A PATHOLOGICAL STUDY OF THE INTESTINE AND OTHER ORGANS OF WEANLING PIGS WHEN FED A RATION OF NATURAL FEEDSTUPFS — LOW IN PANTOTHENIC ACID By Girdhari L. g^arma A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Pathology 1951 ProQuest Number: 10008424 AN rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008424 Published by ProQuest LLC (2016). Copyright o f the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 DEDICATED TO Dr. Prank: Thorp Jr. — an inspiring teacher and sincere friend with an ideal philosophy for directing graduate studies. ACKNOWLEDGMENT S I express my deep and sincere feelings of gratitude to Dr, Frank Thorp Jr. for his valuable guidance and constant supervision in this investigation. Indebtedness is due Mr, M. L. Gray for help in taking photomicrographs and for the valuable suggestions in preparing the manuscript. Grateful acknowledgment is due Dr. R. L. Johnston for help in necropsies of the animals studied and to Dr. W. D. Baten for assistance in the statistical analysis of the data. I am grateful to Drs. R. W. Luecke, M. Lois Calhoun and R. A. Runnells and to Miss Sylvia Lane for their helpful suggestions and assistance. I wish to thank all my friends in the Depart­ ment of Animal Pathology and others for their assistance in one way or the other. The financial support from the Government of India and from Michigan State College, which made this study possible, is highly appreciated. ^ jjcjjf^ ^ ^ aft** jjgpjcsjc * *** ***** * Girdhari L. Shamoa candidate for the degree of DOCTOR OF PHILOSOPHY Final Examination, August 15, 1951, 1:30 P. M., Room 314, Anatomy Building Dissertation: A Pathological Study of the Intestine and Other Organs of Weanling Pigs When Fed a Ration of Natural Feedstuff s — Low in Pantothenic Acid, Outline of Studies Major subject: Animal Pathology Minor subjects: Microscopic Anatomy, Animal Breeding Biographical Items Born, September 3, 1919, Gurdas Pur, East Punjab, India Undergraduate Studies, Sanatana Dharama College, Lahore, India, 1936-1938 The Punjab Veterinary College, Lahore, India, 1938-1942 Graduate Studies, Michigan State College, East Lansing, 1948-1951 Experience: Research Assistant, Indian Veterinary Research Institute Tlukteswar-Kumaun, India, 1943-1948, Graduate Assistant, Department of Animal Pathology, Michigan State College, Bast Lansing, 1950-1951 Member of The Society of The Sigma Xi TABLE OF CONTENTS PAGE INTRODUCTION,............................................... 1 REVIEW OF LITERATURE......... 3 A* Pigs *.......... B.... Dogs............ C. Chickens,............ D. Rats....... 3 12 14 15 MATERIAL AND METHODS....................................... 20 RESULTS........... 23 A*Statistical Analysis.......... B. Clinical Picture...................... C, Post Mortem Findings................ 23 * 45 46 DISCUSSION........................................... 58 SUMMARY AND CONCLUSIONS..................................... 63 LITERATURE CITED............................................ 64 LITERATURE CONSULTED BUT NOT CITED.............. 69 FIGURES. ............... 71 1 INTRODUCTION Vitamins have assumed a position of great importance in the field of nutrition during the last two decades. It is, therefore, necessary to recognize that the type as well as the ratio of different vitamins are of as great importance as the quality and quantity of proteins, carbohydrates, fats and minerals in the ration of animals. Unless a feed contains the proper balance and amounts of various neces­ sary vitamins, it will be inadequate in serving the purpose intended. It is therefore apparent that more emphasis should be given to the vita­ min make-up of the ration, to promote the health and development of animals and to insure sound agricultural economy. The proper amount of the necessary vitamins in the ration of the young growing pig is of vital importance; this was brought out by Cunha (1951); who stated that about 40 per cent of all pigs farrowed, die before they reach market age. swine producer. This is a tremendous loss to the It is generally agreed that much of this loss in the swine industry is attributable to faulty nutrition. Morrison (1940) stated that pigs excel all other farm animals in the econony with which they convert feed into edible flesh. Hogs re­ quire less than 400 pounds of concentrates for each 100 pounds of gain in weight, whereas fattening calves and lambs require 800 pounds or more for a similar gain. It should also be recognized that the rate of growth of pigs is much greater than that of cattle, horses, or sheep, and that the pig reaches puberty at a relatively young age. This brings 2 out the fact that the vitamin requirements of the pig are as important as that of other farm animals. It has been shown by various workers that the response of the pig to a synthetic ration may not be the same as to a diet of natural feed­ stuff s and that a ration which according to accepted ciirrent standards is adequate, does not necessarily meet the nutritional requirements of young growing pigs. It has further been shown that deficiencies of certain B vitamins may have a marked effect on the intestinal tract of the pig. The pur­ pose of this study was to determine whether or not a diet composed of natural feedstuffs low in pantothenic acid would have a similar effect. 3 REVIEW OF LITERATURE A. Pigs Since the isolation of pantothenic acid by Williams et al (1938), considerable work has been done regarding the role of this vitamin in domestic animals. In swine, pantothenic acid deficiency has been shown to interfere principally with the physiology of the digestive system and nervous system. This has been demonstrated by numerous authors by the use of purified rations in which this particular con­ stituent of the B vitamin complex was withheld. Kinsley (1934) remarked that rtinfectious necrotic enteritis’1 was widespread and was second only to hog-cholera in economic importance. The disease usually designated as wfilth disease” was thought to be caused by B. suipestifer. That necrotic enteritis was not always due to an infectious agent, was brought out by McEwen (1937). This author reported necrotic enteritis in young pigs in a herd where swine fever was unknown and where Salmonella could not be detected. The hygienic condition of the herd was described as superior. The pigs showed diarrhea which, in later stages, was blood tinged. There was no rise in body temperature. Non-lactose fermenting gram-negative bacilli could not be isolated. Selective media seeded with intestinal material failed to indicate the presence of Salmonella nor was there evidence of other bacteria to which the disease could be attributed. It was concluded that necrotic enteritis could occur without Salmonella infection. 4 Birch et al (1937) showed that nutritive failure in rearing young pigs, was not due to defects in the amount or quality of the proteins, but to a deficiency of some of the constituents of the thermostable fraction of vitamin B complex other than lactoflavin. Wintrobe and Mitchell (1938), Hughes (1939) and Wintrobe (1939) in­ vestigated further the requirements of vitamin B^ for pigs and studied the pathological picture caused by the deficiency of one or more known factors of the thermostable vitamin B^. Wintrobe and Mitchell (1938) fed young pigs with a ration "adequate" in proteins, carbohydrates, fats, unsaturated fatty acids, minerals, vitamins A, C, D and 11ad equate” amounts of yeast* The latter was gradual­ ly reduced and substituted by thiamine and riboflavin* There was pro­ duced a severe degeneration of the posterior columns of the spinal cord, the dorsal root ganglion cells and the peripheral nerves* It was con­ cluded that these changes were due to the deficiency of one or more components of the vitamin B complex, other than thiamine or riboflavin* Chick et al (1938), in a study of the role of B vitamins, other than aneurin, nicotinic acid and riboflavin in the nutrition of the pig, re­ ported that at least two more water soluble factors were necessaiy for the pig* These authors observed that two vitamins were contained in the filtrate and eluate fractions (Bg) of Edgar and Macrae (1937)* The determination of the respective function of vitamin Bg and of the fil­ trate factor was left until the pure form of the vitamins were available* Hughes (1939) reported slow growth and a crippled condition of pigs fed a riboflavin deficient diet* Addition of riboflavin improved the 5 growth of the animals but there was no recovery from the crippled con­ dition. This was thought to be due to a permanent injury to the nerve tissue. Thiamine deficiency in the ration produced slow growth but no beriberi paralysis. When a ration, deficient in factor 1 (vitamin Bg) and factor 2 (filtrate factor), was fed to pigs, the pigs lost weight and one of them showed "goose stepping". Addition of the factors to the ration improved the condition of the animals. Wintrobe (1939) in a further study of the nutritional requirement of the pig, fed a purified diet of casein, fat and carbohydrates, and small amounts of yeast. A mineral mixture, containing salts normally present in the sows milk, was supplemented with iodized iron. Synthetic thiamine chloride, riboflavin and nicotinic acid would not correct the failure to grow. Synthesis of pantothenic acid by Williams (1939) introduced a new approach to the understanding of the nature of the filtrate factor. So far the elucidation of the influence of pantothenic acid, contained in the filtrate factor, had been attempted rather indirectly by withholding the filtrate factor from the ration. With the availability of synthetic pantothenic acid, various authors used purified diets to evaluate direct­ ly the role of the vitamin. Wintrobe, et al (1940) fed three week old pigs with a basal ration "adequate" in carbohydrates, proteins, fat, minerals. The ration was supplemented with cod liver oil, ascorbic acid and varying amounts of yeast. In one group, yeast was gradually reduced and replaced either by synthetic thiamine, riboflavin and nicotinic acid or by liver, yeast 6 extract and "wheat germ oil. All the pigs fed little or no yeast and given thiamine, riboflavin, nicotinic acid and even the filtrate factor or "wheat germ oil as supplements, showed changes in the nervous system. Hone of the animals fed liver developed neural changes. It seemed that liver contained a substance, the absence of "which produced changes in the nervous system. Thiamine, riboflavin, and nicotinic acid were not considered to contain the protective factor. These authors stated, Mour observations do indicate, however, that yeast affords some protection. Neural involvement was less frequent •when yeast was fed in large amounts, as compared with the changes ob­ served in animals given little or no yeast. Furthermore, the changes in the animals given filtrate factor, made from yeast, on the whole were less extensive than in those not treated in this way.” Edgington et al (1942) attempted to find whether or not nicotinic acid exerted an influence on the prevention of infectious necrotic enteritis. There was no significant improvement shown by the pigs receiving nicotinic acid over those which did not receive this vitamin. The authors pointed out the possibility of a nutritional factor in enteritis under field conditions* Wintrobe et al (1942) observed degeneration of sensory neurons when either pyridoxine or pantothenic acid or both were lacking in the ration. Ho such change occurred when both the vitamins were supplied. These authors did not support the view that thiamine deficiency could damage the nervous system. Thiamine deficiency, on the other hand, 7 was characterized by anorexia, vomiting and elevation of pyruvic acid level in the blood. Hughes (1942) fed young pigs a pantothenic acid deficient diet. The animals developed a subnormal appetite, grew slowly and became emaciated, showed ”goose stepping” and lost hair. itis of the large intestine were also observed. Gastritis and enter­ The conclusion was that pantothenic acid was necessary for normal growth and well being of young growing pigs. Hughes and Ittner (1942) fed young pigs a ration with varying con­ centrations of calcium pantothenate. The minimum requirement of panto­ thenic acid for a 100 pound pig was found to be 7.8 to 11.8 milligrams per day. According to Wintrobe et al (1943), pantothenic acid deficiency produced the most striking degree of malnutrition encountered in a study of B vitamin deficiencies. Doyle (1943) showed that swine dysentery was a disease distinct from necrotic enteritis. When the economic importance of pantothenic acid was well estab­ lished in young pigs and the vitamin could be obtained in pure form, Wintrobe et al (1943) studied the gross and microscopic abnormalities produced in pigs fed synthetic diet deficient in pantothenic acid. The basal ration consisted of "Sheffield Hew Process” casein, sucrose, lard and salt mixture. Comparison of the effects on growth of lack of thiamine, nicotinic acid, pyridoxine or pantothenic acid revealed that the last mentioned vitamin produced by far the most severe impairment 8 of growth* Pantothenic acid deficiency was characterized by diarrhea, dysentery, marked impairment of growth, excessive nasal secretion and cough* There was patchy alopecia — center of the back* most pronounced over the rump and Extensive colitis was present. At necropsy the changes varied from diffuse hyperemia, enlarged lymphoid follicles, small ulcers to extensive inflammatory changes involving the large in­ testines* Atrophy of the cells lining the glands of the intestinal mucosa, abcess formation and ulcerations constituted the main histological changes of the mucous membrane* Moderate normocytic anemia, fall in serum chlorides, increase in carbon dioxide combining power of the blood, a terminal rise in non-protein nitrogen and occasional hyper­ glycemia were associated with the syndrome. The abnormal gait, which was associated with well defined histologic changes in the nervous sys­ tem, improved following administration of calcium pantothenate, but complete restoration of function did not occur. Wintrobe et aJL^ (1944) produced chronic thiamine deficiency in pigs, by feeding a purified diet that had been supplemented with a mixture of crystalline vitamins, from which thiamine was omitted* vomiting, poor growth and cardiac failure developed. Loss of appetite, Symptoms and histo­ logic changes in the nervous system, were, however, not observed* These authors also pointed out that autoclaving destroyed not only thiamine but reduced the pantothenic acid content of yeast as well. Wintrobe at al (1942) established that degeneration of the sensory neurons could be produced in swine when pyridoxine and/or pantothenic 9 acid were excluded from the diet. In continuation with this work, Follis and Wintrobe (1945) used a synthetic ration to compare the effects of pyridoxine and pantothenic acid deficiency in the nervous tissue of swine. The ration was supplemented by thiamine hydrochloride, riboflavin, nicotinic acid, choline chloride, inositol and para-aminobenzoic acid. Varying amounts of pyridoxine and pantothenic acid were added to compare the influence of the two factors. Pyridoxine deficiency produced suspicious disturbance in gait after a three to six week period on the diet. Pathological changes were characterized by denyelinization of peripheral nerves and marked de­ generation of axis cylinders. root ganglion. There was no chromatolysis in the dorsal The cells of the gray matter of the spinal cord did not show any change and the ventral roots were normal. In pantothenic acid deficiency, ”goose stepping” was observed in the fourth week. There were classical signs of chromatolysis of dorsal root ganglion cells and later on, the neurons became necrotic and were removed by macrophages. The authors concluded that pyridoxine deficiency was nyelinoclastic because uyelin degeneration occurred first, and then the degeneration of the neurons. According to this classification, pantothenic acid deficiency was referred to as polioclastic because changes were first noted in the neurons. The authors could not detect lesions of the nerves in thiamine deficiency in swine, and could therefore not support the common accept­ ance of thiamine as an ”antineuritic vitamin”. Colby et al (1948) studied the relationship between pantothenic acid and biotin in the nutrition of the pig. The authors used six 10 week-old pure bred Chester T/Hhite pigs. diet. The animals were fed a purified Pantothenic acid deficient animals showed anorexia, slow growth and developed ’’goose stepping”. Addition of biotin, prolonged the life of the pantothenic acid deficient pigs, but caused the pantothenic acid deficiency symptoms to appear in half the time. A probable interrela­ tionship between pantothenic acid and biotin, was concluded. Experimental data collected thus far had put forth convincing evidence of the importance of some of the factors of the vitamin B complex in the nutrition of the growing pig. This together with the inherent economical loss, due to faulty nutrition in the swine industry, percipitated the pressing need to bring together and evaluate, in a compact workable form, the results of the researches. to this objective was made by Luecke et al (1949). A direct approach The authors initiated an intensive study of swine enteritis under field conditions, so that the role of nutritional deficiencies in the disease could be evaluated. Pigs with diarrhea, absence of any appreciable fever, a history of low protein cereal grain ration and a lack of green forage, were selected for the investigation. Large doses of thiamine, riboflavin, calcium pantothenate, niacin and pyridoxine were injected intraperitoneally. The five vitamins along with vitamins A and D were incorporated in the ration. Sixty-nine out of eighty pigs were completely cured* The pigs necropsied before treatment showed inflammation of the large intestine. The intestinal wall was edematous. adhered to the mucosa even when washed with water. Fecal material Mucoid degeneration, 11 necrosis and ballooning of the glands, were the principle histopathological changes of the intestinal wall# Luecke et al (1949) attempted to determine if pantothenic acid deficiency could be produced by feeding a ration made up largely of corn# Hog cholera immunized pigs were used# In lots where the basal ration was not supplemented by pantothenic acid, the animals showed diarrhea, followed by locomotor incoordination# The sciatic nerve showed swollen fibre tracts which were granular in appearance# eration of the myelin sheath was also detected. Degen­ It was concluded that an insufficient quantity of pantothenic acid in the basal ration could not prevent symptoms of locomotor incoordination and myelin degenera­ tion# McMillen et al (1949) reported on B vitamins for weanling pigs, "Many natural feedstuffs are low or borderline in one or more of these factors# The grains, especially com, vary 100 per cent or more in B vitamin content# Little is known about the availability of niacin, pantothenic acid and riboflavin in natural feedstuffs for small weanling pigs* For these reasons a supplemental allowance of about two times what appears to be the minimum requirement seems to be desirable#" McMillen et al (1949a) showed that a basal ration of corn, oats, etxpeller soya bean meal, meat scraps, alfalfa meal, and mineral mixture was deficient in pantothenic acid, nicotinic acid and riboflavin for weanling pigs# The basal ration, when supplemented with calcium panto­ thenate, riboflavin and nicotinic acid brought about a significant increase in daily gain in weight and prevented deficiency symptoms. 12 Luecke et al (1950) studied pantothenic acid deficiency in wean­ ling pigs fed basal ration consisting of corn, soybean and complex mineral mixture# cent. The crude protein content of the ration m s 16*4 per The addition to the basal ration of a mixture of synthetic B vitamins, containing no calcium pantothenate produced very severe symptoms of pantothenic acid deficiency. No symptoms of incoordination were produced when an unsupplemented c o m soybean diet was fed to the animals. The reason advanced m s that the pigs receiving unsupplemented ration grew so poorly that the pantothenic acid content of the ration m s sufficient to prevent incoordination. On the other hand, the sup­ plemented ration increased the rate of growth and with that an increase in the requirement of pantothenic acid. On the basis of this observa­ tion the authors warned against the addition of riboflavin and nicotinic acid as protein supplements to rations containing minimal levels of pantothenic acid. This was parbictilarly undesirable if such a feed m s used in dry feed lots. B. Dogs Fouts et al (1940) fed adult dogs with a synthetic casein diet supplemented with thiamine chloride, riboflavin, nicotinic acid and vitamin Bg. The deficiency state m s characterized by loss of weight, intermittent diarrhea, moderate anemia and death. The diet m s deficient in a factor or factors contained in a purified liver extract other than nicotinic acid. It was not determined whether the deficiency state m s due to the lack of factor II (chick antidermatitis factor) alone or to the lack 13 of this and some other unisolated compounds of the vitamin B. complex. Scudi and Margaret (1942) stated that pantothenic acid deficiency in the dog produced lowering of blood cholesterol, cholesterol esters, lipoid phosphorous and total lipoids. in weanlings than in adult dogs. The deficiency was more critical Dogs receiving pantothenic acid and whole dried liver in addition to the stock food, showed minimal liver damage. Pantothenic acid deficient dogs developed extremely fatty livers. Schaffer et al (1942) studied pantothenic acid deficiency in dogs. Weanling mongrel puppies and older growing dogs were fed on a basal ration consisting of sucrose, acid washed casein, cotton seed oil, cod liver oil and salt mixture. The ration was supplemented by thiamine, riboflavin, nicotinic acid, pyridoxine hydrochloride and choline chloride. Pantothenic acid deficiency manifested itself suddenly by prostration, coma, and rapid respiratory and cardiac rates. Necropsy of the deficient dogs revealed fatty livers, mottled thymuses, hemorrhagic renal degen­ eration, gastritis or enteritis and intussusception. The plasma calcium and inorganic phosphorus were normal in the deficient dogs, while the blood glucose and chlorides were lowered and the non-protein nitrogen values were raised. These values returned to normal after recovery from the deficiency. Silber (1944) fed a purified diet, lacking pantothenic acid, to pups and adult dogs. Control animals received calcium pantothenate or calcium pantothenate and liver. Within three weeks depleted pups showed anorexia and a decrease in urinaiy excretion of pantothenic acid. Collapse, which occurred in 14 two months, could bo prevented by calcium pantothenate administration. Adult dogs showed a delayed effect on appetite and a less severe de­ crease in urinary excretion# C* Chickens Ringrose and ITorris (1936) showed that dried pork liver contained a factor which could prevent a pellagra-like syndrome in chicks fed an eggwhite diet. There was another factor, in dried pork liver, essential for the growth of chicks fed a purified casein diet# This growth pro­ moting factor, soluble in an alcohol-water solution, was stable to heat­ ing in a dry atmosphere and was relatively stable to autoclaving at pH 11. The pellagra-preventing factor, on the other hand, insoluble in an alcohol-water solution, was destroyed by heating in a dry atmosphere and by autoclaving at pH 11 but not at pH 9* The growth promoting factor was labeled as vitamin G# Jukes and Babcock (193B) studied a factor promoting growth and preventing paralysis in chicks# paralysis# Chicks fed a simplified diet developed It was found that soybean oil would prevent the paralysis but alfalfa meal was more active in this respect than soybean boil# The activity of alfalfa meal was largely destroyed by autoclaving for five hours at 120°# The active factor in alfalfa meal was not extracted by hexane, but could be readily extracted by warm water. The active factor was soluble in 40 per cent ethanol, but insoluble in 90 per cent ethanol# From aqueous solutions at pH 5, the factor could be adsorbed on fuller*s earth and was eluated from the fuller* s earth by a mixture of water, acetone and ammonia. 15 Phillips and Engel (1939) reported myelin degeneration of myeli­ nated nerve fibres distributed widely throughout the white matter except in the posterior region. nized epithelium. The skin showed dry sloughing of the kerati­ These authors were of the opinion that pantothenic acid was essential for the proper maintenance of the spinal cord. Nicotinic acid, riboflavin or vitamin were not effective in prevent­ ing the spinal cord lesions in chicks suffering from dermatitis. Williams (1943) reported that pantothenic acid deficiency in ani­ mals and fowls, was characterized by dermatitis, keratitis, adrenal hemorrhage and atrophy, depigmentation of hair or feathers, failure to grow and alopecia. The author further stated that these and other wide­ spread effects suggested the fundamental function of pantothenic acid in cellular physiology. Shaw and Phillips (1945) found that pantothenic acid deficiency in the chick caused a wide-spread myelin and axon degeneration in the spinal cord, without any accompanying degeneration in the peripheral nerves. No such lesions were observed in chickens suffering from a mild biotin deficiency or an acute deficiency of the folic acid complex. D. Rats Morgan et al (1938) used three similar basal diets for rats with sucrose, lactose or cornstarch as carbohydrate constituent. arrived at the following conclusions: The authors "lactose favors the production in the intestine of rats, probably by micro-organisms, of both flavin and vitamin Bg, but not the filtrate factor. 16 Cornstarch either carries with it or favors the production of the filtrate factor only. Sucrose neither carries nor produces any of the vitamin Bg factors and is therefore the carbohydrate of choice for studies of these factors.” Mills et al (1940) fed rats with a synthetic ration containing sucrose, alcohol extracted casein, c o m oil and salts. The ration was supplemented with thiamine chloride, vitamin Bg, riboflavin, nicotinic acid, choline hydrochloride and haliver liver oil. Rats receiving basal ration alone showed hemorrhagic necrosis of the adrenals. this condition. Inclusion of calcium pantothenate in the ration prevented Addition of choline however, aggravated the symptoms. Daft et al (1940) reported a high incidence of adrenal necrosis in rats which received adequate amounts of pyridoxine but no ”filtrate factor”. Rats which were given a crude fullerTs earth filtrate from liver or rice polishings, did not have adrenal necrosis. Adrenal hemorr­ hage, atrophy and necrosis were prevented by including 100 micrograms of synthetic pantothenic acid every day for six to fourteen days. Salmon and Engel (1940) studied the role of pantothenic acid and adrenal necrosis in rats. A synthetic diet, used for the rats, was supplemented by thiamine, riboflavin, choline, calciferol and carotene. Pyridoxine was given to some animals from the start of the experiment and in others after severe skin lesions had developed. It was concluded that pantothenic acid prevented the adrenal hemorrhage and necrosis which was frequently observed in filtrate factor deficient rats. 17 TTnna (1940) fed rats a diet consisting of dextrose, casein, hydro­ genated cotton seed oil, salt mixture and cod liver oil. supplemented with thiamine, riboflavin, vitamin o The diet ■was and choline chloride# Three to four weeks later the animals ceased to grow and developed scant coarse fur# There was inf lamination of the nasal mucosa and the whiskers were blood stained# At necropsy, the rats showed hemorrhages of the subcutaneous tissue and in the adrenal cortex* The rats could not utilize beta-alanine or alpha-hydroxy beta, beta-dimethyl-gammabutyrolactone, singly or when mixed together, but needed the intact pantothenic acid molecule to recover from the deficiency# Lippincott and Morris (1941) studied the morphologic changes asso­ ciated with pantothenic acid deficiency in the mouse# atrophic and desquamatic dermatitis was observed* Hyperkeratotic, Myelin degeneration was found in the sciatic nerves and spinal cord. Unna ejb al (1941) fed young black rats a purified diet, supple­ mented with thiamine, riboflavin, nicotinic acid, pyridoxine and choline# The rats developed marked greying of the fur within three to seven weeks# The achromotrichia was associated with the syndrome of pantothenic acid deficiency# A daily supplement of 100 micrograms calcium pantothenate prevented the occurrence of grey hair# Sullivan and Nicholis (1942) fed forty-five twenty-one-day-old rats with a purified diet low in vitamin B complex and supplemented with synthetic thiamine, riboflavin, pyridoxine, and nicotinic acid# Dilation of hair follicles and disintegration of sebaceous glands in the areas of alopecia, suggested pantothenic acid deficiency# 18 Henderson et al (1942) worked on the role of pantothenic acid in the rat. Rats, twenty-one days old, were fed a purified ration con­ taining sucrose, purified casein, salts, corn oil, thiamine, pyridoxine, riboflavin, nicotinic acid and choline. were given per week. Two drops of halibut liver oil The rats, on this pantothenic acid deficient ration, became grey in four to six weeks. acid cured this condition. Administration of pantothenic Rats on a heated grain ration showed greying which was likewise prevented and cured by synthetic calcium-pantothenate. Greying produced by copper deficiency did not respond to pantothenic acid administration but was rapidly cured by copper. Para-aminobenzoic acid was ineffective against greying of hair produced by feeding a synthetic ration. Wright and Welch (1943) added succinylsulfathiozole in a purified but adequate diet for rats. Severe pantothenic acid deficiency was produced and a marked reduction in pantothenic acid content of the liver was observed. Both conditions were corrected by supplementing the diet with crystaline biotin and ’’folic acid” concentrates. It was concluded that the utilization of pantothenic acid depended on the availability of biotin and ’’folic acid” which were ordinarily supplied in feedstuffs and synthesized by intestinal bacteria. Emerson and IVurtz (1944) investigated whether or not there was any interrelationship between biotin and pantothenic acid in the nutrition of rats. Biotin deficiency was aggravated by superimposing a deficiency of pantothenic acid upon the animals. Inclusion of biotin in the puri­ fied ration, lessened the severity of the syndrome due to lack of 19 pantothenic acid# Inclusion of pantothenic acid decreased the symptoms of biotin deficiency. West et ad (1944) fed rats a basal diet containing calcium panto­ thenate. The diet of the control rats was not supplemented by calcium pantothenate. All the animals were infected with pneumococcus. About 50 per cent of the rats on the basal diet alone died after showing acute symptoms of pneumonia. None of the rats of the pantothenate group died. Dean and McKibbin (1946) studied pantothenic acid deficiency in the rat by the use ©f a purified diet containing all the essential con­ stituents but not the vitamin under study. The animals on the deficient diet ceased to gain weight and showed diarrhea. At necropsy, the intestines were found to be distended with gas* During the first two weeks of the experiment, the adrenalcortex comprised a smaller proportion of the gland in the deficient rats than in the controls. After this the interrelationship became reversed so that the cortex represented a greater proportion of theglands in the deficient rats than in the controls. In the adrenals of the deficient rats, foci of complete necrosis and hemorrhage were found in the ihscicular and the reticular zone. 20 MATERIAL AND MBTHODS Approximately eight-week old weanling Duroc pigs were used in this study. method* They had been immunized against hog-cholera by the serum-virus The pigs were self fed a basal ration composed of natural feed- stuffs as outlined below: Com 74.0 per cent Soybean oil meal 23.0 per cent Mineral mixture Dicalcium phosphate Limestone Iodized salt Trace minerals (pre-mixed) 3.0 1.0 1.0 0.9 0.1 per per per per per cent cent cent cent cent The ration contained 18 per cent crude protein and 4.53 milligrams of pantothenic acid per pound of feed. This basal ration was supplemented by: Vitamin A 2000 international units per pound of feed Vitamin D 200 international units per pound of feed Niacin 10.0 milligrams per pound of feed Riboflavin 2.0 milligrams per pound of feed Merck* s A. P. P. 0.5 milligram per pound of f eed 6.75 micro grams per pound of feed Vitamin B The animals were housed in a concrete-floored b a m and had access to clear running water from an automatic fountain. Eight pigs in lot 1 were fed the ration described above. 21 The same number of control pigs in lot 2 received the c o m soybean ration supplemented by B vitamins in the following amounts per pound of feeds Thiamine 2.5 milligrams Riboflavin 5.0 milligrams Niacin 10.0 milligrams Calcium pantothenate 10.0 milligrams Pyridoxine 1.5 milligrams Choline chloride was also added at the level of 0.5 per cent. It should be observed that pigs of lot 1 received a ration which was not supplemented by pantothenic acid, whereas the control pigs of lot 2 were fed the ration which had pantothenic acid as one of the supplements. The pigs in lot 1 were weighed on the 14th, 18th, 20th, and 26th day after being placed on this ration. On each of these days two pigs on the deficient ration were sacrificed by electrocution for study. The remaining feed was weighed back to determine the amount of feed consumed during the preceding interval. Before being sacrificed, a sample of blood was taken from the anterior vena cava using potassium oxalate as anticoagulant. Blood was obtained directly from the animals in order to prepare smears for the differential leukocyte count. Hemoglobin concentration was determined by the photo electric method, using a Sheard-Sanford photelometer. Total erythrocyte count was made using Leake and Guy's (1925) solution as diluent and for total leukocyte count, the diluent was one-tenth normal hydrochloric acid. Hemoglobin 22 determination, total erythrocyte and total leukocyte counts were made within a few hours after collection of the blood sample. Hemoglobin concentration, total leukocyte count and differential leukocyte count of blood from the control pigs, were made on three different occasions. Sections of small intestine, large intestine, kidney, liver, and adrenals were taken and immediately fixed in Zenker*s fluid. Paraffin sections were cut four to five mi era in thickness and were stained with hematoxylin-easin for histopathological examination (Mallory 1938). 23 RESULTS The results of the investigation are presented under three head­ ings; A. Statistical analysis: 1. Average daily food consumed. 2. Average daily gain in weight. 3. Amount of food consumed per pound of gain in live weight. B. Clinical picture C. Gross and microscopic pathology of the digestive tract, adrenal gland, kidney and liver. A. Statistical analysis To study the development figures, of the deficiencysyndrome, thesethree outlined above, were calculated for the control andfor the deficient pigs, from the data collected on the Oth, 14th, 18th, 20th, and 26th day after the beginning of the experiment. On these four days, all the available pigs on the experiment and the unconsumed feed were weighed back. The weight of the animals, on the deficient ration, on the five days are presented in Table I. The procedure adopted to calcu­ late weight gained and total weight gained, is illustrated by the following examples Weight of the eight animals on the Othday Weight of the animals on the 14th day Weight gained - 160 pounds = 200 pounds = 200-160 = 40 pounds Weight of the surviving six pigs on the14th day Weight of the six pigs on the 18th day Weight gained = 154 pounds = 151 pounds = 151-154 = -3 pounds Total weight gained from the Oth to the 18th day s 40 t (-3) = 37 pounds 24 Weight of the control pigs, weight gained and total weight gained, for the five days, are presented in Table II. Average daily food consumed, average daily gain in weight and the food consumed per pound of gain in live weight have been tabulated in Tables III and IV. Average daily food consumed by the pigs, on the deficient ration, was 1*52 pounds, whereas the control pigs on an average, consumed 1*99 pounds of food per day* Average daily gain in weight of the pantothenic acid deficient pigs was only 0*31 pounds, whereas the control pigs made an average daily gain of 0*68 pounds. The deficient pigs consumed 4*8 6 pounds of feed per pound of gain in weight; control pigs required 2*94 pounds of the ration for similar gain* The figures for the control pigs given in Tables II and IV were adjusted from the weekly records of the animals for ten weeks* Weights of pantothenic acid deficient and control pigs on the Oth, 14th, 18th, 20th, and 26th days, were compared by analysis of variance (Tables VI - XV), and were illustrated in Graph 1* From Table VII it was noted that the difference between average weights of the deficient and control pigs on the Oth day was not significant* There was, however, a significant difference between the average weights of the two groups on the 14th, 18th, and 20th days (Tables VIII - XIII). Table XV showed that average weight of 30*0 pounds of the two deficient pigs was not significantly different from the average of 39*1 pounds of the control pigs. This could be due to the fact that on the 26th day there were only two pantothenic acid deficient pigs to be compared to eight control animals • 25 TABLE I WEIGH? OF PANTOTHENIC ACID DEFICIENT PIGS Pig No. Oth day Weight in Pounds 14th day 18th day 2 0 th day 21.6 F IB 16 IB *7 F 22 30 Pigs Sacrificed 26th day On 14th day 14th day Vi 19*7 F 20 28 28 18th day 21.9 F 21 24 21 18th day 21.5 F 19 20 19.9 F 19 29 26 19.4 M 23 28 29 154 * 23 102 * 24 2 0 th day 30 s. 28 2 0 th day 31 26th day 29 2 6 th 56* 18 17.5 F 160 Total Average 20.0 Weight gained Total weight gained 25 24 X 28 / 151 110 60 25.0 25.1 27.5 30.0 200-160 = 40 151-154 r -3 40 -3 200 + day J 60-56 = 4 110-102 = * 8 8 f 4 = 49 pounds F 5 Female M = Male * Weight subtracted from the total weight of the animals in the succeeding column to determine the weight gained during each interval* 26 TABLE II WEIGHTS OF CONTROL PIGS Pig No. Oth day 14th day Weight in Pounds 18th day 2 0 th day- 26th day B21 M 27 36 38.3 39.4 42.9 B41 M 20 23 24.7 25.6 27.4 B39 F 20 26 27.1 27.7 29.4 C52 M 21 32 37.1 39.7 46.0 C57 F 24 35 37.3 38.4 40.4 C43 M 23 34 37.4 39.1 44.3 C47 M 19 27 30.4 32.1 38.0 B19 F 25 36 39.0 40.3 44.6 179 249 271.3 282.3 31.1 33.9 35.3 249-179 = 70.0 271.3-249 = 22.3 Total Average Weight gained Total Weight gained F - Female M - Male 22 70.0 4■ 22.3 313 39.1 282.3-271.3 313.0-2B2 = 11.0 = 30.7 4“ 11 •^ 30.7 = 134.0 pounds 4 27 TABLE III INFLUENCE OF THE DEFICIENT RATION ON GAIN IN WEIGHT AMD FEED REQUIREMENTS No. of Days Ration Fed No. of Pigs Fed No. of Days On The Basis Of One Pig Weight Average Gained Daily Weight Gained Feed Consumed Per Pound of Weight Gained Food Con­ sumed Average Daily Food Consumed 137 137 4 112 = 1.22 40 40 7 112 = 0.357 137 7 40 “ 3.425 14 8 18 6 l!2+(6 x 4) = 136 190 190 ? 136 = 1.39 37 37 r 156 = 0.272 190 f 37 = 5.135 20 4 136+(4 x 2) = 144 203 203 r 144 - 1.40 45 45 t 144 = 0.312 203 7 45 = 4.511 26 2 144+<2 x = 156 238 238 4 156 = 1.52 49 49 7 156 = 0.314 236 7 49 = 4.857 14 x 8 = 112 Notes 6 ) Weights expressed in pounds. 28 TABLE IV GAINS IN WEIGHT AND FEED REQUIREMENTS OF CONTROL PIGS No. of Days Ration Fed No.of Pigs Fed No. of Days On The Basis Of One Pig 14 8 14 x 8 = 112 18 8 20 26 Food Consumed 183 Average Weight Dai ly Gained Food Consumed Average Daily Weight Gained Food Consumed Per Pound Of Weight Gained 183 V H 2 = 1.63 72 72 t 112 I 0.64 183 72 r 2.54 18 x 8 = 144 183 4 69 252 t 144 = 252 = 1.70 95 95 r 144 = 0.66 252 ~ 95 = 2.65 8 20 x 8 = 160 183 *103 286 t 160 - 286 = 1.77 106 106 r 160 = 0.66 286 r 106 = 2.69 8 26 x 8 = 208 415 141 141 7 208 = 0.68 415 V 141 = 2.94 Notes 415 t 208 = 1.99 Weights expressed in pounds 29 TABLE V COMPARISON OF THE TWO LOTS OF PIGS Item Average initial weight Pigs on Deficient Diet Control Pigs 20 22 Average daily food consumption 1.52 1.99 Average daily gain in weight 0.31 0.68 Feed consumed per pound of weight gained 4.86 2.94 30 TABLE VI COMPARISON OF WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE Ottf1 DAY Total X X2 Y Y2 18 324 27 729 22 484 20 400 20 400 20 400 21 441 21 441 19 361 24 576 19 361 23 529 23 529 19 361 18 324 25 625 160 3224 179 4061 1Y or t2 £ Y2 or tx Average Weight* ( i.X 2 + £ X2 22.4 20 iY2 ) - ( t x <• i . Y )2 nl + n2 3224 + 4061 - (160 4 179)2 16 7285 - (339.)2 = 7285 - 7182 = 103 16 A t2 - t2 (± +±y W i C -T - ^ (25,600 -f 32,021 - 7182 = 7202.6 - 7182 = 20.6 B. B 5 X 2 Weight, in pounds, of pantothenic acid deficient pigs. Y S Weight, in pounds, of control pigs. C.T. or correction term = ( £X * i. Y)2 nl +’n2 ni = Number of the pigs on the deficient diet. n2 B Number of the control pigs. 31 TABLE VII ANALYSIS OF VARIANCE OF THE 'WEIGHTS OF PANTHOTHSNIC ACID DEFICIENT AND CONTROL PIGS ON THE 0^h M Y Source of Variation Total Between lots Within lots Degree of Freedom Variation or Sum of Squared Deviation 15 103 1 14 20.6 103 - 20.6 = 82.4 Mean Square 20.6 F. 20.6 - 3.4 5.9 82.4 - 5.9 14 P. at 1 and 14 degrees of freedom - 4*60 at 5 per cent level. F. at 1 and 14 degrees of freedom - 8.86 at 1 per cent level. Conclusions Difference between the two averages is not significant. 32 TABLE Till COMPARISON OF WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE 14th DAY Total X2 Y 16 256 36 1296 30 900 23 529 28 784 26 676 24 576 32 1024 20 400 35 1225 29 841 34 1156 28 784 27 729 25 625 36 1296 200 5166 249 7931 ^ X or t^ IX2 Average Weights 25 iX2 + £Y2 £ Y or tg 31 •1 - ( j X + ^ Y )2 nl t n2 5166 + 7931 - (200 •+ 249)2 8 + 8 13.097 - 201,601 16 13.097 - 12,600 = 497 / A 4-2 - ^ % C -T * n2 ' 40,000 + 62,001 8 102,001 8 y2 X - C.T. 8 201,601 = 12,750 - 12,600 = 150 ....B 16 £ ys table ; ix ANALYSIS OF VARIANCE OF THE WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE 14th DAY Source of Variation Total Between Lots Within Lots Degree of Freedom Variation or Sum of Squared Deviations 15 497 1 150 14 497 - 150 = 347 Mean Square 150 F. 150 = 6.05 24.8" 347 z 24.8 14 P« at 1 and 14 degrees of freedom = 4.60 at 5 per cent level. F. at 1 and 14 degrees of freedom - 8.86 at 1 per cent level. Conclusions Significant difference between the two averages. 34 TABLE X COMPARISON OF WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE 18th DAY Total X X2 Y 28 784 38.3 1466.89 21 441 24.7 610.09 23 529 27.1 734.41 26 676 37.1 1376.41 29 841 37.3 1391.29 24 576 37.4 1398.76 30.4 924.16 39.0 1521.00 151 £ X or Average weights 122 f 3847 £ X2 25.1 271.3 £ Y or t2 Y2 9423.01 £Y2 33.9 1Y2 - ( IX 4 jY)2 ni + ng 3647 + 9423 - (151 + 271.3)2 14 13.270 - 178,337.6 14 13.270 - 12,738.4- = 531.6 .....A . t? ~ t« v - C.T. ( *) 4 4 22,801 -f 73,603.9 6 8 - 12,738.4 (3800.1 -h 9200.5) - 12,738.4 = 13,000.6 - 12,738.4 = 262.2 .... B 35 TABLE XI ANALYSIS OF VARIANCE OF THE WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE 18th DAY Source of Variation Total Between Lots Within Lots Degree of Variation or ISum Mean Freedom of Squared * Square Deviations 13 531.6 1 262 #2 12 531.6 - 262.2 - 269.4 F. 262.2 269.4 - 22.4 12 262.2-11.2 22.4 F. at 1 and 12 degrees of freedom s 4*747 at 5 per cent level# F. at 1 and 12 degrees of freedom - 9#647 at 1 per cent level# Conclusions Highly significant difference between the two averages# 50 TABLE XII COMPARISON OF WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE 20th DAY Total X2 24 576 39.4 1552.36 30 900 25.6 655.36 28 784 27.7 767.29 28 784 39.7 1576.09 38.4 1474.56 39.1 1528.81 32.1 1030.41 40.3 1624.09 3044 IX2 110 £ X or Average Weights: £ X2 4 3044 4 1 Y2 - 27.5 282.3 or t2 35.3 ( I X j £Y)2 nl 4 n2 10,208.97 - (110 -+ 282.3)2 nl + a 2 13.252.97 - (392.3)2 12 13.252.97 - 12,826.3 = 426.7 ...... A. t2 - t2 -II 4, -L. nl ”2 Y CM X - C.T. (110)24 (282.3)2 - 12,826.3 4 8 12,100 4 79,603.5 - 12,826.3 4 8 12,975.4 - 12,826.3 = 149.1 ..... B. 10,208.97 £ y2 37 TABLE XIII ANALYSIS OF VARIANCE OF THE "WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE 20th DAY Source of Variation Total Between Lots Within Lots Degree of Freedom Variation or Sum of Squared Deviations 11 42 6.7 1 149.1 10 426.7-149.1 = 277.6 Mean Square F. 149.1 277.6 = 27.76 10 149.1- 5.37 27.76 F. at 1 and 10 degrees of freedom = 4.965 at 5 per cent level. F. at 1 and 10 degrees of freedom - 10.044 at 1 per cent level. Conclusions Significant difference between the two averages. 38 TABLE XIV COMPARISON OF WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE 2 6th DAY X X2 31 961 42.9 1840.41 29 841 27.4 750.76 29.4 864.36 46.0 2116.00 40.4 1632.16 44.3 1962.49 36.0 1444.00 44.6 1989.16 313.0 12,599.34 60 Total 1802 £ X or t-^ Average Weight: £x2 + £ Y or t2 30 39.1 £Y2 - ( 1 X 4 - £ Y )2 nl + ng 1802 -4 12,599.34 - (60 + 313.0)2 10 14.401.34 - 139,129 10 14.401.34 - 13*912»9 c 488.44 ...... A. ■ 2 ,2 tl ~ 2 nl ”2 3600 + 2 - C.T. 97,969 8 *7 • (1800 4" 12,246.1) - 13,912.9 14,046.1 - 13,912.9 = 133.2 .... Y B. Y2 39 TABLE XV ANALYSIS OF VARIANCE OF THE WEIGHTS OF PANTOTHENIC ACID DEFICIENT AND CONTROL PIGS ON THE 26th DAY Sourfce of: Variation Degree of Freedom Variation or Sum . '©f.Squared Mean Deviations Square Total 9 488.44 Between Lots X 133.20 Within Lots 8 488.44-133.20 - 355.24 133.2 355.24= 44.4 8 F. 133.2 Z 3*° F. at 1 and 8 degrees of freedom - 5*317 at 5 per cent level* F. at 1 and 8 degrees of freedom z 11*259 at 1 per cent level* Conclusions Difference between the two averages is not significant. 40 1 CO O rH CO *iH c3 xPH : o o O o O o CM rH CM rH CM rH CM to rH CM i— 1 i— I © rH to © © © rH 03 CO CO CO CJ © O 'cH © CO O sH CM CO © © iH t- •M* © © i— 1 © O cI>- o o rH O © i— 1 O O o o CM PQ © to I Oj r©H p H *H "S © w si O O PH of CM Pi 09 (D Ph © CO © ■ e. o CO c i —iji Ph % M O M pH g Q M O » © CO © © © © © © O © CM CO © Q © CM © O © © O © O CM © CM © CM O © C•k oCM E~ © © © 03 ia 1 Pi © - co O M |2" Eh O fe < P Q pH o u • © pq 0 "ti o • o CL, g H PQ O o LO 03 •k CO CM O o CM * o © CM © Oo •I ■sH CM O o O 2 © o- © CM CM © © © CO o o © Cv © © co •» «k •> •i •V •t © © © •k © CM CM o F) O P-i PQ o 0 *iH Pi o Pj rH © • O PQ *iH (— ) pH i— 1 rrH © • • t— • LO © © LO o O 03 • CM rH CO CO * to CM • © • © o © © ■ • © © • © © • © © • © © 03 • to c• CM rH © © • t- PQ O o o *-q pq .3 rO ■«*! © • O PH rH rH 0 rtf hD (O O o o £f erfo © i—I © K Pi rH PQ • o rH • rH rH 3 rH to • CM i— 1 * rH rH © • CM rH CM • 03 © © • o rH 1— i © CO • © t—i bD bD • *j-! 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Js; P o •h ■§ fc © CO XI O 05 C O fcJ * t - * C M * I > - PQ O 60 • C- • CM • £H • « ^ ^ ^ ^ L O ^ I O ( xh 60 p O O O O O O O • 05 D- • CM • OrH S IP s tD (564 O O 0 * PQ P ctf P CD & & © TJ Xl -P O X3 P O CM gS u © <3 T3 P P 05 <*« rH © feO © 6lD gJ CtS P P © © }> Graph 1 Average Weights of Pigs on Pantothenic Acid Deficient Ration and of Control Pigs 42 W E IG H T in POUNDS K> Cm n o> DAYS on EXPERIMENT m TH Graph 2 Hemoglobin Concentration of Pigs on Deficient Ration and Erythrocyte Counts of Pigs on Deficient Ration 43 w rco 3 o o Z ^ 12. "o' hZ LU O a: CU CL iO cr? 5: < Of! o & X 16 1 14 22 DAYS on EXPERIMENT 26 8r z o -J O O JZ O _» c Hi H > VJ O m H > cr u; y 7 C0 .. o £ E 6 •j OtZ ij.j Cl C 14 DAYS X X 18 22 on EXPER IM EN T 26 Graph 3 Leukocyte Counts of Pigs on Deficient Ration 44 LEUKOCYTES COUNTS « THOUSANDS 54 D A Y S on E X P E R I M E N T 45 B. Clinical picture Some of the pigs showed loose feces ten to twelve days after being placed on the deficient ration, and by the 14th day, all the pigs showed diarrhea* anorexia. The animals were dull and listless and developed The fecal material was a homogeneous dirty yellowish fluid and soiled the hind quarters of the animals. By the 18th day, small flakes of blood could be detected in the fecal material. The hair was not as shiny and smooth as that of normal pigs. Dur­ ing the period of observation there was no indication of alopecia nor of ”goose stepping”. Some of the pigs showed a slight discharge from the eyes. At first this was clear and serous but later it became viscous and produced matting of the eyes. There were no visible symptoms suggestive of inflammation of the respiratory passages. Blood pictures Reference to Table XVT showed that the concentra­ tion of hemoglobin dropped, on the 20th day, to 10.55 grams per 100 milliliters of blood and rose again towards the end of the experiment. It would, however, be noted that the hemoglobin concentration values of the deficient pigs did not show marked difference from the corres­ ponding average value of the control pigs. The erythrocyte counts corresponded with variation in the concen­ trations of hemoglobin. <2 per mm On the 20th day, the count was 5.65 million but went up as did the hemoglobin concentration towards the end of the period of observation (see Graph 2). 46 Examination of blood smears stained with Wright* s showed the presence of a few normocytes in some instances indicating normocytic anemia* Anisocytosis was observed in blood smears from two animals* Leukocytosis was observed throughout the course of the investiga­ tion. This was, however, much more marked on the iBth day, when the total leukocyte count was 32770 per mm^ of blood (see Graph 3). The differential white blood cell count given in Table XVI ex­ hibited a close correspondence between neutrophil percentages and the total leukocyte counts. On the 18th day, udien the number of white ■Z blood cells was 32 770 per mm , the neutrophil percentage was 54 — the highest in the series. There were 38 per cent lymphocytes on the 14th day, and the figure rose subsequently until it was 56 per cent at the end. C. Post mortem findings Gross changes: The carcases were under-developed and emaciated. In most of the pigs the tail and hind quarters were soiled with dirty yellow diarrheal feces. contained some mucus. This fecal material from the 14th day onwards By the 26th day, after the commencement of the experiment, the feces became a little firmer in consistency. In the peritoneal cavities of the first pigs examined, 40 to 60 milliliters of clear transudate was found. of peritonitis. There were no indications The pigs sacrificed on the 20th and 26th days, however, did not show appreciable quantities of this transudate. The buccal cavity and the esophagus failed to show changes during the period of investigation. 47 The stomach appeared normal. The small intestine did not reveal as marked macroscopic changes as the large intestine. In the later stages, acute inflammatory changes were seen affecting the posterior parts of the ileum. changes extended anteriarly for some distance in the ileum. These Occasional­ ly, however, inflammatory foci were detected in the duodenum and the jejunum. These changes were less severe than those observed towards the ileocecal valve and in the large intestine* In the earlier stages of the experiment, the large intestine showed diffuse congestion, which, in some cases, was more intense on the crests of the folds of the mucous membrane. There was an excessive discharge of mucus and the intestinal wall was thick and edematous. As the disease progressed, the congestion in the intestine became much more intensive and in the rectum produced "zebra markings". There were ecchymotic hemorrhages particularly in the neighborhood of the ileoceal valve (Figure 1). These lesions were followed by the development of small rounded discrete ulcers approximately 0.5 centimeters in diameter with a slightly depressed center. Sometimes fecal material was observed adhering to the inflamed mucosa but this material could easily be washed off by a gentle stream of water. There was hypertrophy of the lymph nodules in the wall of the intestine which could be easily palpated. One of the pigs sacrificed on the 26th day, showed both healed and unhealed ulcers. The congestion and hemorrhage in the intestinal mucosa had decreased in intensity and the enlarged lymph nodules could still be palpated in the intestinal wall. 48 Throughout the course of the investigation, peritonitis and in­ testinal parasitism were not seen. The adrenal glands and the kidneys did not present macroscopic lesions. In a few instances there was, however, some hypertrophy of the adrenal glands. Towards the end of the experiment, the cortex constituted a greater proportion of the gland when compared to the thickness of the cortical layer in the earlier stages. Microscopic changes Pig 1. Sacrificed on the 14th day. Large intestines The epithelium of the mucous membrane showed various stages of degeneration and in some areas, coagulation-necrosis (Figure 2). There were a few areas where the necrosed epithelium was desquamated, exposing the subepithelial tissue to the lumen of the intestine. The crypts of Lieberkuhn showed accumulation of mucus, which flowed out of the glandular openings. The epithial cells lining the crypts showed retrogressive changes and were distended with mucus (Figure 3). Hyperemia of the lamina propria was markedly prominent. There were isolated areas where the capillaries, lying immediately below the surface epithelium, had been markedly distended with blood. At such places, the blood was separated from the lumen of the intestine only by a single layer of columnar epithelium with its basement membrane and the capillary endothelium. At some places, denudation of the columnar epithelium had reduced the partition between the blood and the intestinal lumen to only a layer of capillary endothelium and the 49 basement membrane (Figure 4)* Infiltration of serous fluid into the lamina propria was not marked. Cellular infiltration in this layer was uniform, and lymphocytes, macrophages and plasma cells were the chief infiltrating cells. tion by the leukocytes. There was a marked periglandular infiltra­ The latter had not yet penetrated the lumina of the crypts, but some were incarcerated in the walls of the crypts (Figure 4). Changes in the submucosa were not as marked as those of the mucous membrane. There were, however, focal infiltration of lymphocytes and macrophages. Small intestine; In the ileum there was marked congestion in the lamina propria and some of the capillaries were dilated to accommodate six to ten red blood cells. Cellular infiltration in the lamina propria was not so heavy as in the large intestine. The submucous layer showed interstitial edema, but no cellular infiltration. On the surface of the epithelium, there was an accumulation of mucous exudate containing a few leukocytes. Adrenal glands There was congestion of the glomerular zone. intercellular space in the zona fasciculaiis was increased. The In the outer part of the medulla, parenchymatous cells showed degenerative changes, and in some areas hemorrhagic foci were detected (Figure 20). Pig 2. Sacrificed on 14th day. Large intestine* The histopathological picture was about the same as that of the intestine in pig 1. Caught in the mucus, on the surface 50 of the mucous membrane, were a few Balantidium coli (Figure 6). There was desquamation of the mucosal epithelium and congestion in the lamina propria* Goblet cells were conspicuous, and light bluish stained mucus was streaming out of the crypts of Lieberkuhn. Lymphocytes, macrophages, and plasma cells were the principal infiltrating cells of the lamina propria* Some of these cells showed degenerative changes characterized by pyknosis and karyorrhexis of the nuclei (Figure 5)* The submucosa showed capillaxy congestion and edema. One of the crypts, in its submucosal portion, showed accumulation of mucus and de­ generating neutrophiles* Immediately surrounding this crypt, the cellu­ lar reaction was characterized by lymphocytes and a few macrophages. Small intestine: The ileum and the jejunum showed mucoid exudate containing a few leukocjrfces. The number of goblet cells was increased* The lamina propria showed congestion and a relatively mild cellular infiltration. The submucosa showed edema and hyperplasia of lymph nodules, some of which were congested. Adrenal gland: There were a few foci of congestion in the zona glomerulosa but in the remainder of the cortex and the medulla, no marked change could be detected. Pig 3. Sacrificed on the 18th day. Large intestine: quamated. The epithelium of the mucous membrane was des­ Covering the denuded membrane, was an exudate which consisted of degenerated and necrotic tissue, desquamated epithelial cells and mucus. 51 A majority of these cells in the exudate had lost their outline and stained faintly# In the exudate, degenerated polymorphonuclear leuko­ cytes and a few Balantidium coli could also be detected (Figure 7)# The lamina propria was markedly hyperemie and in some ar’eas this re­ sulted in hemorrhage# There was also a considerable amount of edema# Cellular infiltration was prominent with lymphocytes, macrophages and plasma cells# Polymorphonuclear leukocytes were not very common. was an increase in the number of goblet cells# There As a result of this, there was an accumulation of mucus in the intestinal glands and the mzcus was streaming out from the glandular pores. Some of the glands showed hyperplasia which was apparent even below the muscularis micosae (Figure 9)# The glands were filled with mucus and degenerated poly­ morphonuclear leukocytes# glands, producing cysts# This plugged the opening of some of the The epithelial cells lining these cysts showed various stages of degeneration and due to pressure atrophy, had assumed a relatively flat morphology and were surrounded by a zone of lympho­ cytes (Figure 10). The submucous layer was edematous and showed a cellular infiltra­ tion as in the lamina propria, though not as heavy# The lymph nodules were congested and infiltrated with plasma cells. Small intestines The ileum showed marked catarrhal inflammation characterized by the accumulation of pale blue staining mucus on the epithelial surface. observed# Desquamation of the surface epithelium was not Congestion of the lamina propria, however, was marked# was edema of the submucosa, and hyperplasia of the lymph nodules. There 52 Adrenal gland: The cells of the zona fascicularis were atrophied and this produced relatively wide intercellular spaces. of this zone appeared to be undergoing degeneration. The cells These degenerated cells tended to separate from the adjoining tissue and were agglomerat­ ing in isolated masses (Figure 21). The resulting spaces had no endo­ thelial lining and therefore excluded the possibility of being sinusoids. The medulla did not show hemorrhage. Pig 4. Sacrificed on 18th day. Large intestine; The histopathological picture was approximately the same as in the large intestine of pig 3. At several places there was desquamation of the columnar epithelium. The exudate, on the sur­ face of the membrane, consisted of degenerating epithelial cells, mucus and serous fluid (Figure 8). The latter was indicated by albumin granules. The lamina propria showed marked congestion and in some cases hemorrhages were present. Cellular infiltration was mainly lymphocytic; there were however, a few macrophages, neutrophils, eosinophils, and plasma cells. The cypts of Lieberkuhn showed the same changes as in­ dicated in pig 3. The submucous layer showed edema and hyperplasia of lymph nodules. Small intestine; The sections from the jejunum and the ileum showed histopathological changes similar in nature to those described for the corresponding organs of pig 3. 53 Pig 5# Sacrificed on 20th day. Large intestines The mucous membrane showed hemorrhage much more markedly than in the corresponding lesions described for the other pigsThere was desquamation of the epithelium and at two places the desquama­ tion had extended to about half the depth of the lamina propria, pro­ ducing superficial ulcers (Figure 12). The exudate on the surface of the membrane was muco-serous in nature without evidence of fibrin forma­ tion and contained desquamated epithelial cells and tissue debris from the lamina propria. The epithelial cells lining the crypts of LieberMihn were swollen with mucus which was streaming into the lumina of the crypts, some of 'which had developed into cysts. The suscularis mucosae did not show marked histopathological changes except for a slight lymphocytic infiltration. Parts of the cystic dilatation of the crypts of Lieberkuhn were prominent below the muscularis mucosae. The epithilial cells lining the cysts were pressed down by the accumulating exudate and showed retrogressive changes (Figure 10). The cysts were surrounded by marked lymphocytic infiltrations. A few macrophages containing engulfed leukocyte and tissue debris, were also observed among the infiltrating lymphocytes (Figure 11). Small intestine: The lamina propria showed marked congestion, edema, and a few hemorrhagic foci. The submucosa displayed edema and hyperplasia of the lymph nodules. These nodules contained macrophages which were observed in various stages of phagocytic activity. 54 Adrenal gland; The fascicular zone contained a focus of lympho­ cytic infiltration* In some of the cells of the reticular zone, the nuclei stained faintly and only an outline of the nucleus could be de­ tected* Some of the medullary sinusoids showed degeneration of the endothelium and the adjoining parenchymatous cells had separated from the healthier tissue to form masses* This process, on one side of the gland, had extended as far as the outer third of the fascicular zone (Figure 21)* Pig 6* Sacrificed on 20th day* Large intestine: Mucus exudate containing tissue debris, was found on part of the epithelial surface* The epithelium, although showing coagulation necrosis, was intact. The lamina propria was uniformly infiltrated with lymphocytes and a few neutrophils* The congestion was not as severe as reported in the previously necropsied animals. The crypts of Lieberkuhn were filled with mucus and some were dilated but none penetrated the muscularis mucosae* The cellular reaction surrounding these dilated crypts was not as marked as in the corresponding lesions described before. ulcerative areas were detected. Two They were relatively narrow but ex­ tended to the muscularis mucosae* The submucosa showed infiltrating serous fluid* The muscularis externa did not show alterations. Small intestine: The ileum showed histopathologic changes similar to those described for pig 5. There was catarrhal exudate on the 55 mucous membrane. The lamina propria showed congestion and hemorrhage. The submucosa showed edema and enlarged lymph nodules. The latter con­ tained macrophages some of which had more than one nucleus but the number of nuclei did not exceed three. Adrenal glands There were areas of degeneration and prominent interstitial spaces in the cortical layer. Pig 7. Sacrificed on the 2 6th day. Large intestine; The retrogressive histopathological changes were the same but were less intensive than in the pigs previously necropsied. Edema in the lamina propria was not prominent. The mucous membrane showed a few crypts of Lieberkuhn which were relatively slightly ballooned. At one place, however, there was a marked hyperplasia of an intestinal gland. The gland had ballooned and was markedly prominent in the sub- mucosa. The hyperplasia progressed to the extent that secondary and tertiary cystic dilatations were produced from the sides of the primary cyst. The cyst contained only a small amount of mucus and a few degen­ erating cells. Faintly blue stained mucus was streaming from the goblefc cells in the lining epithelium. A few shallow ulcers were detected on the mucous membrane. Small intestines Catarrhal exudate on the mucous membrane, a few- hemorrhagic spots in the lamina propria and the presence of macrophages in the hyperplastic lymph nodules were the principal changes in the ileum and the jejunum. 56 Adrenal gland: The capillaries of the cortex, particularly of the fascicular and reticular zones were dilated and the parenchymatous cells appeared atrophied* The endothelial lining of some of the sinusoids of the medulla as well as the adjoining parenchymatous cells had under­ gone degeneration and agglomerated in irregular masses in the remnants of the sinusoids* This degenerative process was usually along the longi­ tudinal axes of the sinusoids involved, and invaded the cortex sometimes as far as thefhscicular zone* Pig 8. Sacrificed on 26th day* In this animal both repair and degeneration were present* For some areas there was complete destruction of the mucosa and marked hyper­ plasia of intestinal glands* The ballooned cysts were very prominent below the submucosa (Figure 13). Along with this retrogressive process, were detected foci of re­ pair where young fibroblasts replaced the necrotic tissue* blasts were seen in various stages of proliferation* The fibro­ Some were oval with vesicular nuclei whereas others were more or less spindle shaped with deeply staining elongated nuclei (Figure 14)* were seen in these areas* capillary endothelium* Very few crypts There were endothelial buds forming from the Columnar epithelium was seen bridging over the area of fibroblastic activity* Small intestine: Congestion and hemorrhages of the lamina propria and cellular infiltration were not as prominent as previously described* 57 Adrenal glands the fascicular zone* There was a focus of lymphocytic infiltration in The endothelial lining of the sinusoids was normal* Kidneys There was congestion of the medulla in the early stages# One of the pigs sacrificed on the 14th day showed a few hemorrhages in the medullary zone# The transition of hyperemia into areas of dif­ fuse hemorrhage, was detected in one animal sacrificed the 20th day and in both destroyed the 26th day (Figures 15 and 16). In some animals a few erythrocytes were detected in the urinary tubules while in others there was intratubular albuminous material# One of the pigs sacrificed on the 18th day and one from the 26th day showed marked shrinking of the glomerular tufts. Some were lying free in the cavities lined by the parietal layer of Bowmans capsule# stitial edema (Figures 17 and 18)# This was accompanied by inter­ In a few pigs there was focal periglomerular leukocytic infiltration. Livers Congestion, parenchymatous degenerative changes and occasional interstitial edema were the principal histopathological changes. 58 DISCUSSION Until recently, enteritis in weanling pigs was believed to be in­ fections in nature. The belief by the end of the third decade of this century was so firm that the infectious nature of swine enteritis was taken for granted. Kinsley (1934), however, stated that enteritis in swine could occur under proper sanitary conditions. McEJwen (1937) suggested the possibility of a clinical syndrome due to faulty nutri­ tion, similar to that of ’’infectious enteritis”. Birch fffc al (1937), Hughes (1939), Wintrobe and Mitchell (1938), and Ellis and Madsen (1941) suggested the importance of the vitamin B complex in the nutrition of the pig. After the synthesis of pantothenic acid by Williams (1939), the nutritional importance of this vitamin was shown by the use of syn­ thetic diets from which pantothenic acid was withheld. Cunha (1951) reported that the response to a synthetic ration may not be the same as to natural feedstuff s. McMillen et al (1949a) demonstrated an increase in the rate of weight gained and absence of deficiency symptoms by sup­ plementing a basal ration of com, oats, expeller soybean meal, meat scraps, alfalfa meal and complex mineral mixture with niacin, riboflavin and pantothenic acid. In this investigation, it was shown that a commonly used diet, con­ sisting of com, soybean oil meal and mineral mixture supplemented with niacin and riboflavin, produced deficient symptoms which did not develop in the control animals fed the same diet supplemented with pantothenic acid. 59 The pigs on the pantothenic acid deficient ration, showed a de­ crease in appetite, significant decrease in the rate of weight gained and inefficient utilization of feed* There was no marked difference between, hemoglobin concentration values of the deficient pigs and those of the control pigs* Erythrocyte count was decreased and the lymphocyte- neutrophil ratio was reversed. Diarrhea developed after about two weeks on this diet* At necropsy, examination of the large intestine showed marked congestion, superficial necrosis and shallow ulcers which involved about half the thickness of the lamina propria* The fecal material ad­ hering to the intestinal wall was localized in small discrete areas and could be easily washed off by a gentle stream of water* This was in marked contrast to the observations of Dunne et al (1949) in an in­ vestigation of niacin deficiency in pigs. These authors reported that the size of adhering fecal masses varied from a few millimeters in dia­ meter to complete coverage of the intestinal wall, for as much as three feet* Pantothenic acid deficient pigs as compared to niacin deficient animals, did not show as nuch adhering of the fecal material. Appar­ ently this was due to less extensive necrosis of the intestinal mucous membrane. Marked hyperemia of the lamina propria was the chief feature in the early stages of pantothenic acid deficiency* In the niacin deficiency, reported by Dunne eb al (1949), hyperemia was described as ’’mild to moderate” depending on the stage or type of lesion. The exudate on the intestinal mucous membrane in pantothenic acid deficient pigs was mostly 60 mucous in nature* The amount of fibrin in the exudate tms at its minimum, 'which might have been one of the factors contributing to the absence of large fecal masses* The cellular infiltration in the in­ testines of the deficient pigs was mainly lymphocytic in nature* Dunne et al (1949) observed neutrophilic infiltration in those areas which showed bacterial invasion of the tissue* Wintrobe et al (1943) observed polymorphonuclear as well as mononuclear leukocytic infiltra­ tion in the periglandular interstitial tissue* In the present study, polymorphonuclear cells infiltrating into the tissues were few in number* In the exudate and in the ciypts of Lieberkuhn, however, these cells were occasionally seen* Balantidium coli were detected in the exudate and in the superficial parts of the lamina propria in the early stages of the disease. Dunne et al (1949) also reported the presence of this in niacin deficient pigs. Morgan and Hawkins (1948) and Hagen (1948) reported that Balantidium coli was commonly present and was apparently of little importance* The number of Balantidium coli in the intestine of the pigs studied, was troo small to produce the intestinal changes observed. It was felt that the incidental presence of Balantidium coli in no way minimized the in­ fluence of pantothenic acid in the production of the lesions. Under the conditions reported, the adrenal gland showed hemorrhages in the medulla and degeneration of the endothelial cells of the medullary sinusoids and the adjoining parenchymatous cells. There was atrophy of the cells in the zona fascicularis and zona reticularis accompanied by increased intercellular spaces. Wintrobe et al (1943), however, did 61 not observe microscopic changes in the adrenal gland in the study of pantothenic acid deficiency in swine. The relatively higher qualita­ tive and quantitative level of protein in the ration of the pigs, studied by Wintrobe et al (1943), might be one of the factors respons­ ible for the absence of microscopic changes in the gland of the deficient pigs. The mechanism of pantothenic acid deficiency was difficult to ex­ plain. Apparently there was stimulation of the goblet cells to secrete excessive mucus, which probably plugged the crypts of Lieberkuhn and produced pressure atrophy and degeneration of the lining epithelium. This was preceded or was accompanied by marked hyperemia. Degenerated epithelium and the subsequent possibility of disturbed absorption of nutrients including the B vitamins, were some of the complicating factors. Therefore it was difficult to attribute the syndrome exclusively to pantothenic acid deficiency. Williams et^ al^ (1950) pointed to the possible role of pantothenic acid as part of coenzyme A, in acetylation processes* These processes were believed to include the acetylation of choline and aromatic amines and the condensation of oxalacetate to form cis-aconitate. This would disturb the detoxification mechanism of the organism and could lead to abnormal physiology of the crypts of Lieberkuhn, adrenal gland, kidney and liver. The role of pantothenic acid in carbohydrate metabolism could be a contributary factor. ■When the intake of B vitamins falls below the nutritional require­ ment for a given animal, there is a gradual decrease in the B vitamin 62 content of the organism as a "whole* In view of the important role played by pantothenic acid in the normal physiology of the body, de­ ficiency symptoms and alterations could be produced by disturbed cellu­ lar physiology* a colitis* The disturbed cellular physiology manifested itself as This might be explained on the basis that organs and tissues may vary in their ability to fend for themselves under such adverse conditions (Williams et al (1950))* The results of this investigation showed that a ration consisting of corn, soybean oil meal and the mineral mixture with niacin, riboflavin, vitamin Merck1s A* P* F*, vitamin A and vitamin D as supplements, produced deficiency symptoms. There was a marked decrease in the daily weight gained, inefficient utilization of feed consumed, and colitis accompanied by diarrhea* Inclusion of pantothenic acid in the ration of the control animals pre­ vented the development of the deficiency* 63 SUMMARY AND CONCLUSIONS Eight Duroc weanling pigs were fed a ration of natural feedstuffs, low in pantothenic acid* The pigs developed anorexia and voided diarrheal feces two weeks after placing the animal on the deficient diet* There was a signifi­ cant 4ecr<3ase in the rate of weight gained and in the utilization of the feed. The large intestine showed congestion and a few ecchymotic hemorr­ hages and in the later stages small superficial discrete ulcers* The fecal material adhering to the mucous surface could be easily washed away by a gentle stream of water* The columnar epithelium showed degenerative changes and there was marked hyperemia of the lamina propria. The exudate contained mucus, cellular debris and infiltrating leukocytes. The cellular reaction in the lamina propria, and the submucosa was mainly lymphocytic* The crypts of Lieberkuhn showed cystic dilatations and there was hyperplasia of the lymph nodules. One of the pigs, sacrificed on the 26th day, showed fibroblasts proliferating in the lamina propria. Control pigs fed on the same ration supplemented with pantothenic acid did not show the deficiency symptoms. 64 LITERATURE CITED Birch, T* W., H. Chick, and C. J* Martin* Experiments with pigs on a pellagra-producing diet* Jour. 31: 2065-2079, 1937. Biochem. Chick, H., T* F. Macrae, A. T. P. Martin and C. J. Martin. The water soluble B vitamins other than aneurin (vitamin B^), riboflavin and nicotinic acid required by the pig* Biochem* Jour. 32s 2207-2224, 1938. Colby, R. W., T. J. Cunha, C. E. Lindley, D. R* Cordy and M. E. Ensminger. Biotin-pantothenic acid interrelationship and enteritis in the pantothenic acid deficient pig* Jour. Amer. Vet. Med* Assoc. 113s 589-593, 1948. Cunha, T • J . Vitamin requirements of swine. 26 (3): 10 passim, 1951. American Hampshire Herdsman, Daft, F. S., W. H. Sebrell, S. G. Babkock Jr. and T. H. Jukes. Effect of synthetic pantothenic acid on adrenal hemorrhage, atrophy and necrosis in rats* U. S. Public Health Rep. 55s 1333-1337, 1940. Deane, H. W* and J. M* McKibbin. The chemical cytology of the rat’s adrenal cortex in pantothenic acid deficiency. Ebadocrinology 38: 385-4-00, 1946. Doyle, L. P. Swine dysentery. 1943. Jour. Amer. Vet. Med. Assoc. 102: 449-450, Dunne, Howard W*, R. W. Luecke, W. IT. McMillen, M. L. Gray, and F. Thorp,Jr. The pathology of niacin deficiency in swine. Amer. Jour. Vet. Res. 10s 351-356, 1949. Edgar, C. E* and Macrae, T. F. Water soluble B vitamins. Essential dietary factors for the rat present in autoclaved yeast extracts in addition to lactoflavin. Biochem. Jour. 31s 886-892, 1937. Edgington, B. H., W. L. Robinson, W. Buroughs and R. M. Bethke. Tests with nicotinic acid for the prevention of infectious swine enteritis. Jour. Amer. Vet. Med. Assoc. 101: 103-108, 1942. 65 Ellis, N. R. and L. L. Madsen* Relation of diet of swine to development of locomotor incoordina­ tion resulting from nerve degeneration. Jour. Agric. Res. 62s 303-316, 1941. Emerson, G. A. and E. Wurtz. Biotin-pantothenic acid interrelationship in rats fed succinylsulphathiozole. Proc. Soc. Expt. Biol, and Med. 57: 47-49, 1944. Follis, Richard H. and M* M. Wintrobe. A comparison of the effects of pyridoxine and pantothenic acid deficiencies on the nervous tissue of swine. Jour. Expt. Med. 81: 539-551, 1945. Fouts, P. J., 0. M. Helmer and S. Lepkovsky. Factor II deficiency in dogs. Jour. Nutr. 19: 393-400, 1940. Hagan, William Arthur. The Infectious Diseases of Domestic Animals with special reference to Etiology, Diagnosis and Biologic Therapy* Comstock Publishing Company, Inc., Ithaca, New York, 483, 1948. Henderson, L. M., J. M. Mdntire, H. A. Waisman and C. A. Elvehjem. Pantothenic acid in the nutrition of the rat. Jour. Nutr. 23: 47-58, 1942. Hughes, E. H. The role of riboflavin and other factors of the vitamin B complex in the nutrition of the pig. Jour. Nutr. 17; 527-533, 1939. Hughes, E. H* Pantothenic acid in the nutrition of the pig. 64: 185, 1942. Jour. Agric. Res. Hughes, S. H. and N. R. Ittner. The minimum requirement of pantothenic acid for the growing pig. Jour. Animal Sci. 1: 116-119, 1942. Jukes, T. H. and S. H. Babcock Jr. Experiments with a factor promoting growth and preventing paralysis in chicks on a simplified diet. Jour. Biol. Chem. 125: 169-181, 1938. Kinsley, A. T. Infectious necrotic enteritis. Vet. Med. 29: Leake, C. D. and E. F. Guy. A diluting fluid for platlet counting. 84: 890-891, 1925. 27, 1934. Jour. Amer. Med. Assoc. 66 Lippincott, S. W. and H. P. Morris. Morphologic changes associated with pantothenic acid deficiency in the mouse. Jour. National Cancer Inst. 2s 39-46, 1941-1942. Luecke, P. W., F. Thorp Jr., W. N. McMillen, H. W. Dunne and H. J. Stafseth. A study of B. vitamin deficiencies in pigs raised on farms. Michigan Agric. Expt. Station, East Lansing, Technical Bull. 211, 1949. Luecke, R. W., F. Thorp Jr., W. N. McMillen, and H. W. Dunne. Pantothenic acid deficiency in pigs fed diets of natural feed­ stuff s. Jour. Animal Sci. 6s 464-469, 1949. Luecke, R. W., W. IT. McMillen and F. Thorp Jr. Further studies of pantothenic acid deficiency in weanling pigs. Jour. Animal Sci. 9s 78-82, 1950. Mallory, F. B. Pathological technique. London, 72, 1938. W. B. Saunders Company, Philadelphia and McBwen, A. D. Necrotic enteritis of young pigs not associated with Salmonella infection. Vet. Rec* 49: 1507-1509, 1937. McMillen, W. N., R. Wi Luecke and F. Thorp Jr. B. vitamins for weanling pigs. Michigan Agric. Expt. Station, Quarterly Bull. 32s 191-195, 1949. McMillen, W. N., R. W* Luecke and F. Thorp Jr. The effect of liberal B vitamin supplementation on growth of wean­ ling pigs fed rations containing a variety of feedstuff s. Jour. Animal Sci. 8: 518-523, 1949a. Mills, R. C., J. Ii. Shaw, C. A. Elvehjem and P. H. Phillips. Curative effect of pantothenic acid on adrenal necrosis. Proc. Soc. Expt. Biol, and Med. 45s 482-486, 1940. Morgan, Banner Bill and Phillip,A. Hawkins. Veterinary Protozoology. Burgess Publishing Company, 426 South Sixth Street, Minneapolis 15, Minnesota. 74, 1948. Morgan, A. F., B. C. Cook and H. G. Davison. Vitamin B? deficiencies as affected by dietary carbohydrate. Jour. Nutr. 15: 27-43, 1938. Morrison, F. B. Feeds and Feeding, ed. 20* The Morrison Publishing Company, Ithaca, New York, 807, 1940. 67 Phillips, P. H. and R. W. Engel* Some histopathological observations on chicks deficient in the chick antidermatitis factor or pantothenic acid. Jour. Putr. 18: 227-232, 1939. Ringrose, A. T* and N. C. Porris. Differentiation between vitamin G and an insoluble factor pre­ venting a pellagra-like syndrome in chicks. Jour. Putr. 12: 535, 1936. Salmon, E. D. and R. W. Engel. Pantothenic acid and hemorrhagic adrenal in rats. Expt. Biol, and Med. 45: 621-623, 1940. Proc. Soc. / Schaffer, A. E., J. M. McKibbin and C. A. Elvehjem. Pantothenic acid deficiency studies in dogs. Jour. Biol. Chem. 143: 321-330, 1942. Scudi, John V. and Margaret Hamlin. The effect of pantothenic acid deficiency on the blood lipoids of the dog. Jour. Putr. 24: 273-282, 1942. Shaw, James H. and P. H. Phillips. Peuropathologic studies of pantothenic acid, biotin and folic acid complex deficiencies in the chick. Jour. Putr. 29: 107-112, 1945. Silber, R. H. Studies of pantothenic acid deficiencies in dogs. 2 7: 425-433, 1944. Jour. Putr. Sullivan M. and J. Picholis. Nutritional dermatoses in the rat. The effect of pantothenic acid deficiency. Arch. Derm, and Syph. 45: 917, 1942. (Reference cited by Wintrobe, M. M., R. H. Follis Jr., Raul Alcayaga, Moses Paulson and Stewart Humphreys. Pantothenic acid deficiency in swine with particular refer­ ence to the effects on growth and on the alimentary tract. Johns Hopkins Hosp. Bull. 73: 313-341, 1943.) TTnna, K • Pantothenic acid requirements of the rat. 1940. Jour. Putr. 20: 565, Unna, K., G. V. Richards and W. L. Sampson. Studies on nutritional achromotrichia in rats. 553-563, 1941. Jour. Putr. 22: West, P. D., M. J. Bent, R. S. Rivers and R. E. Tisdale. The influence of pantothenic acid upon the susceptibility to pneu­ monia (with a note on the mechanism of the action of sulphapyridine in pneumococcic pneumonia). Arch. Biochem. 3; 321-324, 1944. 68 Williams, R. J • Pantothenic acid — a vitamin. Science 89: 486, 1939. Williams, R. J. The chemistry and biochemistry of pantothenic acid. Advances Eneymol. 3: 253-287, 1943. Abstracted in Biol. Abst. 18s 2 60 (2356), 1944. Williams, R. J., R. E. Ealcin, E. Beerstecher. Jr. and W. Shive. The Biochemistry of B. vitamins. Reinhold Publishing Corpora­ tion. 330 West Forty-Second St., New York 18, 395, 424, 1950. Williams, R. J., J. H. Truesdale, H. H. Weinstock Jr., E. Rohmann, C. M. Layman and C. H. McBurney. Pantothenic acid. Its concentration and purification from liver. Jour. Amer. Chem. Soc. 60: 2719-2723, 1938. Wintrobe, M. M* Nutritive requirements of young pigs. 126: 375-387, 1939. Amer. Jour. Physiol. Wintrobe, M. M., R. H. Follis Jr., Raul Alcayaga, Moses Paulson and Stewart Humphreys. Pantothenic acid deficiency in swine, with particular reference to the effects on growth and on the alimentary tract. Johns Hopkins Hosp. Bull. 73: 313-341, 1943. Wintrobe, M. M., R. H. Follis, Jr., Stewart Humphreys, Harold J. Stein and Marjorie Lauritsen. Absence of nerve degeneration in chronic thiamine deficiency in pigs. Jour. Nutr* 28: 283-288, 1944. Wintrobe, M. M. and D. M. Mitchell. Sensory neuron degeneration in vitamin deficiency. Med. 68: 207-220, 1938. Jour. Expt. Wintrobe, M. M., J. H. Miller and H. Lisco. The relation of the diet to the occurrence of atonia and de­ generation in the nervous system of pigs. Johns Hopkins Hosp. Bull. 67: 377-406, 1940. Wintrobe, M. M., M. Mitchell, H. Miller, R. H. Follis, Jr., Harold J. Stein, Cecil Mashatt and Stewart Humphreys. Sensory neuron degeneration in pigs. Protection afforded by calcium pantothenate and pyridoxine. Jour. Hutr. 24: 345-366, 1942. Wright, C. D. and A. D. Welch. The role of ’’folic acid” and biotin in the utilization of panto­ thenic acid by the rat. Science 98 (2550): 425-427, 1943. (Abstracted in Biol. Abst. 18: 1414 (12939), 1944). 69 LITERATURE CONSULTED BUT NOT CITED Beeson, Vf. M., E. T. Mertz and D. C. Shelton* Effect of tryptophane deficiency in the pig# 8: 532-540, 1949. Jour# Animal Sci# Cunha, T. J#, D# C* Lindley and M* E. Ensminger. Biotin deficiency syndrome in pigs fed desiccated egg "white. Jour. Animal Sci. 5s 219-225, 1946. Daft, F. S. and W. H. Sebrell. Hemorrhagic adrenal necrosis in rats on deficient diets. U. S. Public Health Rep. 54s 2247-2250, 1939. Davis, G. K., V. A. Freeman and L. L. Madisen. The relation of nutrition to the development of necrotic enteritis in swine. Michigan Agric. Expt. Station, Bast Lansing, Technical Bull. 170, 1940. Ellis, N. R. The vitamin requirement of swine. 1946. Elvehj em, C. A. The vitamin B complex. 1948. Nutr. Abst. and Rerv. 16: Jour. Amer. Med. Assoc. 138s 1-10, 960-971, Heitman, H., Jr. and A. G. Hogan. Vitamin deficiencies in rations of natural feedstuff s. Missouri Agric. Expt. Station, Columbia, Missouri, Res. Bull. 432, 1949. Hogan, A. G. and G. C. Anderson. Vitamins required by swine for growth, with some observation on reproduction. Jour. Nutr. 36s 437-449, 1948. Lepkov sky , Samu el • Components of the vitamin B« complex. 363-375, 1941-42. Nutr. Abst. and Rev. 11s Lindley, D. C. and T. J. Cunha. Nutritional significance of inositol and biotin for the pig. Jour. Nutr. 32: 47-59, 1946. Lipmann, F., N. 0. Kaplan, G. D. Novelli, L. C. Tuttle and B. M. Guirard. Coenzyme for acetylation, a pantothenic acid derivative* Jour. Biol. Chem. 167: 669-870, 1947* 70 Luecke, R. W., W. N. McMillan, F. Thorp Jr. and Carolyn Tull# The relationship of nicotinic acid, tryptophane and protein in the nutrition of the pig. Jour. Nutr. 33: 251-261, 1947. McMillen, W. N., R. W. Luecke and F. Thorp Jr# Pantothenic acid deficiency in swine on diets of natural feedstuffs# Jour. Animal Sci# 7: 529, 1948. Nelson, M# M. and M# E. Herbert# Pantothenic acid deficiency and reproduction in the rat# Jour. Nutr# 31: 497-507, 1946# Pearson, P# B. Pantothenic acid content of the blood of mammalia# Chem. 140; 426-432, 1941# Jour. Biol#. Piccioni, M., A. Rabbi and 0. Moruzzi. Animal protein factor in crude casein and its vitamin B,g rela­ tionship. Science, 113: 179-181, 1951# Ram Tulsa. A histopathological study of chicks deficient in pantothenic acid# Poult. Sci. 28: 425-430, 1949. Rasmussen, R. A., H. J# Stafseth, Y. A. Freeman and Mariane, J# Miller. Influence of B vitamins, liver and yeast on induced necrotic enteritis in swine. Yet. Med. 39: 421, 1944. Rosenberg, H. R. Chemistry and physiology of the vitamins# Inc., New York., 1942# Interscience Publishers, Supplee, G. C., R. C. Bender and 0. J. Kahlenberg. Inter-related vitamin requirements: Kidney damage, adrenal hemorrhage and cardiac failure correlated with inadequacy of pantothenic acid. Endocrinology, 30: 355-364, 1942. Williams, R. J., H. K. Mitchell, H. H. Weinstock Jr. and E. E. Snell. Pantothenic acid. Partial and total synthesis studies. Jour# Amer# Chem. Soc# 62: 1784—1785, 1940. Wintrobe, M. M., M. Sanrfcer and H. Lisco. Morphologic changes in the blood of pigs associated with deficiency of water soluble vitamins and other substances contained in yeast. Johns Hopkins Hosp. Bull. 64: 399-423, 1939# Zwemer, R. L. A study of adrenal cortex morphology. 107-114, 1936. Amer. Jour. Path. 12: Figure 1 Pig 3 Large Intestine 1. Edema of the intestinal wall# 2 • Marked congestion# 3# Ecchymotic hemorrhages. 71 Figure 2 Pig 1 Large Intestine 1. Entire epithelium. 2. Congestion of lamina propria. 3. Lymphocytic infiltration. Hematoxylin and eosin stain. 190X 72 ^ m m r . *y- . Jr- "•'?*•*$&.<. - * - -J. -3* V*C£?3*»• 'si .^. *„ ^j^Va.<*•'jw yj* .. v .•.-^«" . 7*.*» '*1^1i 9 ; > £ • Figure 3 Pig 1 Large Intestine 1* Desquamation of the epithelium* 2. Mucus accumulating in the cypsts of Lieb erleuhn# Hematoxylin and eosin stain. 190X 73 ''-SMS' » i-■ . ■kV , ' * * A •? -M* P- »A* *xW * v * jx % \*CJ i '!» ,. v & l . * : ' •'- ■ ^ . • l. y.#^ V ' ^ C v v .; • s*.% • • 1 # 1 ; ? $ , . . . . „ s a p : v , > S B K w ^ s^SS E ^ r a ^ I s v ' v * •••-j . -«<,.,..; J_ " v . „•* v * % . - , S < r j M | v, 2. S Jll *& •> f WHjaPBBM pfef^ . x *MfcO k r % r \ JL • Figure 4 Pig 1 Large Intestine 1* Desquamation of the epithelium* 2* Engorgement of the blood vessel exposed to the lumen of the in­ testine* 3* Periglandular infiltration -with leukocytes. Hematoxylin and eosin stain* 190X 74 Figure 5 Pig 2 Large Intestine 1* Infiltration of the lamina propria with lymphocytes and macrophages» a. lymphocyte b. macrophage Hematoxylin and eosin stain. 1150X 75 II 'VT&*ZWF£MZfr © o © •• 1 ♦ •:p *.. L > ^ n Figure 6 Pig 2 Large Intestine 1. Baiantidium coli in the mucous exudate* Hematoxylin and eosin stain* 100X 76 Figure 7 Pig 3 Large Intestine 1* Accumulation of mucous exudate on the surface* 2 • Balantldium coli in the superfi­ cial parbs of the lamina propria* Hematoxylin and eosin stain* 140X 77 Figure 8 Pig 4 Large Intestine 1* Mucus, degenerated epithelial cells and leukocytes forming exudate on the surface* Hematoxylin and eosin stain 80X 78 ■'V't V Figure 9 Pig 3 Large Intestine 1# Early ballooning of the intestinal glands. Hematoxylin and eosin stain. SOX 79 Figure 10 Pig 3 Large Intestine 1* Mucus and degenerated leukocytes accumulating in the intestinal glands (Figure 9)# 2. Degeneration and pressure atrophy of the lining epithelium of the glands. 3. Periglandular lymphocyte infiltration. Hematoxylin and eosin stain. 80X 80 Figure 11 Pig 5 Large Intestine Macrophages containing engulfed leuko­ cytes and tissue debris# These macrophages were seen in the periglandular lymphocytic infiltration and in the lymph nodules showing hyper*plasia# Hematoxylin and eosin stain. 1150X 81 9J* «kl * < •1 ea * r A Figure 12 Pig 5 Large Intestine 1. Desquamat ion of the surface epithel­ ium. 2. Superficial ulcer extending to about half the depth of the lamina propria. Hematoxylin and eosin stain. 100X 82 3 . g ••;< w i T r ' ’l /<-'«' ■->' v ~/ t /;'.... ■■-: -fcfc > ■ < K-- V. ‘ . < , t- , '• . < * * . V ' , J'V- v ■ V ::V' c>» # * a ^ A $ j/,.!>'> r, y ,j' •'■& o>;.',. ^ v *? v v.' •rv;A v,* 'v, a.|: >‘;'.^ V %££$*** ■ ’* Figure 15 Pig 8 Large Intestine Marked hyperplasia of the intestinal glands* Hematoxylin and eosin stain* SOX 83 figure 14 Pig 8 Large Intestine 1* Fibroblasts proliferating in the lamina propria. 2* Engorged capillaries in the subepithelial area* Hematoxylin and eosin stain* 1150X 84 7 Figure 15 Pig 6 Kidney Congestion and hemorrhage in the medulla# a. congestion b. hemorrhage Hematoxylin and eosin stain# 125X 85 !L. £•> r5vk‘ . Wv 3 * & W \ V iv Pigure 16 Pig 7 Kidney Hemorrhage in the medulla* Hematoxylin and eosin stain. 12 5X 86 r .V * f A ' *ii*>, ‘• > A M :4 ■ ? A V* Figure 17 Pig 5 Kidney 1* Edema in the cortex. 2# Shrinking of the glomeruli. Hematoxylin and eosin stain* 170X 87 > .% , . r * L* * V v - < -• • - O ’ : o ■**. v l l&?eCL ** in* -ZS^ s*” . . l ♦ > rv D 3 1 ? • - '** ' • / V < *> ...* 4 ^ U. ^ ^ *S^ fcT? L 5 f t " « jp»*i O ri?L Pig 5 Figure 18 Kidney Edema of the cortex and shrinking of the glomerulus. Same area as Figure 17* Hematoxylin and eosin stain* 725X 88 ^ ■* W « 4 ^ .2 n x P b 4'*^* ^>ur 1>* f **v> »% >5^ A* >JF w» \ 4 \ t V n* ,*•> C * fie ^ 4 ) \ I O v • „ • * • i f ^ » "▼Vi: * * t I#V. .fifcs «<%* t ** Figure 19 Normal Adrenal Gland 1. Central vein. 2. Medullary sinusoids. Hematoxylin and eosin stain. 13 OX 89 Figure 20 Pig 1 Adrenal Gland Hemorrhagic foci in the medulla# Hematoxylin and eosin stain* 125X 90 figure 21 Pig 3 Adrenal Gland 1. Degeneration of the parenchymatous cells of the medulla and of zona fascicularis* 2. Degenerated cells agglomerating in masses* Hematoxylin and eosin stain* 130X 91