixth day the leukocyte counts returned or were showing a trend to return to manual values. Pigs infected with T68 and fed a 32 per cent casein ration. had a decline in hemoglobin values on the third day following inoculation and had even lower values on the sixth and ninth days after inoculation (TABIES XXVII and XXVIII .,‘. Infected and control pigs fed a six per cent casein ration had low hemoglobin values before inoculation and both groups continued to decline after inoculation: however. the pigs infected with TGE had hemoglobin values below those 0! contra]. pigs e 67 TABLE XXIII. Weights of infected and control pigs on 32 per cent casein ration. Experiment II. (lbs.) Pig __ Age in 3E5? J 5:. 19 2:": 33 47 ill-— 6}.— Qantrol 1 11.2 14.7 18.4 24.0 29.0 35.5 43.0 8 7.0 10.2 13.5 19.0 24.0 28.0 35.0 14 5.9 8.1 10.3 15.5 19.9 24.2 30.0 18 8.8 10.0 13.5 18.2 26.3 33.5 22 8.9 10.3 14.2 18.7 25.3 34.5 ' cted 5 7.8 10.2 14.5 19.6 25.3 30.5*‘ 6 9.4 12.6 17.4 22.8 27.0 31.0" 7 6.5 8.5 11.9 17.5 21.1 26.8“I 11 7.8 8.2 10.5 16.0 20.5 23.5** 15 10.5 11.3 15.0 19.0 7.8‘ 28.4 16 9.7 10.3 13.4 17.8 21.5‘ 22.4 19 10.9 11.0 14.2 18.2 27.4” 32.0 20 8.5 8.? 12.0 15.0 23.3* 25.0 23 8.3 8.7 12.4 17.0 26.0‘ 27.5 24 4.9 ..7 8.5 12.5 23.0‘ 24.8 W 7 ' 3.4 10.7 14.0 19.1 24.3 27.8 35.2 Lnfected Mean 8.4 9.5 13.0 17.5 23.5 26.1 26.. ‘Infected on 55th day. “Infected on 54th day. TABLE XXIV. Weights of infected and control pigs on 6 per cent casein ration. Experiment II. (lbs.) gfiml 1? 26 33 ' 1 2+ El 68 20 3 10.6 12.1 11.8 13.6 15.0 16.3 20.0 0 10.9 15.0 16.6 19.3 21.5 23.8 28.0 13 5.4 7. 7.5 9.0 10.0 11.5 12.0 30 11.0 12.2 13.0 14.8 17.0 18.9 21.2 21.3 20.3 31 11.0 14.2 14.4 15.1 17.2 19.0 21.1 21.2 21.1 Infected 2 10.8 14.6 16.5 18.5 21.0 23.2" 9 9.3 11.8 13.4 15.4 17.0 18.0** 10 6.3 8.9 9.9 11.5 13.0 10.0.. 12 5.8 6.5 6.9 8.2 9.0 9.3“ 25 11.6 12.7 13.0 15.0 17.0 18.0 22.2‘ 19.7 19.5 26 11.2 13.” 14.0 15.9 18.0 19.8 22.3* 17.1 16.3 27 we 14.0 11.7 12.5 14.2 15.4 17.4 18.4* 17.1 16.3 28sit 11.5 13.5 14.8 16.5 19.1 19.7 23.0‘ 21.2 19.0 29‘1”“l 9.0 10.9 10.2 12.1 13.0 14.0 15.4' 14.2 12.9 32... 10.9 14.1 15.2 16.2 19.0 20.4 20.7" Egihtrol Mean 9.8 12.2 12.7 14.4 16.1 17.9 20.5 21.3 20.7 W ‘l 10.0 11.8 12.6 14.4 16.2 17.4 20.3 17.9 16.8 'Infected on Elst day. '— "Infected on 54th day. “‘Pig number 32 died two days after inoculation and pigs number 2?. 28. and 29 died nine days after inoculation with ms. Figure 8. Control pig fed '32 per cent and six per cent casein ration. fire 9._M_Pig fed 32 per (zen: and 21x per cent casein ration and infected with TGE. 70 TABLE XXV. Hemocytometer leukocyte counts of infected and control pigs on 32 per cent casein ration. Experiment II. (cells/cum.) Pig Daxs fite; 1.33252; gtion <3 -5A 31 91 15.650 10.850 8.850 9.250 8.700 8,550 6.600 10.600 14 13.650 13.350 10.250 17.1400 18 10.050 11.800 12.300 14.700 22 1a.000 12.650 13.100 10.750 W 16.850 10.800 11.800 10.250 6 9.200 9.750 10.050 11.100 7 8. 500 8,950 6.700 9.000 11 11.050 6.950 7.600 12.550 15 11.100 9.250 12.050 11.250 16 13.750 7.700 18,050 1.5.300 19 6.750 0.250 10.100 111.000 20 11.700 6.200 14.900 13.600 23 16.600 8.650 13.600 21.050 24 8.650 7.050 12.800 19.150 :§E§;1t:glee§g ,1 ~— 12.450 11.nu0 11.020 13.300 W 11.955 7.995 11.805 13.725- w— TABLE XXVI. 71 Hemocytometer leukocyte count: of infected and control pigs on 6 per cent casein ration. Experiment II. (cells/cum.) I?ig s t culat £24 -5 J it 9 gyontrol 3 13.900 9.300 6.650 8.700 4 10.800 10.850 9.200 10.250 3.3 10.750 10.050 10.900 11.850 30 18.150 25.050 23.000 13.900 31 11.000 15.550 19.500 15.250 e ted 2 17.600 6.250 8.650 114.050 9 11.900 8.850 10.200 16.150 10 9.14150 6.150 7.1450 11.800 .12 16.h50 6.800 7.800 16.550 25 16.250 8.350 11.100 18.550 26 13.000 (-3-. 8.650 14.1450 2'7 22.700 9.550 13.700 28 18.300 18.950 14.1400 29 16.000 18.050 11.150 19.900 32 21.100 <3 t 1 Me 13.720 1h.160 13.850 11.990 @acted new 16.275 9.850 10.3““ 15.922 TABLE XXVII. Hemoglobin levels of infected and control pigs on 32 per cent casein ration. Experiment 11. (Cum/100 m1.) Pig 29.13 After Inoculation E2. 1 -5 3 I 9 gamma}. 1 16.7 12.3 15.0 14.0 8 14.3 17.1 12.6 12.6 14 14.0 13.8 13.0 14.7 18 12.3 14.7 12.3 12.6 22 11.2 15.0 13.8 13.0 M 5 13.8 11.2 12.0 13.4 6 15.8 14.0 13.4 15.4 7 14.0 12.0 10.5 12.6 11 16.7 13.4 12.3 13.4 15 13.8 16.0 14.3 13.4 16 8.4 9.6 8.8 7.8 19 13.0 13.4 13.0 12.3 20 14.7 15.4 15.4 12.6 23 13.0 14.7 14.3 12.0 214 11.5 14.0 15.0 12.3 W 11 13.7 14.6 13.3 13.4 W 13.5 13.4 12.9 12.5 73 TABLE XXVIII. Hemoglobin levels of infected and control pigs on 6 per cent casein ration. EXperiment II. (Gm./100 ml.) Pig Daxs Arte; Lnogglgtion __fi £2, 25. 3. 9__ gontgol’ 3 13.0 12.0 13.0 13.0 11.5 11.5 11.5 9.6 13 11.5 12.3 12.0 12.0 30 12.7 10.8 11.4 9.1 31 11.7 10.8 10.4 10.8 Inghcted 2 13.4 12.3 10.8 10.5 9 13.0 12.3 13.0 12.0 10 1C.5 10.0 10.0 10.5 12 12.0 10.5 10.0 10.0 25 11.7 13.7 10.4 7.5 26 9.8 3.3 4.6 4.9 28 10 o 8 70 5 8° 5 29 13.4 13.7 12.1 12.7 32 5.2 font—r31 kg 12.1 11.5 11.7 10.9 greeted Mean 11.4 10.4 10.0 9.7 74 Hematocrit values (TABLES XXIX and XXX) for control and infected pigs fed 32 per cent casein ration were not significantly different. but values for control and infected pigs fed six per cent casein ration progressively became lower from five days before to nine days after inoculation. Pigs infected with TEE and fed six.per cent casein ration. howevera decreased at a more rapid rate than the control piss. There were several interesting changes presented in differen- tial leukocyte counts (TABLE XXII) but no definite trends were ascertained. The data for distribution of serum proteins, presented in TABLES XXXII and XXXIII. revealed that pigs infected with T68 and fed 32 per cent casein ration. had a slight decline in per cent of albumin nine days after inoculation. Serum samples from control pigs fed six per cent casein generally had lower_than normal percentage and higher than normal alpha globulin levels. Pigs infected with TGE and fed six per cent casein ration had a progressive increase of gamma globulin from the time of inoculation to nine days after inoculation and a corresponding decrease in albumin levels. Alpha globulins uniformly ‘were higher than values considered normal. Total serum protein of control pigs and pigs infected and fed 32 per cent casein rations remained near normal values throughout the experimental period. Pigs fed six per cent casein ration had lower than normal values and there were no significant differences between the control and infected lots (TABLES XXXIV and XXIV), 75 TABLE XXIX. Hematocrit values of infected and control pigs on 32 per casein ration. Experiment II. (packed cell volume per cent) M o t o ugg_f _. =5 3_ _g SL1 §2¥££21 46.0 35.0 41.0 38.0 8 40.5 37.0 36.5 36.0 14 41.5 41.0 40.0 41.5 18 38.5 38.0 34.0 37.0 22 34.0 40.0 38.5 30.0 10222221 5 39.0 35.0 35.0 39.0 6 43.5 41.0 38.0 42.0 7 39.0 37.0 28.5 34.0 11 42.0 41.0 37.0 37.0 15 42.0 41.0 42.5 38.0 16 26. 5 24.5 29.0 23. 5 19 38.0 34.0 35.0 36.5 20 43.5 37.0 40.5 34.5 23 39.0 37.5 39.0 30.0 24 35.0 35.5 38.0 34.0 Was 1 t 40.1 38.2 38.0 36.5 38.8 36.4 36.3 34.9 76 TABLE XXX. Hematocrit values of infected and control pigs on 6 per cent casein ration. Experiment II. (packed cell volume per cent) Pig. s t In up. -5_ 3_ _53 9 gontggl 3 39.0 33.5 35.0 37.5 4 34.0 36.0 32.0 25.0 13 34.0 30.0 35.0 32.0 30 36.0 37.0 34.5 29.0 31 31‘05 3&05 32.0 3300 W 2 36.0 37.5 30.5 28.0 9 35.5 34.0 37.0 32.0 10 30.5 29.5 28.5 28.0 12 36.0 32.0 27.0 26.0 25 35.0 40.0 33.0 25.0 26 28.0 14.0 16.0 19.0 28 31.5 26.5 27.0 29 39.0 38.0 38.5 38.0 32 18.0 35.5 34.2 33.7 31.3 e t c 32.8 31.7 29.8 28.0 77 TABLE XXXI. mean differential leukocyte counts.‘ Experiment II. (per cent) m _..i lot 231s After Inogglation ,_ lashssxisa -5 3 A. Neutrophils Segmented 35.8 33. 34.6 36.4 Nonsegmented 5.6 6. 8.4 7.2 Lymphocytes 5n. 2 550 530 0 5h. 6 Monocytes 4.0 2. 3.0 1.6 Eosinophils 1.0 2. 1.2 0.8 Basophils 0.8 1. 0.6 0.0 B. Neutrophils Segmented 29.3 26.4 24.6 '.3 Nonsegmented 4.7 10.8 8.8 10.3 Lymphocytes 61.4 60. 5 62.0 51.4 Monocytes 3.9 2.1 3.3 2.6 EosinoPhils 1.4 1.4 1.2 2.0 BasOphils 0.4 0.2 0.7 0.4 C. Neutrophils Segmented 28.6 30. 34.8 _2.0 Nonsegmented 6.6 7. 8.6 7.7 Lymphocytes 61.8 58. 53.2 58.3 Monocvtes 0.8 1. 2.2 2.3 EosinoPhils 2.6 2. 1.4 0.7 Bascphils 0.2 0. 0.2 0.0 D. Neutrophils Segmented 34.? 26.1 31.6 46.0 Nonsegmented 8.0 8.8 8.8 12.0 Lymphocytes 53.1 61.7 55.1 39.0 Monocytes 2.4 1.0 1.8 2.5 Eosinophils 1.9 3.1 2.7 2.0 Basophils 0.4 0.8 0.8 0.5 'Pigs fed (A) Control 327: (fij‘xnreczed‘3ii:’(01 Control 6%. and (D) Infected 6§ casein ration. 78 Oman...“ " 0M Human! meN—anm. " 0&5. III 1 a.os m.aN w.qH m.NH e.m: 6.6N e.mH o.mH m.ms o.sN m.mH s.NH e.Ns c.1N s.mH o.mH 3.4: s.eN p.mH 6.0H o.om m.HN m.nH m.MH 6.5: a.NN m.nH m.mH o.Hn «. N 6.:H m.HH o.Hs c.0N n.6H o.aN H.am N.oN n.0H m.sN n.3m m.MN N.NH n.6N m.os N.6H N.NH 6.“ 3N H.es N.eN 0.6H m.wH m.Ns m.oN H.HH p.6H 0.6: 6.0N H.NH H.HN c.ma 1.1N “.4H .eH mN H.mm o.aN H.3H :.NN H.6m m.Nm o.HH m.mH m. m o.¢N n.4H o.pN w.sm o.mH m.nH e.mN oN 4.“: m.mN e.NH o.mH m.ms o.mN m.NH s.m m.Nm w.NN N.mH N.w N.ms m.NN 6.6H s.HH 6H o.nN H.Hm H.8H w.mN H.NN m.om s.sH m.mN N N N.NN o.HH m.Nn N.NN e.Hm a. H n.eN H 0.6: m.mN m.m m.. w.os s.mN m.NH N.HN a m N.¢N H.mH m.ON m.He ..JH H. H a.rN H 2.0: a.rm 5.8H H.m N.mm s..N m.«H m.s N .1 m.uN m.HH 6.0 .mm .. a m.mH m.s HH m.om m.N« m.uH w.OH o.we N.NN 6.:H «.m m as H.om o.mH w.m H.H 4.x: «.mH m.m N o.sm m.eN N.0N H.3H s.ms N.nN H.5H s.NH a m m.oH N.mH o.aH o.om .mH N.nH 0.6 o m.ae “.mN c.3H N.oH H.m: «.mN o.mH H.HH N m m.eN N.:H s.a .H@ 0.6 U.NH A.m n n.Nm H.mN m.NH a.HH a.em o.NN s.NH H.a m on N.NN o.sH 6.NH s.os o.mN H.NH e.HH NN N.as c.3N e.mH m.nH 6.“: s.sN H.sH o.eH N as m.NN N.:H «.mH n.6s .HN N.HH m.ON mH s.ae 6.3N n.8H ,.oH m.oe m.¢H m.mH m.HH a an m.oN o.mH :.m o.m. m.4N m.mH m.» 4H o.mm m.eN ~.m1 m.ON o.me o.NN n.mH w.mH o o: m.NN m.:H N.mH 4.0“ m.HN s.sH o.eH m m.mm H.NN w.,H N.mH m.mn N.mH a.mH 0.9 m as m.mN H.wH N.NH N.af .gH n.6H u.m H .1 11 Hoummgu .nHa .1 soon .m .naa .. upon any: .pHa .a some hurl .nHa .a mwum11¢u11111qz nuaHmmmHu acaHeQOHu 11. 1maaHepoHo .mnaHasOHu mam Ll m. m m: :4 1 1 co ueHsoocH your? a I! 1 l1 111...... 3:3 .33 .HH coca—Rhos .539.» fleece uses .Hom NM co swam Hoaucoo use mecca: Mo 24395 Egon we eduoaonnoauuode some“ .Hg WEE 063%? n ed fig “gm fl cw! m.oN n.6m 6.NH H.oH H.Hm m.nm m.aH 0.6H m.mN 5.4m m.mH «.mH m.am o.Nm m.aH m.mH mmumuuuqquqnu o.mN m.Hn N.NH m.HN o.mm o.aN H.5H o.mH H.Nm m.mN a.eH o.HN N.os o.mN e.m N.mH r5458 3.8m m.Hm m.MH N.mH Nm N.mn H.6m m.nH e.nH m.mm o.Hm m.nH “.mH m.Hm m.sm n.6H 6.5H N.oN N.Ns o. H 5.6H 6N N.mN e.mm m.aH m.NN m.sN n.2m s.nN N.NH m.aN m.mN N.6N 6.5H mN a.nN H.Nm m.NH s.NH e.mN m.sm “.mH H.mH H.mN 3.3m o.MN m.eH «.mm m.aN N.mH “.mH “N a.MN N.Ns o.mH n.eH s.mN o.ms o.mH s.mH w.MN H.ee m.mH s.HH o.oN a.Ns N.wH o.nH 6N s.mN o.eN 6.NH H.6N m.Hm m.mm o.mH m.mH 6.6N p.3m m.HN 6.NH mN 6.NH m.Ne o.HN m.aH m.Nm m.sm a.6H a.mH m.MN m.am m.mH «.mN H.mm m.mm o.mH m.mH NH m.Nm n.6m N.6H o.mH m.Nm H.6m 6.5H 6.0H H.6m H.Nm m.:H N.eH m.am m.sN H.6H s.mH oH N.ON N.wm H.6H H.mN 6.6m m.sm H.mH “.mH m.Hm e.mN 6.6H m.mN H.oa 6.5N o.oH s.mH m :.Nm m.Hm o.nH N.oN 6.5m 6.6m m.aH n.eH N.mm 0.0m m.MH o.sH s.Ns m.nN o.mH e.MH N N.mN n.3m H.mH o.mN 6.0m 6.6m N.mH “.mH “.mN 0.3m N.mH N.HN 5.5m m.mm 6.0H N.NH Hm m.eN n.2m m.mH o.mH s.Nm H.Hm N.mH m.aH N.mN e.Nm n.0N N.mH o.sm :.Hm N.aH m.oH on m.Nm m.eN m.aH N.5N N.Hm N.aN m.nH m.nN 6.6N m.Hm m.mH 0.6N N.ss o.nN 6.3H N.nH MH m.nN 0.5m n.0N o.NH m.He H.sN m.HN m.m H.mm N.oN n.6H N.NH N.ms e.oN e.nH m.sH a m.en 5.6N m.nH N.HN m.wm N.mN n.6H s.HN 3.6m m.sN e.mH e.mN N.He m.mN m.MH o.mH n .J - nun .. 3.2 m 6.09 H A: m camp H .H. .. an. - L .5286 i mfiHBoE ”names . 23%on rt mm 1 m m H... “a ll 11 i l l apnea soaw .HH “caafiuomxm .832 538 “=8 .3 o 5 ”ma H9550 as concouaH mo scHopoaq sense as «Humaosa oppooflo aoagm .HHHuNN women TABLE XXXIV. 80 (Gun/100 111.) Total serum protein of infected and control pigs on 32 per cent casein ration. Experiment II. Pig s Aft ML --5 3 9 Qontrgl 1 6.0 6.0 6.9 5.9 8 3.8 7.9 5.9 6.3 14 4.1 5.2 5.8 6.0 18 6.1 5.0 5.0 5.1 22 6.4 5.6 6.6 5.8 Infgcteg 5 4.6 5.3 5.3 5.6 6 5.0 6.5 4.4 4.2 7 5.3 5.0 5.1 2.8 11 5.6 5.5 4.7 6.1 15 6.4 6.5 6.7 5.7 16 6.1 5.5 6.6 5.4 19 5.5 5.6 4.6 5.2 20 6.9 5.9 6.7 4.9 23 5.3 5.0 6.6 5.0 24 6.0 5.5 6.3 5.4 w 5.3 5.9 6.0 5.8 W 5.7 5.6 5.? 5.0 81 TABLE XXXV. Total serum protein of infected and control pigs on 6 per cent casein ration. Experiment II. (Gm/100 ml.) ‘13 w s t on __ £91 -5 3 a £832; 5.4 5.2 5.2 4.8 4 4.1 6.8 4.5 3.5 13 4.1 3.5 5.0 4.3 30 3.2 3.3 4.1 3.2 31 3.4 4.0 3.9 3.5 igggggggg 4.1 4.9 4.8 4.6 9 4.6 5.2 4.7 4.2 10 3.6 4.2 5.0 4.8 12 4.7 3.6 3.1 3.6 25 3.7 3.6 3.3 26 3.3 3.6 4.0 5.0 27 3.3 3.2 4.2 28 3.2 3.7 3.4 29 3.7 3.3 3.6 “-8 32 3.6 W 1 t 4.0 4.6 4.5 3.9 3.8 3.9 4.0 4.3 1.; 82 Nitrogen determinations of excreta from infected and control pigs on 32 and six per cent casein rations covered a period of 12 days and consisted of four. three-day collection periods. One three-day collection period was before and three. three-day collection periods after inoculation with the virus. The data for the three-day collection periods are presented in TABEES XXXVI and XXXVII and the data on a 24- hour basis in Figure 10. Control animals fed 32 per cent casein remained in strong positive nitrogen balance throughout the 12 days. Pigs infected and fed 32 per cent casein decreased in amount of nitrogen retained during three-day collection periods ending on the third. sixth and ninth days following inoculation but remained in positive balance. Control pigs on six per cent casein maintained a positive balance throughout the experiment. The values were low but consistently low. Annals infected with 1‘08 and fed six per cent casein ration had decreased nitrogen retention and went into negative nitrogen balance during the Periods ending on the third and ninth days after inoculation but were 313- ghtly positive during the second three-day collection period. The data for sodium balances are presented in TABLES XXXVIII and XXXIX. and collection times were the same intervals as those reported f 01‘ nitrogen. There were no significant differences between control and infected pigs fed 32 per cent casein ration. Pigs infected with TGE and fed six per cent casein ration had a negative sodium balance for the first but had strong positive sodium balances for the second and third °°llection periods after inoculation. Potassium balance data are presented in TABLES XI. and XLI. P188 infected with m2 and fed 32 per cent casein had a decrease in TABLE XXXVI. Nitrogen balance of infected and control pigs or 32 per cent casein ration.’ Experiment II. €.'.1r-':./3-day collection) Pig _ __ 31:11 - ~5 ‘;2§2£2. 30.65 40.30 40.47 35.13 5 24.6? 31.52 40.36 31.45 14 29.30 32.81 33.70 30.74 .3 3:.9L 3‘.20 27.70 22 40.50 42.90 49.40 34.90 infected ‘ 34.62 27.78 25.9» 28.26 h 27. 4 22.70 15.70 18.05 7 25.3v 38.07 47.72 23.52 11 4C.8: 14.30 26.19 26.11 .15 35.30 20.5C 15.90 v.10 10 29.60 11.30 12.20 23.80 19 3%???) 734.20 21.53 30.51:. 2'0 7.7.60 18.20 10.“ 24.70 2 3 45,. 4c 14. 30 10. .312 1; , 5c 27:; 19.10 31.90 22.53 71.00 ‘ 3:1 incl M - A ' ~_ -11 “u" 32.20 36.95 40.98 32.38 3.3.1? ted Me 34.75 25.30 20.11 23.76 *Tntake Gm. nitrogen - (urinary Gm. nitrogen + fecal 13m. 7‘51 trogen) = ha.T .ce. 3 "Number of days after inoculation denotes the last day of a -"day collection period. 84 32 1 Inf. 6 ‘1: Cent. \\\\i— &\ Days after inoculation with the TGE virus. Figure 10. Nitrogen balances. Thirty-two and six per cent refer to the percentage of casein in the ration. Infected refers to animals infected with TGE virus. Intake is plotted downward from the zero line; fecal and urine outputs are then plotted upward from the point of intake. When total output touches the zero line, equilibrium is represented; when output fails to reach the zero line, positive balance is represented; when output extends beyond the zero line, negative balance is represented (Reifenstein at al., l9h5). (_1) kn TABLE XXXVIT. Nitrogen balance of infected and control pigs on 6 per cent casein ratior.? Experiment II. {Gm./3-day collection} Pig Daxs After inoculation“ _ 'n __ .5 3 _ n i"_ _....- 22%222;' 7.68 6.03 8.07 8.66 h 7.#3 6.6l 6.87 5.h1 13 7.#9 5.26 6.u5 6.60 30 9.15 5.32 “.72 4.6h 31 10.83 11.02 11.05 12.20 zaégztgg, 6.39 ~16.13 l.hi 0.12 9 6.28 3.92 3.59 7.28 to 8.65 - 5.63 4.78 1.67 12 6.63 5.1a 5.50 6.u1 25 9.§3 3.19 - 1.2L 5.73 26 8.1 - 3.65 - 2.08 - 7.17 27 h.24 1.81 3.13 0.79 28 7.0% 2.32 - 2.89 6.52 29 7.“? 7.36 3.23 6.47 32 - 2.99 - 3.91 P trol e V? —*f —*~ -_-“- 8.51 c.8b 7.“? 7.52 f ct M 6.1% - 0.76 1.71 0.32 ‘Intake Gm. nitrogen - (urinary Gm. nitrogen + fecal Gm. nitrogen} = balance. "Nunber of days after inoculation denotes the last day of a 3-day collection period. TABLE XXXVIII. Sodium balance of infected and control pigs on 32 per cent casein ration.‘ Experiment II. {mg.!34day collectioni M Pig 3 . - Ng‘:fi -§ 3 9 gontggl 1 217u.7* 2092.05 2032.86 2235.12 8 232.11 2299.70 3163.33 1876.11 lb 1661.09 1865.Q§ 22L3.62 1676.48 18 2026.97 28a6.88 2394.13 22 2347.2» 263h.31 3439.33 2335.56 7-¢gzsed 5 -82'..1 ’ioo.2~ 2599.72 765.18 6 2246.49 1768.6 198.67 7 Lb90.l? 2656.16 2626.16 1&16.2h ll 26Jh.87 i619.89 2637.01 1372.27 1‘ “328.?"{ -qu.05 1766.13 788.u6 16 1552.69 636.56 1872.78 232$.7R 19 881.5“ 2570.01 3a28.0? 1757..7 20 2088.7“ l?17.50 1139.87 2835.92 23 230b.51 2400.92 1895.71 2351.1L 2“ 2605.01 1956.“? 2b56.9% 3726.59 nt 1 7 1688.21 2737.67 2972.28 2103.67 t 1905.a0 2 €1.u8 2219.96 1753.64 *Intake mg. sodium - (urinary mg._sodium + fecal mg. sodium? = balance. NNumber of days after inoculation denotes the last day of a 3-day collection period. “5" TABLE XXKTX. Sodium balance of infec-ed and control pigs on 6 per cent casein ration.‘I Experiment II. img./3-day collection) ======================================================================== Pig =% 5: _ - ___ l0; ": 5. Control 3 1317.71 1793.32 387.31 3372.79 6 176.6% 1823.01 ”81.1< 1557.16 13 900.61 1797.81 2055.01 1&23.Zh 30 1066.12 -610.91 715.10 1689.28 31 1681.73 1007.86 1692.80 1399.06 fa ted 1657.09 .j? 8029.08 2273.19 9 522.39 228.72 1799.12 1h6fi.96 10 190.70 75.99 2907.11 1861.95 12 578.29 763.L8 2370.25 2919.37 25 1526.7° 1810.89 2102.48 1379.1? 26 L973.10 - 930.16 - 358.22 - 30:.u1 8 2212.27 ~5312.10 - 611.22 383.91 29 1733.89 :375.5u 1652.59 lb7l.92 32 688.55 19.09 gentrol Mean .“ —_ __ .— 1327.31 2010.58 ile.37 2286.30 infected fleag 1179.61 - 288.92 1519.72 1h27.1u ‘Intake mg. sodium - Turinary mg. sodium + fecal mg. sodiuml 5— balance. "Number of days after inoculation denotes the last day of a 3-day collection period. TABLE XL. Potassium balance of infected and control pigs on 32 per cent casein ration.‘ Experiment II. (mg./3-day collection) Daxs After Inoculation" -5, 3 4688.93 2291.21 3029.36 3828.39 4785.18 0887.64 31h5.61 “101.00 3079.18 432u.05 5169.23 101.75 3574.23 5955.09 9 0518.11 3722.69 2639.28 2907.59 3321.60 , 3917.66 7648.07 3962.16 2613.73 6 3528.94 1579.70 1555.27 7 2753.85 3335.87 9373.99 2222.39 11 5615.93 2306.1u 3h51.82 2773.18 15 3626.12 1089.72 972.39 1326.23 16 2899.57 1691.96 1610.86 2726.39 19 3318.91 3598.73 2721.80 39b2.63 20 2298.22 2135.66 1478.41 5561.85 23 3505.38 1093.72 1617.52 2270.26 zu 5020.52 2806.85 3318.50 1332.23 gontzol Mega 372u.6i 3907.99 4950.07 3421.84 Infected Mean 3884.02 2663.57 2508.21 2938.01 'Intake mg. potassium - {urinary mg. potassium + fecal mg. potassium) = balance. **Number of days after inoculation denotes the last day of a 3-day collection period. TABLE XLI. Potassium melance of infected and control pigs on 6 per cent casein ration.‘l Experiment II. (mg./3-day collection) Pig __ Days After Inoculat1on" N0. -5 3 9 gontrol 3 3078.09 3777.90 1330.98 5978.96 9 1763.72 3665.92 405.56 329.58 13 2383.33 3067.60 3106.3 23“4.69 30 2538.26 2973.90 1780.82 2575.91 31 2652.82 5600.12 2750.35 5682.82 Enfected 2 3710.99 -1720.4h £016.93 3171.h7 9 2891.57 686.87 1783.93 20b3.37 12 2367.77 1392.88 2195.65 2995.34 25 2593.76 3022.69 2917.08 1679.98 26 2215.58 -2203.17 550.99 1‘53.56 27 1918.23 - 20h.52 1677.99 1520.36 28 3297.24 -6928.79 283.53 41.96 29 3259.01 3112.19 2966.93 2052.89 32 ‘- 36a. 55 "' 175077 Egntrol mean —— “ .1 2983.29 3716.98 1873.61 3382.21 Iggegted Mean 'Intake mg. potassium é—Zhrinary mg. potassium + fecal mg:¥ tassium) = balance. ’*Number of days after inoculation denotes the last day of'a 3—day collection period. 90 potassium retention shortly after inoculation with the virus but remained strongly positive: and this decrease was evident during the three. three- day collections after inoculation. Pigs infected with the virus and fed six per cent casein ration had a negative balance during the first three-day collection period but returned to a positive balance during the second and third three-day collections following inoculation. 3. Symptoms Infected pigs fed six per cent casein ration had symptoms of vomition as early as 16 hours and watery diarrhea as early as 20 hours after inoculation. Some of the pigs of this group, however. had a longer incubation period and began symptoms as late as 48 hours following inoculation. Infected pigs fed 32 per cent casein had symptoms of vomition and diarrhea no earlier than 36 hours after inoculation. The above correlation is similar to that found in Experiment I. With the onset of symptoms of TGE animals refused feed and usually consumed not more than one-half of feed offered. The symptoms of vomition and diarrhea persisted for five to six days, after which the feces were more firm and the animal resumed eating. The infected pigs fed six per cent casein ration went off feed with the onset of symptoms and commonly remained off feed for a longer period of time than described for those animals fed 32 per cent casein ration. Temperatures were taken twice daily and no correlations were made or trend determined between control and infected animals. 91 h. Grosg Lesions Growth again was the most obvious change noted between live animals fed 32 and six per cent casein rations. When the pigs remained on the ration for over four weeks the skin would become dry. scaly, and the hair thin and long (Figure 11). Approximately one-third of the animals on the six per cent casein ration deve10ped hyperke ratotic lesions in the skin. Two animals in the six per cent infected group developed extensive skin lesions which were characterized by raised, pigmented. focal areas measuring roughly two to five centimeters in diameter. They were uniformly distributed over the surface of the body (Figure 12). Due to the fact that infected animals were not euthanized for necr0psy until ten days after inoculation, only those animals still showing symptoms of diarrhea had distended stomachs which contained curdled casein and gas and had catarrhal gastroenteritis (Figure 13 3, Small, raised parakeratotic-like lesions were noted in the cardia of . the stomach in less than one-third of both groups of animals on six per cent casein ration. Nearly one-half of the animals infected with ’I‘GE had. at necropsy, approximately 15 ml. of serous fluid in the pericardial sac. ‘The most outstanding lesions were in the liver, with extensive changes in animals infected with TGE and fed six per cent casein ration (Figure 16) Control pigs on six per cent casein ration had similar but less extensive lesions (Figure 15) The liver tissue was mottled with depressed, irregular. dark red areas ranging in size from one millimeter to one centimeter in diameter. These lesions extended rather uniformly distributed throughout the parenchymatous tissues in all lobes of the Figure 11. A cont rel pig fed 91:: per cent. cssr-ir. Note the long. silky-appearing hair. *9 L2. f-t‘:-.n lemons of a pig fed six per cent. casein . and infected wi 11 T1133. O1 ..: _’ _ figb hi I" firrrn 13. :bduminal organs of a pig led six per cent . f n '- ‘- l 318-1n ratio" 1rd nested ten davs f.s lflOuUTJKlOn with 2;. “are liver necrosis. which =upear as light crev a-r-a- . Figure 14. Abdominal organs of a pig fed milx and posted two days after inorulation th TGF. Note the creatlv distended 51cmach. "17.4. .- I . 7” “ “-1 s‘x ~..~--:- we. FEsurw 15. The liver 9 casein rafinn. Hate the . . . as aurwered I?fl?&3£flu are u’er n-s:r:=is wh7cn appears h flielbmrofagngim fefl.edwnh'm5smi per Cent casein raiion. Compare with Figure -5 eased amount of nezrosis. P" :5 95 liver. The damaged liver tissue appeared dark red in color. while the more normal tissue appeared lighter. grayish-brown in color. No gross changes were noted in livers of control or infected animals on 32 per cent casein ration. 5- 11122212212211.2122 There were no microscOpic lesions observed in the skin of control pigs or pigs infected with T68 and fed 32 per cent casein ration. The skin of control and infected pigs on six per cent casein had hyperkeratosis. similar to that of Figure 2, Experiment I. The sweat glands contained large and active secretory cells in both control and infected animals on six .per cent casein ration (Figure 17). Twa animals infected with TGE and fed six per cent casein had scattered. circumscribed pustules in the skin which measured approximately two millimeters in diameter. The pustules contained large numbers of neutrophils, arranged between layers of stratified squamous epithelium. The squamous epithelium. below the pustule. had increased thickness of prickle-cell layer (Figures 18 and 19). Control animals fed six per cent casein had slight increases in numbers of Anitschkow wocytes between normal appearing myocardial cells. Six per cent infected animals had several foci of mocardial necrosis and myocardial edema. into which Anitschkow myocytes and fibroblasts had infiltrated (Figures 21. 22, 23, and 21‘). An organized thrombus was found in a small artery of one pig (Figure 20). Degenerative changes occurred in new areas in the pericardial fat. Control animals and animals infected with T68 and fed six per cent casein ration had small circumscribed lesions of the esophagus as :“ a 'Iin gland" Hematox. ive sweat rafion. .e. 7.5.,» .w Hematoxylin and ion. a. v case'n ra. x per cent : A ,9 fed S 74 THE and osin. x F qur'e 1'7. F“ uni-7 of thr- skin. Higher power of Figure 1’. Z} aoan‘nJeis. 1?? layers sf stratified squamous nn“ alfiuw. an: '1‘ predominantly neutrOphils. Hematoxylin Figure Pi. ’n orgeii. ed -hronbus in a blood vessel of . . T“E and fed six per ion. uematcxylin an‘ Eosin. x 187. the heart. . cent casein ra s n. '1. _--_ . - .__,_,,-.‘-§..“_, .a 7.1 ' '5’. ‘ I... u ' Fi_ire 22. Focal myocardial necrosis. Higher power of Figure 21. 11‘ necrotic myocardium. {2! normal myocardium. and ’3) two Anitschkow myocytes. Hematoxylin and Basin. x 750. 7?:-~= 23. Hyena. ial edema. Pig was infected with TGE L&; Ted six per cent casein ration. Note the infiltration a .-: ’31 . . hrsbiasts and Anitschkow myocytes in the edematous areas. Hematoxylin and Basin. x 187. \ !. Figure 2h. Anitschkow myocytes in the edematous area illustrated in Figure 23. Hematoxylin and Eosin. x 1875. 100 which were characterized by many small foci of neutrophils embedded in a greatly thickened parakeratotic epithelium (Figures 25 and 26). These lesions contained hyphae which were visible on.sections stained with hematoxylin and eosin. When these fungi were stained with Gomori's methenamine-silver nitrate. they appeared morphologically similar to Candida s2, Many small intra-epithelial abscesses'were present that had not incited parakeratosis. The lamina propria had an excess of infiltrating lymphocytes. neutrophils and plasma cells. One animal infected with TGE and fed six per cent casein had. at the Junction of the stratified squamous epithelium and the simple columnar epithelium of the stomach. an intra-epithelial abscess similar to those described above in the esophagus. One animal infected with TOE and fed six per cent casein ration had parakeratosis of the cardia of the stomach. and in this section there was no evidence of microabscess formation (Figure 27). Control and infected animals on six per cent casein had slight edema in the lamina prOpria and submucosa in the cardia. fundus and pylorus of the stomach: and in one case edema was extensive (Figure 28). There was an increase of submucosal fat in approximately one-third of the animals fed the lowbprotein ration. The mucus producing glands of the cardia and pylorus of infected animals fed six per cent casein ration had mucinous degeneration and there occurred an excess of mucus adhering to the gastric mucosa (Figures 29 and 30). Control and infected animals fed six per cent casein ration had slight excess of mucus adhering to the surface of the intestinal mucosa and an increase in submucosal fat. One animal had a small ‘ Us. ‘ , l r’;- ‘I tfv’ Fleure E-. Intra-epithelfal focal abscess of the esophagus. Pig was infected with TGE and fed six per cent casein ration. 71‘ parakeratosis, (2! abscess. and (33 infiltra- tive neutrcnhils. Lvmphocytes and plasma cells in the lamina propria. Hematoxylin and £951.. x 137. Z Intra-enitheliel focal abscess of the esnrhhgus. 'c"=r power of Figure 25. Note the presence of fungal thnae. Hemetoxylin and chin. x ?50, .. .44.. z~zre 27. Parakeratosis of the :9rdi? of th stomach. :7 Ha: infected wi?h TEE and fed =Ex ner oent eneein ratlnn. {l} parakeratonic epithelium. an: (2! ballooning degeneration. Hematoxylin and Rowin. x 187. V1 '3'] ' I'V' . .-' ,‘JI’. . ,u -- .. I» of th? stomach. Control pig fed sLx per cent casein raticu. Hematoxylin and Eosin. x 75. rirvr: T3. Edema in the submucosa of the glandular cardia ~rW—' :- 103 ." “JR . . . , q . 7’ ‘. — - C. ‘84; i .l IR. .. I’m .31 c. (1A5 2rd; p.44 m _ ‘ Tizare :9. "uzznous degenerarien of :h— pvlorns of the ‘omach. Fig was inzccted With TGE and 1‘ed six per cent a air. raY.i-J. Hematoxylin and Eosin. . - 37. ("121‘ Figure 10. Catarrhal gastritis. TGE and fed six per cent casein ration. Note the mucus and its cor .tents adhering to the surface epithelium of the cardia of the stomach. Hematoxylin and Basin. x 187. Fig was infected with 10b granulomatous lesion in a submucosal lymph nodule of the ileum. TWO animaksinfected with TUE and fed six per cent casein ration had slight edema in the lamina propria of the duodenum. Control and infected animals fed six per cent casein had atrophic acinar cells which contained small numbers of secretory granules. Animals infected with TGE and fed the lowbprotein ration had edema which separated the acini from one another. resulting in disorgani- zation of pancreatic tissue (Figure 31). Parenchymatous tissues of the pancreas were negative for fat and glycogen. Control animals fed six per cent casein ration had occasional and infected animals fed six per cent casein ration had many collecting tubules of the kidney in which the epithelia contained pyknotic nuclei Gigure '32 l. The kidneys of one animal had four areas in which fibrous connective tissue extended from the capsule through the cortex and medulla. and there was a sharp transition between the normal and impaired renal tissues (Figure 3?). One animal had hyaline degeneration around small blood vessels at the junction of the cortex and medulla of the kidney (Figure 3b). All sections stained for glycogen. with Best's carmine stain, were negative. However, the transitional cells of the renal pelvis contained glycogen. Sections stained for fat. using the Sudan IV stain. gave the following results: Control and infected animals fed 32 per cent casein ration and control animals fed six per cent casein ration were either negative or contained slight amounts of fat. Pigs infected with TGE and fed six per cent casein ration had increased amounts of fat in the cytOplasm of cells of the proximal con- voluted tubules, descending branch of Henle's loop and the larger collecting tubules (Figures 35 and 362. This increased fat content was present in approximately one-half of the animals in this group. Figure 31. AtrOphy of pancreatic acini. Pig was infected with T68 and fed six per cent casein ration. (l) island of Langerhans. (2) congestion, (3) edema. and (h) atrophic acini. Hematoxylin and Eosin. x 187. 'Qh' .. . ‘ ‘ ' cap ‘ ,. ..' ..,~ ”an . - . "Afih "No." ‘ . ‘ W" ‘fij'w‘figw‘fi? ‘2'. ' f . .5. ir: ‘--.a , ., (’.'.‘I.lrfi,~’ 8'3 ‘ E‘. Figure 32. Cortex of kidney. Pig was infected with TOE and fed six per cent casein ration. Note the pyknotic nuclei and fat vacuoles in epithelium lining the collecting ‘ tubules. Hematoxylin and Eosin. x 187. a: " , A. 33:33; 't’. Q \'\‘ {ff-ii ‘ if ‘ x . J (/I K 29.. a: m... A. am.‘ ...r. g: :isure “W. Early I'ibrosis of the kidnn:.r. Pis_ was int 'ected with “GE and fed six per cent caaezn ration. _ fibrou$ connective tissue. and ’2: normal rena.i ti 5 .ue . hematcxyiin and Rosin. x 1.87. 9'2." ’"fir’s‘i ‘ I , . ~ ' , 0...... ,_ .. 5w. {‘u‘l in :21; J Figure 15. Hyalin degeneration around a small blomi vessel of the kidney. Control pig fed six per cent casein ration. Hematoxylin and Eosin. x 750. Pig'u‘e ”is. (A'Ecbt-Lles of fat in the cytOplasm of cells Lining the proximal. and collecting tubules of the kidney. fig was infected with T6)? and fed six per cent. casein ration. Sudan IV stain. x 1.87. 4" ‘v Figure '36. Fatty metamorphosis of cells lining the proxi- mal convoluted tubules of the kidney. Pig was infected with TGF. and :‘ed six per cent casein ration. Hematoxylin and Basin. x 750. 108 Control animals on 32 per cent casein ration had a few individual cells in liver lobules that stained distinctly baSOphilic (Figure 37). Pigs infected with T63 and fed six per cent casein ration had baSOphilic granules in the cytOplasm of necrotic hepatic cells. These granules measured from approximately one micron in diameter to the size of the hepatic cell (Figure 38). The granules were positive to von Kbssa‘s stain (Figures 39 and “bi An occasional liver lobule of infected animals fed 32 per cent, and both groups of pigs fed six per cent. casein rations contained small cells in which the cytOplasm stained strongly eosinOphilic. and in these cells the nuclei were pyknotic (Figures #1 and “ZL Control and infected pigs fed six per cent casein had many liver lobules in which the cells toward the periphery were large and contained poorly defined spaces in the cytOplasm. These cells did not contain a ficient quantities of fat or glycogen to account for the spaces. therefore suggesting hydropic degeneration {Figure 43). The control animals fed six per cent casein ration had sinuses in the peripherolobular region that were congested. Scattered throughout the liver tissue were groups of lobules, adjacent to each other. that were undergoing marked degeneration. This degeneration was characterized progressively by a condensation of the cytOplasm around the region of the nucleus in some lobules, while in other lobules there were hepatic cells with uniformly dense and strongly eosinOphilic cytOplasm in which there was either pyknosis. karyorrhexis or karyolysis of the nucleus (Figures uh. #5 and #6; In still more advanced necrosis :‘Eé” {:i1§.:u I. . (355.3. - ‘E’ Figure 3“. an isolated basophilic heratic cell. Control '35:- :‘rfl‘ $2. per cent casein ration. Hematoxylin and Eosin. -' .4». x 72c. 3: . q: Figure 38. Basephilic stippling in the cytOplasm of necrotic liver cells. Pig was infected with TGE and fed six per cent casein ration. Hematoxylin and Basin. x 750. ~_'nre ‘7. Von Kossa porl'ivu rubFfances in ~v’02394n cf necro'1? liver celLs. Pig wee inlecfcd wif* TGE and fed six re: cent casein ration. Nata tne more normal liver “alas. Von Konsa‘s etain. x L87. rfvurs hfi. Vcn Kossa positiv of "errrtlc liver cells. Rig Kcssa'fi staln. x 750. substan~es ir the cytoplasm (3 her Dower 9’ Figure 3?. Vc; 73w, , V3A‘V‘ 6%; ft; , . ’ c \‘l-’ I 5’ ‘ F.r re v.1. Hepatic necrosis. Pig was infected w: n TGE ani r—d 12 per cent casein ration. Note the lack of necrosis in adjacent lobules. Hematoxylin and Easin. x 7:. 4 _ O .. 3 J.) 0.397 1:9 L2. Fepatic necrosis.. 'igher no wer of igure 41. .al liver cells. (2‘ interlobula.r connective tissue, cells with darkly ste -ninj cytoylasm and pyknotic . hematoxy in and Eosin. x 750. 9‘3 F cure 43. Hydropic degeneration of henatic celis. Pig w=s infected with TGE and fed six per cent casein ration. Note the homopiinh!o confer. Hematoxylin and Eosin. x " 3m A'Jl’ ‘- WNWQVQHDMNNWfi‘W#OV‘PFW QWWflJ‘mpm. ,- ,. . ‘i . . ‘. . .,‘ -. ‘4 . , , 1" “.x-P" “if/”‘9“.LW”. V., 54‘“ 'N‘ "g ’ . x 9.. «5 , ~¢ ~ i3: — “~~$—"4}?§£§IH°" " ‘ Y ' .‘h' "m !.:'L‘:;( .' .3 , :- ‘ _..‘f. ' ”1A6 “ n a“. ‘ .. , -;-'f. r' it c_,‘ l\ .' c; A ’ ". , -, .. vhv . fl -,_ .uq w c .1: s‘if’ ,1 “z? a" "I‘m-59} a 'J'fJH .f'u e. ‘ s ‘ \Léik f‘i ‘ '.‘ 0'4 t '» H v a. ‘ . ' - - 75‘. . .‘f‘v ‘ . 1.‘ ‘ fig bit“. 4? fihfigk.re¢nhi}3§,i r» '. 4X“ «3 33’ .- ' 4. ’2‘?- ) Jif'wgfié‘iéfri‘ ” 'f--.‘:+ ;&'\\«;e‘. ‘5“? 4'.~‘ .- ' 7:5 ‘_§’.‘j'.,x".-‘-3.=~.Lffi,' *1) figure “4w liver changes of a control pig fed six per cent casein ration. (l) hemorrhage. (2) perfunclear basa- philic condensed cytoplasm. and (3) hydrcpic degeneration. Hematoxylin and Eosin. x 75. .‘n '. ‘3 4_ G‘ ... . .fi;',‘..lf~ Figure 45. Liver changes of a control pig fed six per cent casein ration. (l) hepatic cell regeneration, (2) uniform ecsinophilic necrotic hepatic cells, and {3} perinucloar basophilic cytoplasm. Hematoxylin and Basin. x 75. 2 .59, FOR 5 O V \ Figure 46. Hydropic degeneration and perinuolear baso- philic condensation of the cytoplasm. Control pig fed six per cent casein ration. Hematoxylin and Basin. x 750. 111. there was removal of hepatic cells. after which the sinusoids were filled with blood. giving the appearance of massive intralobular hemorrhage. There were increased numbers of fibroblasts and increased amounts of connective tissue in interlobular regions. especially abundant in the vicinity of the portal triad {Figures 47, 1+8 and 1&9}. With the death of parenchymatous cells there was gradual replacement of the lobular region with connective tissue. Therefore.the lobular area became smaller. If the necrotic lobules were located near the capsule of Glisson there was an indentation of the capsule due to contraction of the connective tissue fibers (Figure 50). The average number of adjacent lobules undergoing degeneration was from three to ten. There was an occasional lobule that retained its original size and shape: it contained normal appearing hepatic cells, necrotic hepatic cells or blood (Figure 51) . The predominant type of necrosis of the hepatic cell was coagulation necrosis. which later progressed into liquefaction necrosis. Some of the liver lobules had hepatic cells that contained many mitotic figures. enlarged nuclei. and cells containing two or more nuclei which suggested extensive liver regeneration (Figures 52 and 533. Infected animals fed six per cent casein ration had the same type of liver changes as just described for the controls; however. necrosis was more extensive in infected pigs. Most of the infected pigs had livers which contained less that one-half normal hepatic cells, and in some instances contained less than ten per cent normal cells. In some animals there was an excessive increase in interlobular connective tissue in which there were increased numbers of bile ducts. The bile canaliculi were often distended with bile. C...MHW1\§I4:PJJUJ«‘ . ./ 41:16”? Jag . .a .‘ u( a. u. 1. .ZN. .. X.H]l.v l 1:3 Lobular necrosis and cirrhosis of the liver. 7. Figure h C .1 t O nr 1C 08 8n a C\; 2 t( n a r e I pm 1 .lb SO 1 d or £6 V dl ml mm 0 hn t .MG r am 6 ta 0 e\l. f1 n( .1. .h A n w o .1 ant Ha r ematoxylin and Eosin. x 75. u 5 liver lobule. . x 1; u}: {71' . ; a .fi . .v..\. I... an era; In ( 9 ,o. y m” m c f “no. “ally” .1 t .16 “a." uomh .1 u t . conq‘mznlj hrwix nsfl 0.18 0 Pee n esrni V a c.i 5 ion 0 l 18E t u “was. she/\Hulm names 50.. .Wl ix .tw 5.1600 asumm wwwme 1.11an C an” mvl in Elb G O T11 ar hme t0?» unm bile ducts. a n m F L" r " L a: J“ 0"_ . A. ,- »'I ~ ‘:.I .' i. a L 1 J 4:4»? of :ue liVer. Higher power of vylin and Eosin. x 187. Figure 50. Indentation of the Capsule of Glisson by hepatic necrosis. Control pig fed six per cent casein ration. Hematoxylin and Eosin. x 75. Fig-rs S2. A more normal hepatic lob-ale. surrounded by neerctiv liver tissues. Pig was infected with T58 and red six per cent casein ration. Hematoxylin and Ensin. " 0. a ‘ .KMVR Figure 52. Liver regeneration. Pig was infected with TGE and fed six per cent casein ration. Note binucleated hepatic cells and enlarged nucleoli. Hematoxylin and Eosin. x 750. \ O : > r A -e 5? Liver regeneration. Dig was infected with Pl- .- IF. 5 . FE: and fed six per cent casein ration. Note greatly enlarged hepafie cell and its nucleus. Hematoxylin and eosin. x 730. Figure 5L. fanny metamorphosis of a henatic lobule. Pig was infected with TGE and fed six per cent casein ration. Note that most of the liver cells are not present. but the fat remained in its original position. Sudan IV stain. x 187. 119 Glycogen stains (Best's carmine) for all experimental groups were positive with the exception that those liver lobules undergoing necrosis were negative. Fat stains (Sudan IV) were negative for all liver sections of infected and control animals fed 32 per cent casein and were strongly positive in all infected and control animals fed six per cent casein rations that had extensive interlobular fibrosis (Figure 5N, page 118 J. The fat content was greater proportionately with the increase of inter- lobular connective tissue. Fat globules were evident in hepatic cells that were undergoing coagulation necrosis and remained in place even after the cell was destroyed by lysis. Co Will 1. Growth The data on weight gains for infected and control pigs on 2h per cent and six per cent casein rations are presented in TABLES XLII and XLIII. The pigs on both rations made consistent gains from 26 to 68 days of age. The control and infected animals fed six per cent casein ration and transferred to 2k per cent casein ration eight days after inoculation, gained weight rapidly following the transfer and approached the weights of animals that were fed 2h per cent casein ration throughout the experimental period. TABLE XLII. 120 weights of infected and control pigs on 2h per cent casein ration. Experiment III. (Ibs.) Pia W i ' Ha. as 33 9L 7 , Si N 92%;:21' 13.0 16.8 20.7 25.5 7 8.7 10.3 13.1 21.2 19.0 27.5 33.9 2n 11.u 14.3 16.9 25 12.9 15.1 17.9 15.u 26.1 36.5 u5.2 IQ§££E£Q. 13.86 17.1 20.9 24.3"”I 30.3 40.1 “9.6 5 8.4 9.8 11.2 11.u** 8 13.4 15.6 18.6 18.2-t 20.9 30.5 38.4 11 12.9 15.3 18.9 18.7'* 22.1 31.5 38.2 1n 14.9 16.8 19.7 22.0" 27.u 36.6 #5.1 20 12.7 15.8 18.1 21.3.. Sanizsl.fliih. 11.5 14.1 17.2 20.7 22.6 32.0 39.6 0d 12.7 15.1 17.9 19.3 25.2 34.7 uz.9 "Infected on HEth day. TABLE XLII. 120 Weights of infected and control pigs on 24 per cent casein ration. Experiment III. (lbs.) 111:: 2a. 32L m M QEgllfil. 13.0 16.8 20.7 25.5 7 8.7 10.3 13.1 21.2 19.0 27.5 33.9 24 11.4 14.3 16.9 25 12.9 15.1 17.9 15.4 26.1 36.5 45.2 Infested. 13.86 17.1 20.9 24.3“ 30.3 40.1 49.6 5 8.4 9.8 11.2 11.4** 8 13.4 15.6 18.6 18.2** 20.9 30.5 38.4 11 12.9 15.3 18.9 18.7"' 22.1 31.5 38.2 14 14.9 16.8 19.7 22.0** 27.4 36.6 45.1 20 12.7 15.8 18.1 21.3""I W 11.5 14.1 17.2 20.7 22.6 32.0 39.6 IBI2222§_M£52.12.7 15.1 17.9 19.3 25.2 34.7 42.9 "Infected on 44th day. ‘— 121 TABLE XLIII. we: hts of infected and control pigs on per cent casein ration. Experiment III. (Ibs.) a. _-_._______..___..______. m 3.; 31 40 H 3‘2' ’ 51+ M 5222‘s; 13.9 14.4 15.6 16.2 17.9 20.0 23.0 12 12.9 13.6 14.7 15.6 18.5* 27.0 34.0 15 13.7 13.7 14.4 15.0 16.9 20.0 24.0 18 15.1 15.6 17.5 18.6 23.1* 32.0 39.1 23 11.5 11.8 12.6 26 8.4 8.6 9.3 10.1 Ingaskaé. 10.5 10.8 12.4 13.0** 14.3 16.6 18.9 3 14.1 14.9 16.1 16.5" 21.1' 29.7 40.5 9 9.2 9.0 9.6 9.5" 11.6* 16.4 23.4 10 15.3 15.7 17.7 19.3““I 21.3 26.9 31.3 13 10.6 10.9 12.5 12.6** 14.6* 21.8 29.0 16 12.6 13.4 14.3 13.5" 16.9‘ 23.7 32.2 17 10.1 10.3 10.9 11.4.. 19 8.6 8.7 9.2 10.5** 21 10.7 11.9 13.3 13.7"I 15.3 17.6 21.2 22 10.2 10.8 12.5 11.0“I 11.4 12.6 15.6 W 12.5 12.9 14.0 15.1 17.4 20.0 23.5 W 20.8 29.5 36.6 In“flag-Jinan.11.2 11.6 12.9 13.1 15.8 18.4 21.8 W 16.1 22.9 31.3 r8t¥;;.‘;§hanged pigs on 52nd day from 6? ceeein ration {3724i casein _— l"“Infected on 44th day. 122 2. Agalzses Data on total leukocyte counts in TABLES XLIV and XLV indicated that there was not a leukopenia following inoculation, as was described in the results of Experiment I and Experiment II. Hemoglobin levels (TABLES XLVI and XLNII} for animals on six per cent casein ration were lower than those of animals on 24 per cent casein ration. Infected animals fed six per cent casein ration and infected animals changed from.six to 24 per cent casein eight days after inoculation had hemoglobin values that decreased and remained below ten grams per 100 ml. through the twenty-fourth day following inoculation. Hematocrit values in TABLES XLVIII and XLIX indicated that there were no significant differences between control and infected pigs on 24 per cent and control animals on six per cent casein rations. There was only a slight decrease in hematocrit values for infected animals fed six per cent casein ration l7 and 24 days after inoculation. Significant trends were not determined from the data of mean differential leukocyte counts (TABLE L) between the various groups of animals. The data on serum protein distribution of infected and control pigs on 24 per cent and six per cent casein rations are presented in TABLES LI and LII. Infected animals on 24 per cent casein ration three days after inoculation with TGE had increased gamma globulin. and it increased further on the tenth and seventeenth days and declined but remained above normal 24 days after inoculation. Albumin levels were within normal range of distribution. Infected animals on six per cent casein ration had a similar increase in gamma globulin but increased to .I 1| Irllflblulnl' ill! ul 1' . I ...| In; II.I|’.II‘J| TABLE XLIV. Hemocytometer leukocyte counts of infected and control pigs on 24 per cent casein ration. Experiment III. (cells/cum.) Pig ‘28:" .1: .: ’3_: .0710 t O! 1: 24 W 17.500 20.200 31.200 7 25.450 17.650 34.900 23.000 22.450 13.700 24 18.200 13.400 25 18.750 11.150 23.000 19.750 16.450 13,400 1013212; 15.800 16.750 21.300 27.400 18.150 13.850 5 18.550 14.500 15.700 8 17.350 17.000 15.650 25.000 18.000 13.500 11 18.250 24.400 25.550 27.450 25.800 12.700 14 19.550 23.900 13.650 28.950 13.750 12.800 20 20.450 23.900 19.000 W 19. 975 15.600 29.700 21. 375 19.450 13. 550 t 18.325 20.075 18.475 27.200 18.925 13.213 “‘1‘ 1 21.1 TABLE XLV. Hemocytometer leukocyte counts of infected and control pigs on 6 per cent casein ration. Experiment III. (cells/0mm.) 2913 Eng gtgr 3653;151:102 - N2.....—_ .__1 43— *' =%t_ 02—- _1_a12.......alZ........ZE_... 923;:2; 14.050 18.500 21.300 13.000 12.350 13.350 12 27.200 27.950 25.150 16.300- 19.050 10.250 15 33.950 26.700 18.050 20.200 19.200 15.550 18 23.550 26.100 27.450 22.400. 13.750 14.450 23 20.900 18.150 26 17.950 28.700 15.200 IDSEESad. 2 20.175 28.700 15.700 11.350 18.350 17.750 3 24.575 16.650 17.800 25.900* 14.600 12.750 9 35.500 24.200 19.200 23.800* 22.900 9.450 10 22.950 25.100 14.400 22.250 20.250 20.800 13 20.150 23.350 24.450 21.150. 17.700 10.900 16 32.900 27.800 23.800 25.6509 18.650 14.250 17 21.050 23.100 25.000 19 24.500 21.800 15.750 21 20.250 20.000 14.100 13.850 17.800 16.450 22 17.700 23. 300 18.650 20.400 13.450 16. 500 W 22.933 24. 350 21.430 16.600 15.775 14.450 19.350 16.400 12.350 23.975 33.400 18.885 16.962 17.463 17.875 24.125 18.463 11.838 1‘ Stun e ‘C'hanged pigs on 6th day from 6% casein ration to 24? casein TABLE XLVI. Hemoglobin levels of infected and control pigs on 24 per cent casein ration. Experiment III. (Gm./100 m1.) 9.8 9.8 10.1 7 11.3 11.3 11.6 10.7 10.4 9.5 24 11.0 11.0 25 11.0 10.7 11.3 11.3 10.1 10.4 101222.20 1 11.0 11.6 11.3 11.9 9.5 11.6 5 13.7 12.2 11.0 8 13.1 10.7 10.4 10.4 9.5 9.5 11 10.7 10.7 10.7 9.8 9.2 10.7 14 9.8 10.7 9.5 8.9 9.2 10.1 20 12.8 11.0 9.5 antg 1 Eigfiv '7 10.8 10.7 11.0 11.0 10.3 10.0 c 0811 11.9 11.2 10.4 10.3 9.4 10.5 126 TABLE XLVII. Hemoglobin levels of infected and control pigs on 6 per cent casein ration. Experiment III. (Gm. [100 m1.) Pig 4 a s ., - 3051-11- o; No. -8’ :17 3‘ 10 24 92%;:gll 11.0 11.3 11.0 10.4 9.8 10.4 12 11.3 11.6 10.1 9.5* 9.2 10.1 15 11.3 10.7 9.5 9.2 9.2 9.8 18 10.1 11.9 9.2 11.01* 10.7 10.4 23 12.2 11.0 26 12.8 10.1 8.9 M 2 11.0 11.0 9.5 10.7 9.5 10.7 3 10.4 10.7 9.5 9.8‘ 8.6 9.5 9 12.2 10.4 8.6 9.2‘ 8.6 9.5 10 11.3 9.8 8.6 11.0 8.3 9.5 13 10.7 10.4 9.2 8.9‘ 8.3 9.5 16 11.3 11.0 8.9 8.9* 8.6 9.2 17 10.7 9.2 8.0 19 11.3 10.1 9.2 21 11.0 10.4 8.9 9.8 8.6 8.0 22 12.2 11.9 11.3 8.9 8.9 9.2 W ,1 11.5 11.1 9.7 9.8 9.5 10.1 W 10.3 10.0 10.3 In£a£§a§_naan.11.2 10.5 9.2 10.1 8.8 9.4 9.2 8.5 9.4 iChanged pigs—3n 6th day from 6$9casein ration to 24$ casein ration. TABLE XLVIII. Hematocrit values for infected and control pigs on 24 per cent casein ration. Echeriment III. (packed cell volume per cent) Pig o t on 39, 01 '38' ‘:1_ .;1 10 _JEZ_-—— 2812:. £22”.- 35.0 34.0 36.0 7 37.0 38.0 40.0 38.0 36.0 33.0 24 36.0 36.0 25 38.0 37.0 38.0 38.0 35.0 36.0 10§22222. 38.0 40.0 35.0 36.0 32.0 38.0 5 40.0 38.0 35.0 8 40.0 37.0 37.0 34.0 33.0 33.0 11 37.0 38.0 36.0 33.0 33.0 7.0 14 36.0 38.0 34.0 31.0 33.0 35.0 20 37.0 36.5 35.0 W 36.5 36.3 38.0 38.0 35.5 34.5 3810 37.9 359; 33.5 32.8 35.8 TABLE XLIX. 5.4 N I); Hematocrit values for infected.and control pigs on 6 per cent casein ration. Experiment III. (packed cell volume per cent) Pig __.__ t t no. -8 ;1___T 3 19 12... .._____£‘.f___ £222:2;' 36.0 32.5 36.0 33.0 34.0 35.0 12 36.0 39.0 35.0 30.0* 32.0 35.0 15 33.5 37.0 33.0 30.0 33.0 33.0 18 34.0 38.5 31.0 35.0* 37.0 37.0 23 38.0 36.0 26 38.0 35.0 27.0 Integged 2 35.0 32.5 30.0 34.0 33.0 35-0 3 36.0 35.0 33.0 32.0‘ 31.0 34.0 9 35.0 34.0 29.0 29.0‘ 32.0 32.0 10 36.0 33.0 31.0 36.0 29.0 32.0 13 34.0 34.0 31.0 29.0"I 31.0 32.0 16 33.5 39.0 33.0 23.0‘ 33.0 33.0 17 34.0 32.0 29.0 19 37.0 34.0 32.0 21 35.0 39.0 29.0 28.0 30.0 27.0 22 39.0 40.0 36.0 32.0 31.0 33.0 W 35.9 0 236.3 32.4 31.5 33.5 34.0 32.5 34.5 36.0 In{2£§2§.fl§in.35.5 o 35.3 31.3 32-5 30.8 31.8 29.8 31.8 32.8 ration. §Changed pigs on 6th day from 6¥casein ration to 24$ casein 129 TABLE L. Mean of differential leukocyte counts.’ Experiment III. (per cent) Lot , te tio ._ Legom” -8 :1. 3 10 Z 311 A. Neutrophils Segmented 48.8 45.5 55.3 34.3 32.0 24.0 Nonsegmented 0.8 1.8 9.0 0.0 1.5 0.5 Lymphocytes u8e3 48.5 32e0 63.0 64.0 7ue0 Mbnocytes 1.3 0.5 3.0 1.0 2.0 0.5 Eosinophils 0.5 3.0 0.0 1.7 1.0 0.5 Basophils 0.5 0.8 0.0 0.5 0.5 0.5 B. Neutrophils segonted 3907 520 3 5208 5900 $08 38.8 NOHSQMM 1e3 3e5 SeS 1e8 les 2e3 lymphocytes 56.6 43.2 42.7 38.0 41.5 56.0 Mbnocytes 1.3 0.5 1.0 0.5 1.3 2.3 Eosinophils 0.8 0.5 1.3 0.5 0.8 0.8 BasOphils 0.8 0.3 0.0 0.3 0.3 0.0 C. NeutrOphils Segmented 45.7 59.3 50.8 46.5 37.5 30.0 Nonsegmented 1.3 3.2 3.8 1.0 1.0 1.5 lymphocytes 50.5 35.8 44.0 51.3 55.2 66.0 Mbnocytes 0.31.2 0.2 0.5 2.5 1.5 Eosinophils 0.8 0.5 0.6 1.0 2.5 1.0 Basophils 0.0 0.0 0.6 0.0 1.0 0.0 D. Neutrophils Segmented 52. 3 56. 6 54.6 57.8 44.8 49. 3 Nonsegmented 2. 3 .1 10.9 3.5 2.3 1.5 uwmphocytes 43.2 38.0 32.6 38.5 51.0 46.0 Monocytes 1.4 0.6 1.4 0.0 1.5 2.5 Eosinophils 1.2 1.5 0.5 0.3 1.4 0.5 Basophils 0.2 0.2 0.0 0.0 0.0 0.3 E. Neutrophils Segmented 45.5 49.0 26.0 Nonsegmented 1.0 1.0 2.5 lymphocytes 50.5 47.0 70.0 Mbnocytes 1.0 1.0 1.0 EosinOphils 2.0 1.0 0.0 BasOphils 0.0 1.0 0.5 F. NeutrOphils Segmented 68.3 45.5 39.8 Nonsegmented 4.3 3.7 3.3 lymphocytes 27.6 48.3 54.5 Monocytes 0e 3 1. 8 2e 5 Eosinophils 0.5 0.8 0.0 Base hils 0.0 0 0 0 0 ‘Pigs fed (1) Control 32%; (B) Infected 32%. (0) Co ntrol Infected 6%. (E) 6% Control changed to 24% Control and (F) 6% Infected changed to 24% Infected casein ration. 130 1 edSQH-G nu edggsmfle oa.mm Ha.m~ mo.aa Ha.sa Hm.as mn.~m ms.ma mo.aa ma.¢s oa.m~ om.ma mm.aa maumlummmquu sm.mn ma.on mo.ma mm.aa sm.es om.s~ mm.aa mm.oa no.3: em.u~ oa.ea m:.~w o s _muuuuauuummu mn.am mm.sm ms.ea Hm.aa ms.sm mo.~m om.o~ em.ma om.ns mm.em ms.ma o~.~H om sm.mm ma.am 36.64 ao.ea aa.~s m~.mm am.oa sN.oa 64.8: oa.s~ n~.o~ Ha.oa an am.as Hm.a~ sa.ma mm.sa am.as mm.o~ sa.ma am.ma ma.ms mm.m~ sm.ma om.aa Ha m~.Hs om.e~ H~.sa Ho.aa no.6m mm.am os.ma om.ma am.as as.sm mo.ea oo.~a w oa.~s sm.e~ ao.o~ oo.aa 8s.mm sa.a~ s~.mm as.oa ~H.ms mm.n~ am.aa ms.aa n sa.sn an.~m mo.o~ oa.~a aw.ws ~H.¢~ no.8H om.oa s~.as No.nm am.ua mn.m H 8384.3 no.3: m~.em Hm.ea om.ma NH.ss mm.o~ am.oa mo.~a Hm.os mm.w~ om.aa 88.24 mm mm.mm ms.am ao.sa mo.aa mm.ns. em.m~ No.6H oe.aa aw 8m.am 86.4m mn.ma sa.~a No.4: om.8~ mm.ma om.aa ms.mm om.~m om.sa Hw.ma a om.en mo.mm mm.ma mm.oa N~.es om.sm 86.6H am.m o~.am ms.mm 64.na mm.a p . . e 6 .40m .caasnoao meaasnoao .neaaenoau Mme an 1, an “an. 1+- soup see a p » fiasco acmv .HHH unceaaomxm Acheson use eouoemsd mo newepoaa echo» «0 mdmoaoneoppoeae modem Elli! 1.“ .:o«uea :«emeo pace nod am so nmaa .HA mflm