I I I I If I: 'I W THE fifvéFLUENCE OF AN ENTERSC ENFELCWR 53:“! 23M) METABOLISM Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY DELBERT L. WHITENACK 1970 This is to certifg that the thesis entitled THE INFLUENCE OF AN ENTERIC INFECTION ON ZINC METABOLISM presented by Delbert L. Whitenack has been accepted towards fulfillment of the requirements for _&_L degree in My ,7 4;" 7;. fl / /°’ 4 ////"/ ‘ 4[‘ :1 71‘41 rt 11' 1 1 .1 411/ L.’ V~"" '1 fr’ 1"!" " Major professor Date August 6, 1970 0-169 IINNDING IV : qus a SUNS' @995 amnm me. ‘3‘! .Iun'nn I: Egflflitojt. menu] 5“ .. 1 ___ A ABSTRACT THE INFLUENCE OF AN ENTERIC INFECTION ON ZINC METABOLISM By Delbert L. Whitenack Two balance trials, using a total of 28 pigs, were conducted to determine the influence of an enteric infection on zinc metabolism. The deficient purified diet contained 12 ppm zinc, while the adequate diet contained 100 ppm zinc. The influence of the transmissible gas- troenteritis (TGE) virus infection and the antimicrobial drugs, oxy- tetracycline and mycostatin, were evaluated in pigs fed both diets. An enteric infection decreased zinc retention significantly in pigs fed either the zinc—deficient or zinc-adequate diets. Pigs fed the zinc-deficient diet had lower serum zinc values than pigs fed the adequate zinc diet. In infected pigs fed 100 ppm zinc the serum values were decreased but not in infected pigs fed 12 ppm zinc. Zinc deficiency was characterized by anorexia, reduced weight gains, parakeratosis, depleted fat depots, serous atrophy of fat, and atrophy of the thymus. Histopathologically there was depletion of cortical thymocytes, incomplete karatinization on the tongue, esophagus and cardia of the stomach, and the villi and glands of the ileum were reduced in size and number. Cytoplasmic vacuoles were observed in the villous epithelium of the ileum. Signs in TGE-infected pigs were Delbert L. Whitenack vomition, diarrhea, dehydration, and anorexia. Microscopically, there was atrophy of villi with the epithelium at the tips of the villi flat- tened or cuboidal in the jejunum and ileum. One third of the infected pigs fed the zinc-deficient diet died while all of the infected pigs fed the zinc—adequate diet survived. The TGE infection was more severe in pigs fed the zinc-deficient diet. Oxytetracycline and mycostatin reduced the incidence of candida albicans infection in the oral cavity, esophagus and cardia of the stomach. Growth on the diets before infec- tion was slightly better in pigs fed the antimicrobial drugs. THE INFLUENCE OF AN ENTERIC INFECTION. ON ZINC METABOLISM By. ' t Delbert L? Whitenack A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1970 ACKNOWLEDGEMENTS The author wishes to express sincere gratitude and appreciation to Dr. C. K. Whitehair, major professor, and members of the guidance committee for counsel and guidance in the conduct of this research and in the preparation of this manuscript.‘ The author is extremely appreciative to Dr. Elwyn R. Miller of the Animal Husbandry Department for assistance and guidance in the perform— ance of this research. My sincere thanks to Dr. Duane E. Ullrey and Mrs. Betty Schoepke for guidance in the determination of zinc levels. The author wishes to express his appreciation to all members of the Department of Pathology for technical and professional aid and guidance. The writer especially wishes to acknowledge his gratitude to his wife, Gwen, whose sacrifices and encouragement made this study possible. The author is grateful for financial support through a postdoc- toral fellowship awarded by the Division of General Medical Sciences in the National Institutes of Health. 11 . TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE. . . . . . . . . . . . . . . . . . . . . . . . 3 General. . . . . . . . . . . . . . . . . . . . . . . . . . . 3 The Role of Zinc in Animal Nutrition . . . . . . . . . . . . 4 Zinc deficiency in the rat. . . . . . . . . . . . . . 4 Zinc deficiency in swine. . . . . . . . . . . . . . . 7 Zinc deficiency in the chicken. . . . . . . . . . . . 8 Zinc deficiency in human subjects . . . . . . . . . . 9 Zinc deficiency in ruminants. . . . . . . . . . . . . 10 Influence of Dietary Components Upon Zinc Availability . . . ll Phytic acid . . . . . . . . . . . . . . . . . . . . . 11 Calcium . . . . . . . . . . . . . . . . . . . . . . . 13 Chelating agents. . . . . . . . . . . . . . . . . . . 14 The Role of Zinc in Metalloenzymes . . . . . . . . . . . . . 14 General . . . . . . . . . . . . . . . . . . . . . . . 14 Metallothionein . . . . . . . . . . . . . . . . . . . 15 Carboxypeptidase. . . . . . . . . . . . . . . . . . . 15 Alcohol dehydrogenase . . . . . . . . . . . . . . . . 15 16 Alkaline phosphatase 0 O O O O O O O O O O O O O O O 0 Influence of Infectious Diseases on Zinc Requirements. . . . 16 Transmissible Gastroenteritis (TGE) in Pigs. . . . . . . . . 18 Summary. . . . . . . . . . . . . . . . . . . iii MATERIALS AND METHODS . . . . . . . . . . Experimental Animals . . . . . . . Selection and Care of Experimental Diet 0 O O I O O O O O O O O O O Pathogen O O O 0 O O O O O I O O O Hematology . . . . . . . . . . . . Histopathology . . . . . . . . . . Zinc Analysis. . . . . . . . . . . Analysis of Data . . . . . . . . . Experiments. . . . . . . . . . . . RESULTS . . Experiment 1. . . . . . . . Experiment 2. . . . . . . . Experimnt 1 O O O O O O O O O O 0 Clinical signs. . . . . . . Zinc balance and hematology Gross lesions . . . . . . . Histopathologic changes . . Experiment 2 . . . . . . . . . . DISCUSSION. Clinical signs. . . . . . Zinc balance and hematology . Gross lesions . . . . . . . Histopathologic changes . . Growth and Clinical Signs. . . . . Blood Analyses and Serum Zinc. . . Zinc Balance . . . . . . . . . . . Gross Lesions. . . . . . . . . . . iv Page 22 22 22 24 24 24 25 26 26 27 27 27 31 31 31 33 33 35 48 48 48 51 52 57 57 58 59 59 Page Histopathologic Changes. . . . . . General. . . . . . . . 61 SWY O O O O O O O O O O O O 0 REFERENCES 0 O O O 0 O O O O O O O VITA O O O O O O O O O O O O O O O O LIST OF TABLES Table Page 1 Composition of basal purified diet. . . . . . . . . 23 2 Experimental design - Experiment 1. . . . . . . 28 3 Experimental design - Experiment 2. . . . . . . . .q. 30 4 Performance, zinc balance and blood analyses as influenced by dietary zinc and TGE infection during a 3-day balance trial. Experiment 1 I I I I I I I I I I I I I I I I I I I I 34 5 Growth performance and values of serum zinc as influenced by dietary zinc, antibacterial and antifungal agents. Experiment 2 I > I I I I I I I I I I I I I I I I I I I I I I I 49 6 Summary of zinc balance studies and blood analyses after I I I I I I I I 50 infection. Experiment 2. . . . vi LIST OF FIGURES Figure Page 1 Pigs fed 100 ppm and 12 ppm zinc diets . . . . . . 2 AtrOphic thymus from a pig fed the 12 ppm zinc diet for 3 weeks I I I I I I I I I I I I I I I I I I 36 3 Normal thymus from a pig fed the 100 ppm zinc diet for 3 weeks I I I I I I I I I I I I I I I I I I I I I I I I 36 4 Hemorrhage and necrosis of gastric mucosa from infected pig fed the zinc-deficient diet. . . . . . . . . . . . . . . 37 5 Submucosa of stomach from infected pig showing a focus of neutrophils and a thrombus in a blood vessel . . . . . . . . 37 6 Ileum from pig fed the zinc-deficient diet and infected with TGE virus. Note shortened, fused and blunted villi . 39 7 Mucosa of the ileum from TGE-infected pig fed the zinc- Note the cuboidal appearance of the . . . 39 deficient diet. epithelium and loss of the striated border . 8 Squamous epithelium on the dorsal surface of the tongue. Dark staining material in the cornified interpapillary epithelium is fungal and bacterial growth. . . . . . . .-. . 4O 9 Cornified epithelium from dorsal surface of the tongue from a pig fed the zinc-adequate diet for 51 days._ Note larger dark-staining yeast forms of Candida albicans and numerous Gram-positive bacteria. . . . . . . . . . . . . . . 4O 10 Distal eSOphagus from a pig fed 100 ppm zinc diet with keratotic_layers of epithelium containing fungi. . . . . . . 41' 11 High magnification of fungi in cornified epithelium of distal esophagus. Note budding yeast forms and hyphae . . . 41 12 Parakeratosis of zinc deficiency in the esophagus of a pig that had been fed the 12 ppm zinc diet for 21 days. Note the pyknotic nuc1e1 I I I I 'I I I I I I I I I I I I I I 42 13 Parakeratosis of the dorsal surface of the tongue from . 42 a pig that had been on the low zinc diet for 37 days . . . vii Figure 14 15 l6 17 18 19 20 21 22 23 24 25 26 27 28 c deficiency in the thymus after 51 days Lesions of zin Cortex reduced in size. . . . on the zinc—deficient diet. Note of pig fed the zinc-adequate diet. ning crowded thymocytes. . . . . . . g fed the low level Portion of thymus wide cortex contai Nutritional muscular dystrophy in a pi of zinc and infected with TGE virus. . . . . loss of striation and mineral deposits in e from an.infected pig fed Fragmentation, skeletal muscl diet I I I I I I I I I I I eplaced by cellular inate in the exudate, d cells of reticulo— Skeletal muscle fibers partially r exudate. Mononuclear cells predom and probably are macrophages or relate endothelial origin . . . . . . . . . . . Normal skeletal muscle from infected pig fed the zinc- adequate diet. . . . . . . . Portion of testis from infected pig fed the 12 ppm zinc diet. Normal juvenile seminiferous tubule, degenerative knosis of germinal epithelium seminiferous tubules with py and Sertoli cells. . . . . . . rminal epithelium and interstitial cells in Necrosis of ge pig fed 12 ppm zinc. . . . . . . the testis of an infected hat had been on the zinc- Portion of ileum from a pig t Note reduction in size of deficient diet for 4 weeks. glandular tissue and villi . . . villi of epithelial cells at the tips of the Vacuolization C'défiCient diet 0 e o o o o o from a pig fed the zin pig that 4 t the tip of a villus in a Normal epithelium a t containing adequate zinc for had been on the die we eke e. o o o e o o a Note thickened, hyperkeratotic and parakeratotic squamous a1 surface of the tongue from a pig epithelium on the dors fed the zinc—deficient diet and antimicrobial drugs. . . . helium on the dorsal surface of the Normal keratinized epit ving the zinc—adequate diet . . . . tongue from a pig recei c acinar cells from a pig ient diet for 4 weeks. 113 and diminution in ion of pancreati n the zinc—defic acinar ce ules . . . . . . . . . High magnificat that had been 0 Note reduction in size of the number of secretory gran of pancreatic acinar cells from a pig High magnification fed the zinc-adequate diet . . . . I I I I I I I I I I viii the zinc-deficient Page 44 44 45 45 46 46 47 47 53 53 54 55 55 56 56 INTRODUCTION AND OBJECTIVES A voluminous amount of literature has accumulated on zinc deficiency in man and animals since it was demonstrated in 1955 that parakeratosis in swine was due to a zinc deficiency. A summary of current information is that zinc is a dietary requirement for most species. The physio- logical function of zinc is that it is a component of a number of dif- ferent metalloenzymes and a deficiency results in alterations in a wide variety of tissues. There has been less research effort devoted spe- cifically to the histopathologic effects of a zinc deficiency in the various species of animals than the metabolic effects. Likewise, very little experimental work has been conducted on the influence of infectious agents of especially the intestinal tract on zinc requirements. During the past decade reports have emphasized the importance of zinc in human nutrition and health. The importance of infectious agents was also emphasized, and it was suggested that infection is an important factor influencing zinc requirements in man and animals. Experimental work to determine the magnitude of zinc losses during an enteric infection have not been reported. The objectives of this research were: (1) to determine the pathol- ogy of zinc deficiency in the young, growing pig; (2) to study the influence of an enteric infection on zinc absorption in zinc-depleted and zinc-adequate baby pigs; (3) to determine the interrelationship of zinc and infection; (4) to study the effect of an antibacterial and 2 antifungal agent added to the diet; and (5) to correlate lesions of a zinc deficiency with clinical signs and growth. REVIEW OF LITERATURE General Conclusive evidence that zinc is necessary for normal growth and development of the rat was mentioned in 1934 in Wisconsin (Todd at aZ.). On a synthetic diet low in zinc the rate of growth of the rat was accelerated by the addition of zinc salts. In the 1940's, a syndrome characterized by severe dermatitis was recognized in swine in the United States and other countries. In 1953, Kernkamp and Ferrin, at the Minnesota Experiment Station, characterized this syndrome histo— logically as parakeratosis. Tucker and Salmon (1955), at the Alabama EXperiment Station, demonstrated that zinc alleviated the signs and This latter work precipitated a lesions of parakeratosis in swine. number of reports on the importance of zinc in animal nutrition. In recent years zinc deficiency has been identified with an important clinical and public health problem in man associated with dwarfism and hypogonadism (Prasad at al., 1963). The reports of Sandstead at al. (1967) and Prasad (1967) have also emphasized the importance of zinc in human nutrition and health, and they demonstrated interrelationships of zinc deficiency with diseases of nematodiasis, schistosomiasis, and malaria. In swine, Mansson (1964), in Sweden, observed that pigs having a CZostridEum,perfringsns infection had more severe lesions of parakera- tosis. In poultry, zinc-65 absorption rates decreased in birds infected with Eimeria neoatrix (Turk and Stephens, 1966). 3 4 This literature review pertains primarily to the pathologic manifestations of zinc deficiency and the influence of infectious agents on zinc requirements in man and animals. The Role of Zinc in Animal Nutrition Zinc deficiency in the rat. Todd at al. (1934) described the abrupt effect of a decreased growth rate and inappetence in the zinc—deficient rat. In addition, they noted loss of hair, WhiCh occurred over the head and shoulders and-progressed to the ventral portion of the neck and thorax. In 1940, Day and McCollum used a low zinc diet in which rats were estimated to receive 2-4 mg. zinc per day. Young rats ceased gaining weight in 2 to 3 weeks. Thinning of the hair became apparent after the 3rd week and was followed by alopecia. In a subsequent report, Follis et a1. (1941) described histopathologic skin changes in these rats. The alterations were divided into those which were considered specific for zinc deficiency and those which were regarded as nonspecific. The earliest change in the skin was an increase in the number of cell layers of the epidermis. Instead of the normal thickness of 2 to 3 cells, the epidermis increased in thickness so that it consisted of 6 to 8 cell layers. Keratohyaline granules were more numerous. There was keratini- zation of the hair follicles and the basal portions of the hair shafts disappeared. As the epidermis increased in thickness, the nuclei of the more peripheral cells were pyknotic and were retained. During the early stages of the disease, an inflammatory reaction was absent in the corium, but later polymorphonuclear and mononuclear leukocytes were observed, particularly about blood vessels. In the areas where epithelial changes were marked, the hair follicles completely disappeared. In 5 contrast, the sebaceous glands were more prominent and the cells were hypertrOphic. The microscopic appearance of the esophagus was abnormal in rats that had been on the experiment 33 days. Barney at al. (1968) described the earliest changes in the esophagus as hyperkeratosis and parakeratosis, first observed on day 16 of the experiment. The lesions in the esOphagus described by these authors were similar to the earlier reports and appeared to be specific for zinc deficiency. The cells in the basal layer were more numerous and more closely packed than normal. The overlying stratum was increased in width, averaging 6 to 8 cells in thickness, in comparison with 2 to 3 of the control rats. The nuclei were somewhat swollen and the nuclear membranes wrinkled. On the intermargin of this layer, the nuclei were smaller and more deeply stained. These workers concluded in their depletion and repletion studies that the deepest layers of the transi— tional cells of the esophagus were injured in zinc deficiency and the subsequent abnormal differentiation to parakeratotic cells was a mani- festation of this earlier injury. The successive histologic changes following zinc repletion suggested that healing apparently occurred by displacement of the abnormal cell layers above the basal stratum by normally differentiating cells. Miller at al. (1958) reported that weanling male rats on a zinc- deficient diet for 8 weeks developed partial or complete atrophy of, germinal epithelium of the testes. All changes except the testicular atrophy were reversible with zinc repletion. Those rats that had atrOphy of the tubular epithelium also had a reduced size of all other sex organs. At sexual maturity the atrophy of the tubular epithelium and reduction in zinc concentration in the testes was even more evi- dent. It was concluded on repletion studies that there was retardation 6 of sexual development, and it was evident that once degenerative changes had occurred as a result of zinc deficiency, the seminiferous tubules of the testes were not capable of regeneration when zinc was again available. Lesions in the testes were also described by Macapinlac at al. (1966) and Barney et a2. (1968) in that weanling rats fed a zinc- deficient diet for 23 days had degeneration of the seminiferous tubules of the testes characterized by pyknosis of nuclei, exfoliation of cells and formation of multinucleated giant cells. There were few sperm in the epididymis of the zinc-deficient rats compared with the restricted fed controls which received the basal diet supplemented with zinc sulfate to provide an additional 30 ppm of zinc. After zinc repletion for 2 weeks there was no amelioration and tubules appeared to be more severely damaged. The earliest histological change in spermiogenesis appeared to be the transformation of spermatids to spermatozoa. The tubules of the testes appeared immature when examined and spermatogenesis had not been fully established; the younger cells such as spermatocytes and spermatogonia were probably injured early in zinc deficiency despite the absence of demonstrable morphologic abnormality. Cells already damaged before zinc repletion proceeded to degenerate irreversibly and were extruded and lost from the germinal epithelium. In more detailed studies, Whitenack (1968) and Whitenack et a2. (1970) reported similar gross and microsc0pic lesions in rats fed an egg white diet containing 0.9 ppm zinc. Gross lesions were progressively more severe throughout the experiment and were more severe in male rats. The primary changes in the epithelial cells of the germinal epithelium of the seminiferous tubules were similar to those in previous reports. However, on repletion studies the degenerative lesions in weanling male rats were reversible, but were irreversible in sexually mature males. 7 Using a soy protein diet washed with ethylenediaminetetraacetic acid (EDTA), Swenerton and Hurley (1968) described the following zinc deficiency signs: retardation of growth, abnormal posture, alopecia, and dermal lesions around the eyes, mouth and feet. Male rats fed 60 ppm of dietary zinc in the ration had growth rates comparable to stock— fed rats. However, after 12 weeks there were esophageal and testicular lesions similar to those observed in the group of zinc-deficient males. Symptoms were completely reversed by zinc supplementation. In female rats Hurley (1969) reported that a zinc deficiency caused abnormal estrous-cycles, which were determined by vaginal smears. Zinc repletion had an immediate effect on growth rate, and the females returned to normal estrous cycles and were able to produce normal young. In pregnant females zinc deficiency caused early gross congenital mal- formations. The rapid effect of the zinc deficiency during pregnancy suggested that the female was unable to mobilize zinc from maternal stores for the benefit of the fetus. In contrast, Sandstead at al. (1970) concluded that there was increased fetal wastage without gross teratology occurring in pregnant rats fed a zinc-deficient diet high in protein. Fetal size decreased independent of placental size. Zinc deficiency in swine. Kernkamp and Ferrin (1953) first described a condition in swine that was characterized by hard, dry, crusted pro- liferation of the superficial layer of the skin. Microscopically the crusted masses were composed of cornified epithelium, collections of keratin and debris. Large numbers of nuclei were scattered throughout the proliferated and keratinized substance. This condition of the skin was called parakeratosis. Since parakeratosis in swine was a common problem in animals raised under practical feeding conditions, intensive 8 research was carried out in an attempt to elucidate the cause of this disease. In 1955, Tucker and Salmon described the essential role of zinc in the prevention and cure of parakeratosis. At the Michigan Agricultural Experiment Station, Miller at al. (1966) produced an experi- mental zinc deficiency in the baby pig using purified diets containing isolated soybean protein. Diets of this type contained approximately 10 ppm of zinc. Smith and co~workers (1961), at Purdue, established that the zinc requirement of baby pigs fed semipurified diets containing isolated soybean protein was 46 ppm. In further experimentation in Michigan-to determine zinc requirement of baby pigs fed diets which contained casein as the protein source, all pigs fed 10 ppm or less of dietary zinc had signs of zinc deficiency. Zinc retention data indi- cated that a dietary zinc level of between 14 and 20 ppm was required to maintain total body tissue levels similar to those of the nursing pig (Shanklin at aZ., 1968). In additional research, comparisons of biological measures made on baby pigs indicated that zinc deficiency resulted in a reduced rate and efficiency of body weight gain, inappe— tence, parakeratosis and a reduction of serum zinc concentration. Thymus weight was greatly reduced in zinc-deficient pigs (Miller at aZ., 1968). Zinc deficiency in the chicken. Following the discovery of the essen- tial role of zinc in the prevention of parakeratosis in the pig, inves— tigations were extended to other animal species, especially the chick. O'Dell at al. (1958) and Young at al. (1958) described zinc deficiency in young chickens. The signs and lesions were slow growth rate, poor. feather development, rapid respiration, shortening and thickening of the long bones of the legs, and a tendency for the hock joints to enlarge. Microscopically, there were hyperkeratosis and acanthosis of the skin and parakeratosis of the esophageal epithelium. 9 The leg deformities were characterized microscopically by an apparent failure of cartilage cell development in the epiphyseal plate and decreased osteoblastic activity. In the noncalcified epiphyseal regions, the cells adjacent to the blood vessels appeared essentially normal, while those further away from the blood vessels were abnormal. Such cells were larger and more rounded than normal; they were irregularly arranged, and there was more abundant matrix between the cells. Cartilage cell differentiation and development appeared to be delayed and erratic in areas remote from blood vessels (O'Dell at aZ., 1958; Young et 41., 1958; Westmoreland and Hoekstra, 1969). Additional observations were made using histochemical staining procedures on the long bones of zinc-deficient chicks (Westmoreland and Hoekstra, 1969). These workers reported no noticeable effect on alkaline phosphatase staining associated with the centers of calcification. However, in the noncalcified, cartilaginous regions profound effects of zinc deficiency on the enzyme were observed. The cells adjacent to blood vessels exhibited alkaline phosphatase staining similar to the cells of control bones, while those cells further away from blood ves- sels had decreased alkaline phosphatase activity. Zinc deficiency in human subjects. Prasad at al. (1961) described a nutritional deficiency of zinc in man. The deficiency was noted in Egypt and Iran. The clinical manifestations were short stature, marked hypogonadism, hepatosplenomegaly, and anemia. The diet of these people consisted of bread made from wheat flour and the intake of animal pro- tein was low. Early studies indicated that the anemia was due to a deficiency of iron and was corrected by oral iron. With the addition of dietary zinc, the following changes were observed: improved growth rate, appearance of pubic hair in all cases within 7 to 12 weeks, 10 increased size of genitalia, and development of secondary sexual char- acteristics within 12 to 24 weeks. No such changes were observed in the iron—supplemented group nor in the group fed an animal protein diet. In more recent work it was reported that the growth retardation and gonadal underdeveIOpment were primarily related to the deficiency of zinc (Prasad, 1966; Sandstead at aZ., 1967). The most striking result of zinc supplementation to undernourished 12- to 14-year-old schoolboys in Iran was the effect of zinc in accelerating the rate of sexual maturation (Ronaghy et aZ., 1969). Prasad (1966) conducted several biochemical and metabolic tests to demonstrate evidence of zinc deficiency. The zinc concentration in plasma, erythrocytes, and hair was decreased. Radioactive zinc-65 studies revealed that plasma zinc turnover rate was greater, the 24-hour exchangeable pool was smaller, and the excretion of zinc—65 in stool and urine was less than control subjects. Zinc deficiency in ruminants. The importance of zinc in ruminant nutri- tion was demonstrated in a series of experiments at the University of‘ Georgia (Miller at aZ., 1962, 1966, 1968). Experimentally, when ruminants were fed a zinc-deficient diet, there was a fairly rapid but small reduction in zinc content of many tissues. The zinc content of muscle and brain was not reduced. Prolonged feeding of a deficient diet, which caused a clinical deficiency, resulted in a further reduction in zinc content of soft tissue. When animals were fed a zinc-deficient diet, soft tissues, but not bone, developed an increased affinity for zinc. Bone apparently accumulated zinc which was left over after the require- ments of soft tissues were met. Deficiency signs of zinc in calves appeared progressively in the following order: excessive salivation, loss of hair around the eyes and mouth, hyperkeratosis of the skin of 11 the neck, level of the mandible and inside the legs, enlargement of the hock bones, and the appearance of nervous stepping movements of the hind limbs. In England, Mills et a1. (1967, 1969) noted that weight gain ceased abruptly in calves and lambs fed 0.05 mg. zinc per kg. liveweight per day. Plasma zinc concentration decreased from 1.0 ug./ml. to below 0.4 ug./ml. after 1 week on a deficient diet. Growth arrest occurred within 2 weeks on the deficient diet. The calf and lamb apparently have a limited capacity to store zinc in‘a form that can be utilized during periods of inadequate intake. The clinical signs in lambs fed a deficient diet were excessive quantities of frothy saliva. ThiS‘ was a transitory phenomenon lasting from 5 to 10 days. There was pro- nounced pallor of the tongue associated with a marked increase in bac— terial flora. Wool became loose and was easily pulled from the skin. In horned animals there was production of soft, deformed horns lacking the typical surface striations of normal horn. A conclusion was made that if plasma zinc concentration decreased below approximately 0.4 ug./ml. and remained below this level for more than 1 week, growth was arrested and clinical signs of deficiency develOped. Influence of Dietary Components Upon Zinc Availability Phytic acid. Several studies have demonstrated that the source of pro- tein in the diet, whether of animal or plant origin, has an effect upon the utilization of zinc. In 1957 it was reported that the zinc in soy- bean protein was less available to chickens than that in casein (O'Dell and Savage, 1957). Similar observations were made with pigs (Oberleas at aZ., 1962; Smith et aZ., 1962) and rats (Forbes and Yohe, 1960). 12 Pigs fed a casein diet that contained 14 ppm of zinc did not develop signs of parakeratosis and the rate of gain was 780 grams per day, whereas those fed a soybean protein diet containing 25 ppm of zinc developed parakeratosis and gained only 120 grams per day. Rats fed casein or egg white as the source of protein required 10 to 12 ppm zinc in the diet, while those fed soybean protein required 18 ppm. The apparent absorption of zinc by rats fed casein was 842 compared to 44% by those fed soybean protein (Forbes and Yohe, 1960). This phenomenon of soybean protein lowering the percent absorption of zinc from the intestine has been collaborated by other workers (Savage et 41., 1964; Edwards, 1966). It appeared that the zinc in isolated soybean protein was bound so that it was absorbed less efficiently than the zinc in animal proteins, such as casein and egg white. Plant seeds, notably oilseeds and cereal grains, contain phytate, the hexophosphate ester of inositol. Since phytate complexes with metal ions, it was believed that phytate was responsible for the lower availability of zinc in an all-plant diet. Phytic acid was added to a casein-based diet, and there was decreased zinc availability with production of signs similar to those observed in animals fed a soybean protein containing a comparable level of phytate (O'Dell and Savage, 1960). Similar results have been obtained by addition of phytic acid to a diet based on free amino acids (Likuski and Forbes, 1964). Sup- plementation with adequate zinc restored normal growth rate and pre- vented skin lesions. Kratzer et al. (1959) observed that autoclaving soybean protein increased the availability of.zinc to turkey poults. Autoclaving a natural diet fed to pigs decreased the incidence and severity of para- keratosis (Smith at aZ., 1960). The phosphate ester bonds in phytate were broken by autoclaving in an aqueous medium. 13 Calcium. It was demonstrated that excess calcium in the-diet aggravated the signs of zinc deficiency in pigs fed a corn—soy diet (Luecke at aZ., 1956; Stevenson and Earle, 1956; Luecke at aZ., 1957; Forbes, 1960; Hoekstra, 1964). Calcium enhanced zinc deficiency signs when added to diets based on plant protein that have a high concentration of phytate. Diets in which calcium had no effect contained animal protein (Forbes, 1960). The addition of excess calcium in a soybean diet fed to chicks also depressed growth rate unless supplemented with zinc (O'Dell at aZ., 1958). Further studies of the calcium-zinc interrelationship revealed that excess calcium depressed growth rate only in the presence of phytate (Oberleas et aZ., 1962). In the absence of phytate, excess calcium had no detrimental effect. In man, the calcium level, which was varied tenfold, had no effect on excretion or absorption of zinc. The phytate content was probably low because meat furnished most of the protein and cereal grains were highly refined (Spencer et aZ., 1965). It was demonstrated that in vitro systems that at a pH of 6 there was a ratio of 5 zinc atoms per mole of inositol hexaphosphate in the precipitate formed from phytate and zinc. The addition of calcium.ions to make a Ca-Zn ratio of 100:1 increased the percentage of zinc precipi- tated from the in vitro system (Byrd and Matrone, 1965). Oberleas at al. (1966) observed that zinc phytate was least soluble at pH 6 and the addition of calcium decreased the solubility further. Therefore, when animals consume a large quantity of phytate in the diet, an insoluble and nonabsorbable phytate complex containing zinc is formed. Excess calcium accelerates the process not only by mass action.but also by elevation of the intestinal pH. l4 Chelating agents. Kratzer at al. (1959) were the first to report that ethylenediaminetetraacetic acid (EDTA) decreased the zinc requirement when soybean protein was used in the diet. One hundred parts per million EDTA in the diet was equivalent to about 8 ppm zinc, and was approxi- mately the amount of zinc bound by the soybean protein (Forbes and Yohe, 1960). Chelating agents with stability constants in the range of 13-17 were effective in increasing zinc availability (Vohra and Kratzer, 1964; Nielsen at aZ., 1966). Amino acids, such as cysteine, which has a constant of 18.2, will complex with zinc and make it more available. It appears that EDTA binds zinc in competition with phytate and this soluble complex is available to the intestinal mucosa (O'Dell, 1969). Role of Zinc in Metalloenzymes General. Keilin and Mann (1940) isolated carbonic anhydrase and demon- strated a specific biologic function for it which was dependent on the presence of zinc. The enzyme was thought to contain 0.331 zinc. Bovine pancreatic carboxypeptidase was the second enzyme whose function was found to be dependent on the presence of zinc (Vallee and Neurath, 1954). In the next decade more than 25 additional zinc-containing pro- teins were identified (Vallee and Wacker, 1968). Several dehydrogenases, aldolases, peptidases, phosphatases, an isomerase of yeast, a trans- phosphorylase, and a phospholipase contain zinc, attesting to its importance in carbohydrate, lipid and protein metabolism. A recent report (Parisi and Vallee, 1969) suggested that the transi- tion and 113 metals, which include zinc and iron, tend to be stably bound in complexes with organic molecules. This stability appeared to serve their biologic roles where a more fixed association of metals with 15 organic compounds was needed for characteristic biologic functions. The metal atoms of metalloproteins were so stably bound that they were not removed from the protein by isolation procedures. Metalloenzymes were catalytically active metalloproteins that contained stoichiometric amounts of firmly bound, biologically active metal atoms. Metallgthionein. The chemical properties of cadmium and zinc are so much alike that cadmium is always found in association with zinc ores, suggesting that cadmium might occur in proteins either associated with or in place of zinc. The discovery of cadmium in horse kidney led to the isolation of metallothionein from the renal cortex. Metallothionein is the only biological material known to contain cadmium. It contains 5.9% cadmium, 2.22 zinc and small quantities of capper and iron (Kfigi and Vallee, 1961). A similar protein has been isolated from the human kidney. Efforts to define its biological function have not been successful. Carboxypeptidase. Carboxypeptidase A was found to contain zinc (Vallee and Neurath, 1954), and its specific action was the hydrolysis of C- terminal amino acids of proteins, peptides and corresponding esters. The activity of carboxypeptidase A may be inhibited by a variety of complexing agents. Loss of enzymatic activity was directly proportional to the loss of zinc. In more recent work, zinc was not only essential for catalytic function of carboxypeptidase A, but also participated in the binding of peptide and ester substrates (Vallee at aZ., 1966). Alcohol dehydrogenase. The alcohol dehydrogenases from microorganisms and mammalian species are zinc metalloenzymes.' The enzyme catalyzes the oxidation of ethanol or the reduction of acetaldehyde using 16 diphosphopyridine nucleotide as a cofactor. When zinc was removed from the enzyme, activity ceased and the molecule dissociated into 4 subunits (Kagi and Vallee, 1960). Alkaline_phosphatase. Alkaline phosphatase of Escherichia coli, a zinc metalloenzyme, was first recognized in 1962 (Plocke et aZ., 1962). This enzyme was also present in many animal tissues and fluids. Decreased activity of this enzyme has been reported in zinc-deficient animals. Studies on the significant reduction of intestinal alkaline phosphatase activity in the zinc depleted rat was associated with alterations in enzyme behavior that was due to modification in enzyme structure or lowered concentration of the enzyme (Luecke at aZ., 1968). Influence of Infectious Diseases on Zinc Requirements Scrimshaw (1967), in a review publication, emphasized that children in underdeveloped countries of the world were retarded in physical growth and development due to malnutrition and its interaction with infection. Pollack (1968) was also of the Opinion that much of the mal- nutrition was secondary to the increased metabolic demands brought about by disease processes. Interference with absorption secondary to increased peristalsis from diarrhea led to further nutritional deprivation and accentuation of malnutrition. Although the importance of infectious agents has been reported as a factor which increased zinc requirements, there have been only a few studies of zinc metabolism in diseased man or animals. Sandstead at al. (1965) described the clinical findings in 39 Egyptian infants with kwashiorkor. Serum alkaline phosphatase activity was depressed, but increased to normal values following treatment with zinc. This response was similar to that observed in zinc-deficient animals after adequate 17 therapy. Plasma zinc levels were low, and this was suggestive of a zinc deficiency. The low levels of plasma zinc may be due to intestinal losses associated with diarrhea. Tissue and serum zinc values, significantly lower than normal, have been described in individuals with alcoholic liver disease (Halstead et aZ., 1968). Patients with cirrhosis of the liver excreted abnormally large amounts of zinc in the urine. Although the mechanism of this zincuria was unclear, the results were interpreted as indicative of a conditional zinc deficiency. Prasad (1966) reported that patients with hookworm infection and schistosomiasis had a chronic blood loss. Since red blood cells contain iron and zinc, these parasitic infestations were important contributing factors in the production of iron and zinc deficiencies. Sandstead et a1. (1967) emphasized that the daily zinc loss per patient may be as high as 200-25011g. from hookworm infections. Schistosomiasis induced metaplasia of the urinary bladder and intestinal epithelium which resulted in the growth of polyps from which blood losses occurred. Turk and Stephens (1967) found that cecal coccidiosis had a mild and variable influence upon absorption of radiozinc. This was in con- trast to an earlier study (Turk and Stephens, 1966) using Eimeria necatrmr, which infected the middle area of the intestine of chickens; and there was a marked effect on absorption of zinc. In severely affected birds, absorption rates exceeded those of unaffected birds on the first day after infection, decreased on the third day, decreased severely by the sixth day, and then returned to levels observed in unaf- fected birds. Mild intestinal inflammation increased absorption, whereas severe inflammation with hemorrhage decreased or stopped absorption. 18 In a recent report Pekarek and Beisel (1970) demonstrated that serum zinc concentrations decreased rapidly after an initial acute bacterial or viral infection. This alteration of zinc metabolism was mediated by an endogenous humoral factor released by neutrophils. In infected labora- tory animals, the zinc depressing factor appeared in serum.within 2 hours after infection or intoxication and in sufficient concentration to demon- strate its effect upon transfer to normal recipient animals. The endogen- ous zinc depressing mediator was a heat—labile, methanol—soluble, non- dialyzable, low molecular weight protein similar in many respects to endogenous pyrogen. However, the zinc depressing mediator had less species specificity than the endogenous mediator of fever. Transmissible Gastroenteritis_(TGE) in Pigg_ An infectious agent of known pathogenicity for the young pig would be required to study the influence of it on zinc metabolism. The fea- tures of the transmissible gastroenteritis (TGE) virus appeared suitable for use as an enteric pathogen. The virus acts primarily on the epi- thelium of the small intestine. The disease is readily reproduced and has a virulence which can be established to produce an enteric infection and not kill the pig before suitable metabolism trials can be conducted. This disease is common throughout the swine raising areas of the world. Doyle and Hutchings (1946), at Purdue University, first described a gastroenteritis in pigs that was characterized by vomition, diarrhea, rapid loss of weight and a high mortality in baby pigs. On postmortem examination, there was a large amount of fluid intestinal contents having a whitish, yellowish, or greenish color. In a subsequent report, Bay et al. (1949) reported that the disease was readily transmitted to young pigs by a ground suspension of intestine, kidney, spleen, liver, brain 19 or lung from infected pigs. The causative agent was inactivated by heat at 56 C. for 30 minutes, as did 0.52 phenol in filtrates incubated for 30 minutes at 37 C. Since filtrates would reproduce the disease, it was assumed that the causative agent was a virus. Additional extensive studies demonstrated that TGE was an acute viral diarrheal disease of pigs. The first clinical sign of infection was vomition, usually occurring approximately 24 hours after exposure. Vomition was followed within a few hours by profuse, watery diarrhea. Dehydration was rapid and pigs often lost 20 to 252 of body weight within 24 hours. Very young pigs usually died within 5 days (Haelterman, 1963; Haelterman and Pensaert, 1967; Cross and Bohl, 1969). Microscopic lesions in the stomach included necrosis and loss of mucosal epithelium, karyorrhexis and pyknosis, congestion, and infiltra- tion of neutrophils and monocytes into the mucosa. The villi of the small intestine were eroded or club shaped, and the epithelium was necrotic. In pigs that survived the infection, there was a regrowth of the atrophic villi approximately 6 days later with cessation of diarrhea (Bay at aZ., 1951). Maronpot and Whitehair (1967) reported that accomr panying these microscopic changes were histochemical changes characterized by decreased staining intensity of acid phosphatase, alkaline phospha- tase, adenosine triphosphatase, leucine aminopeptidase, succinic dehy- drogenase and malic dehydrogenase in the affected intestinal mucosa. In field cases of TGE, the gastroeintestinal tract may be severely inflamed, but inflammation was not found in experimentally infected antibody devoid pigs (Young at aZ., 1955). The small intestine of germfree baby pigs experimentally infected with TGE virus was charac- terized by shortened and fused villi. The effect on the epithelial cells was hydropic degeneration and some appeared squamous like and others 20 were cuboidal. There was no necrosis, hemorrhage or inflammation (Trapp at aZ., 1966). The lesions of TGE in many respects resemble those of sprue described for man (Maronpot and Whitehair, 1967). Hooper and Haelterman (1966) reported that growth of the TGE virus was limited primarily to the small intestine of the baby pig and was greater in the jejunum than the ileum. Intestinal tissue was shown con- sistently to contain the greatest concentration of virus. The titers were as high as 106 pig infective doses per milliliter of ground intestine in suspension. Young et a1. (1955) reported that the titer was 107 pig infective doses of virus per gm. of duodenal tissue in one pig within 24 hours after infection. Their results indicated that the virus did not replicate in the stomach and colon. The natural and most effective route of infection was by oral administration. A greater amount of virus was necessary to infect pigs parenterally than when it was given orally (Lee et aZ., 1954; Young at aZ., 1955). Reber and Whitehair (1955) experimentally infected 26-day-old pigs that were fed a 352 casein diet. The average amount of water retained by the pigs following infection was half that retained during the pre- infection period. Fecal water was increased 40 times above preinfection levels. Feed consumption was decreased approximately 40%. Cross lesions were a catarrhal gastroenteritis. Immunity studies indicated that there was a transfer of antibody, through the milk of sows that had recovered from either naturally or experimentally induced TGE virus.. Methods of immunity production, other than oral exposure by the virulent virus, were unsuccessful (Nelson, 1954; Bay at aZ., 1953). 21 Several cytopathic TGE isolates-have been grown in pig kidney cell cultures. Characterization of the virus was thus possible. Physical and chemical studies of tissue cultured virus indicated it possessed characteristics common to the Myxovirus group (Sheffy, 1965). Recently Pensaert at al. (1970) studied the effects of the Shizuoka (SH) strain of TGE virus at the 35th to 37th cell culture passage levels. These effects were determined in young pigs and primary pig kidney cells. In young pigs, the disease produced by this strain of virus was similar to that produced by the earlier isolated Purdue strain. There was a longer incubation period, lower mortality and less extensive atrophy of villi in those pigs infected with the SH strain. Summary The reports by Kernkamp and Ferrin in 1953 on the disease, parakera- tosis in swine, and by Tucker and Salmon in 1955 on the role of zinc in the disease, initiated interest and research on the importance of zinc in human and animal health. A large body of knowledge is currently available on many aspects of zinc nutrition, including requirements, sources, availability and various zinc-dependent enzymes. Only a meager amount of information is available that would explain the variability in the incidence and severity of the disease between herds and between swine in the same herd. Enteric infections are rather common in young swine and one of the more important pathogens is the transmissible gastroenteritis virus. The literature suggests this pathogen could be used experimentally to induce a specific enteric disturbance in swine and thus determine its influence on zinc requirements. This would provide information of prac- tical value in livestock production and, in addition, information of basic and biomedical interest. MATERIALS AND METHODS Experimental Animals The baby pig was used as the experimental animal in this study. This animal was chosen because it (1) was of a size that permitted col- lection of tissue material and excreta in quantities for analytical work, (2) was large enough to make clinical observations, (3) was available at minimum expense, (4) was uniformly susceptible to the TGE virus that produced specific enteric pathology, (5) was susceptible to zinc deficiency, and (6) was a desirable experimental animal for application of data to man and animals (Busted, 1965). Selection and Care of Experimental Animals Seven-day-old pigs were selected from sows that had no previous history of TGE infection. One initial experiment was discarded because of a secondary bacterial enteritis. The pigs used in this study were all crossbred pigs from the Michigan State University swine herd. In Experiment 1, 11 male and 1 female pigs were used. In Experi- ment 2, 10 male and 6 female pigs were used. They were fed the basal experimental ration (Table l) for an adjustment period of 2 or 3 days. They were then allotted at random to experimental groups and fed diets as indicated in Tables 2 and 3. They were fed the respective diets for a period of 2 or 3 weeks before exposure to the enteric infection. The infected and uninfected pigs were maintained in separate rooms and were cared for by different caretakers. Records of food consumption, 22 23 Table 1. Composition of basal purified diet1 Isolated soybean protein2 30. DL-methionine O o-cellulose3 3 Lard S Glucose monohydrate4 52 Mineral mixture* 6 Corn oil 1 2 0 Vitamin mixture** Total 10 . 2 *Mineral mixture: KCL Kl FGSO4'2H20 CuSO4 CoC03 MhSOa-HZO MgC03 NaHCO 3 CaHP04-2H20 C8CO3 12.5 Cerelose 13.498 Total 100.0002 H 03*! N O‘U‘lNCOOOOO I I OOOHI—‘l—‘NOO UN **Vitamin mixture: 22% Thiamine-mononitrate 3 Riboflavin 6 Nicotinamide 40 Calcium pantothenate' 30 Pyridoxine hydrochloride 2 Para aminobenzoic acid 13 Ascorbic acid 80 o-tocopheryl acetate 10 Inositol 130 Choline chloride 1300 see. Pteroylglutamic acid 260 Biotin 50 Cyancobalamin 100 2-methyl-1,4-naphthoquinone 40 Vitamin A palmitate 1500 Vitamin D2 12.5 1Mineral mixture was supplemented with 0.42 ZnSOa'HZO to contain 100 ppm zinc. In Experiment 2 the diet was supplemented with 0.1 ppm selenium from NaZSEO3. Soya Assay Protein, General Biochemicals, Chagrin Falls, Ohio. Solka Floc, Brown Co., Chicago, Ill. Cerelose, Corn Products Co., Argo, Ill. 24 urine and fecal excretion, weight changes, and clinical signs for zinc deficiency were maintained throughout the experiment. Also, the pigs were observed after inoculation for signs of TGE infection. ‘Qigt The composition of the low zinc basal diet is given in Table 1. This diet was used by Miller at al. (1968) in zinc deficiency studies at Michigan State University. In the first experiment, 6 pigs were fed the basal diet containing 12 ppm zinc and 6 the same diet supplemented with ZnSO4-H20 to make a diet containing 100 ppm. In Experiment 2 the basal diet was supplemented with 0.1 ppm selenium because myopathy was observed in some of the pigs in Experiment 1. The diets and treatment for each group of pigs are given in Tables 2 and 3. The pigs were fed and watered ad Zibitum except during the 3-day balance trials. The drinking water contained no measurable zinc. Pathogen The transmissible gastroenteritis (TGE) virus was used as the patho- genic agent.* Each pig to be infected was given an oral dose of a phos- phate buffered saline suspension which contained 105 pig infective doses per ml. The virus was obtained by homogenizing infected pig intestine. The homogenate was centrifuged and the supernatant fluid filtered. The filtrate was titered in specific-pathogen-free pigs. Hematology At the conclusion of each balance trial, blood samples were taken from.the anterior vena cave of all pigs for cellular determinations and *Courtesy Dr. E. 0. Haelterman, Purdue University, Lafayette, Indiana. 25 serum analyses. Two milliliters of blood were placed into the disodium salt of ethylenediaminetetraacetic acid (EDTA) to prevent coagulation. Hemoglobin was determined by the cyanmethemoglobin method. Packed cell volumes were determined using microhematocrit tubes. Differential white blood cell counts were made from Wright's-stained blood smears. The remainder of blood (6-8 ml.) was placed in acid-washed centrifuge tubes and allowed to clot. This was centrifuged at 2500 x gravity and the serum.was withdrawn, placed in aciddwashed vials and frozen until zinc determinations were made. Histgpathology' At the end of the treatment period, pigs were necropsied at selected intervals in Experiment 1 (Table 2) or at the termination of Experiment 2. Organs, or portions of organs, from each pig were collected and fixed either in Zenker's solution, Bouin's fluid, and/or in 10% formalin solu- tion containing sodium acetate as a buffer. Bones were decalcified in decalcifying solution.* All tissues were infiltrated and embedded in a paraffin-base mediumI* and sectioned at 61:. Sections were stained with hematoxylin and eosin and, in selected cases, special stains were used to aid in differentiation. The staining procedures followed were accord- ing to the Armed Forces Institute of Pathology Manual of'Histologio Staining Methods (1968). *Cal-Ex, Fisher Scientific Co., Chemical Manufacturing Division, Fair Lawn, N.J. **Paraplast, Sherwood Medical Industries, Inc., St. Louis, Mo. 26 Zinc Analysis Serum samples from each pig were diluted 1:7 with deionized dis- tilled water. Zinc standards used had a zinc concentration of 50, 100, and 150}1g./100 m1. Samples were analyzed in duplicate. Urine samples from each pig were diluted 1:2 with deionized distilled water and were analyzed for zinc concentration without wet ash digestion. Urine samples were analyzed in duplicate. Zinc standards for urine analysis contained 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5 ppm zinc. A 0.2 gm. fecal aliquot was weighed from pigs fed the 100 ppm zinc diet and a 0.5 gm. aliquot from pigs fed the 12 ppm zinc diet. The fecal samples were added to Phillips flasks in duplicate and predigested with 40 m1. of nitric acid and heated until approximately 1 m1. of fluid remained. Final digestion was made by adding 7 ml. of 70% perchloric acid and heating until 1 m1. remained. This was diluted with 100 ml. of deionized distilled water and zinc levels determdned. Zinc standards for fecal analysis contained 0.1, 0.2, 0.5, 1.0, 1.5 and 2.0 ppm zinc. All serum, urine and fecal zinc concentrations were determined with a Jarrell-Ash Model 82-516 atomic absorption spectrophotometer using a single electrode zinc cathode tube with absorption measured at 2133.5 angstroms (Miller at aZ., 1968). The zinc analyses were conducted in the Animal Nutrition Laboratory under the guidance of Mrs. Betty Schoepke and Dr. D. E. Ullrey. Analysis of Data- Data were examined by analysis of variance. The statistical sig- nificance of treatment differences was determined by the F test of Snedecor (1956) or the multiple range test of Duncan (1955). 27 Experiments Experiment 1. Twelve 7-day-old baby pigs from 3 litters were selected. Six pigs were fed the diet which contained 12 ppm zinc and 6 fed the diet which contained 100 ppm zinc. The diets were fed mixed with water to form a slurry. They were continued on these diets for 2 weeks, at which time 3 from each group were transferred to an isolation room and inoculated orally with a 1 ml. suspension of the TGE virus as outlined (Table 2). At this time the pigs were placed in individual stainless steel metabolism cages for a 3-day period of fecal and urinary collec— tions with a constant level of food intake. Pigs were moved from the metabolism cages to the individual rearing cage for each feeding, and their snouts were wiped free of feed before they were returned to the metabolism cages. Each feeding was completed in approximately 10 minutes and this procedure successfully prevented contamination of excreta. Urine was collected in 3 N HCl to prevent bacterial contamination. The feces were collected daily and dried at 50 C. Total urine volume for each pig was measured and a sample stored in the refrigerator in acid- washed plastic bottles. Composite dried feces from each pig were weighed, ground in a Wiley mill and stored in air-tight containers. Urine and feces were later analyzed for zinc levels. Experiment 2. Sixteen 7-day-old crossbred baby pigs from 2 litters were selected. The pigs were reared in stainless steel cages with stain- less steel wire-mesh bottoms. All pigs were fed the basal diet with added selenium, given in Table 1 during an initial 4-day adjustment period. They were then assigned at random to experimental groups, as given in Table 3, balancing for initial weight and sex. Environmental temperature was kept at 80 to 85 F. 27 28 Table 2. Experimental design - Experiment 1 A; Diet and Treatment Pig Numbers Basal diet, 12 ppm zinc 2(37),9(30),10(21) Basal diet, 12 ppm zinc, plus TGE virus S(51),6(l7),7(45) Basal diet supplemented, 100 ppm zinc l(23),5(45),6(45) Basal diet supplemented, 100 ppm zinc plus TGE virus 8(30),ll(51),12(23) ( ) = Days on experiment. 29 Four pigs were fed a diet which contained 12 ppm zinc; 4 pigs fed the 12 ppm zinc diet to which had been added 50 mg./kg. oxytetracycline HC1* and 400,000 units/kg. mycostatin;** 4 pigs fed a diet which contained 100 ppm zinc; and 4 pigs fed the 100 ppm zinc diet with the same anti- microbial agents added. All pigs received 0.1 ppm selenium added to the diets. Animals in all groups were individually fed ad Zibitum. Food intake was recorded daily and weights of pigs were taken weekly. The pigs remained on these diets for 3 weeks, at which time 2 animals from each group were placed in individual stainless steel metabol— ism cages and adapted to feeding 3 times daily for 3 days. The diets were fed mixed with water limited to an amount consumed in 10 minutes. At the end of this 3-day adjustment period, fecal and urine collections were made for 3 days with a constant level of food intake. The same pigs from these 4 groups were transferred to an isolation room and inoculated orally with 5 ml. of a suspension of TGE virus containing 105 pig infect- ive doses per ml. (Table 3). Feces and urine were collected and stored as described in Experiment 1 for later analyses. *Terramycin, Chas. P. Pfizer and Co., Inc., New York. **Nystatin, Squibb and Sons, E. R., 745 Fifth Ave., New York. 30 Table 3. Experimental design - Experiment 2' Diet and Treatment Basal diet, 12 ppm zinc Basal diet, 12 ppm zinc, plus TGE virus Basal diet, 12 ppm zinc, plus antibacterial1 and antifungal agent Basal diet, 12 ppm zinc, plus antibacterial1 and antifungal2 agent, plus TGE virus Supplemented diet, 100 ppm zinc Supplemented diet, 100 ppm zinc, plus TGE virus Supplemented diet, 100 ppm zinc, plus anti- bacterial1 and antifungal2 agent Supplemented diet, 100 ppm zinc, plus anti- bacterial1 and antifungal agent, plus TGE virus 150 mg. oxytetracycline/kg. diet. 2400,000 units mycostatin/kg. diet. Pig Numbers 104-16, 103-3 104-14, 104-7 104-4, 104-12 104-5, 104-3 104-1, 103-2 104-11, 104-6 104-8, 103-1 104-9, 104-2 RESULTS Experiment 1 Clinical signs. All of the pigs fed the 12 ppm zinc diet gained weight during the first 2 weeks of the experiment, but at a slower rate than the pigs fed 100 ppm zinc diet. Food consumption decreased after the first week of the experiment in the pigs fed the zinc-deficient diet. The pigs fed 12 ppm zinc and infected with TGE virus developed a severe diarrhea in 24 to 36 hours. One pig became progressively weak, develOped a diffuse, yellowish, fetid diarrhea, complete anorexia, severe dehydra- tion, and died 4 days after exposure. The 3 pigs fed 100 ppm zinc and infected developed slight to moderate diarrhea but continued to have an appetite. There was no vomition in any of the infected pigs. Four days following exposure of the 6 pigs, the noninfected pigs also developed clinical signs of TGE. The balance trial had been completed at this time and therefore this did not influence the results of the zinc retention studies. One pig fed the zinc-deficient diet developed complete anorexia, dehydration, and a fetid, yellowish diarrhea which contained partially digested food. This pig was killed and necropsied. Growth stoPped in all of the pigs fed the zinc-deficient diet during the third week of the trial, and skin lesions appeared on the feet, ven- tral abdomen, around the eyes, and on the medial side of the thighs. There was excessive sebaceous secretion followed by crusting. The skin adjacent to the crusts was erythematous and wrinkled. It was dry, scaly, and the hair coat was thin (Figure l). 31 32 Figure 1. Figs fed 100 ppm (1) and 12 ppm (2) zinc diets. 33 Zinc balance and hematolggy, Results of zinc balance studies and hema- tology during Experiment 1 are summarized in Table 4. Food consumption was decreased in pigs fed 12 ppm dietary zinc and daily gain was less than those fed 100 ppm zinc. The TGE infection had a marked effect on growth resulting in a weight loss in both infected groups. The decreased food consumption and weight loss resulted in a negative utilization of food (gain/food) which was primarily due to the infection. Daily zinc retention was significantly decreased by low zinc intake and infection. These pigs had a daily zinc intake of 1.22 mg. and were excreting 1.00 mg. Thus they were retaining only 0.22 mg. In the pigs fed the low level of zinc and infected there was more excretion than intake of zinc, resulting in.a negative zinc balance. The pigs fed adequate zinc and infected were retaining significantly less zinc than the noninfected group. Hemoglobin and hematocrit values were decreased in the infected pigs, but not influenced by zinc nutrition. In infected pigs fed the deficient diet there were significantly decreased total leukocytes. In these pigs there was a reduction in segmented neutrOphil count and an elevated lymphocyte count. Serum zinc was significantly decreased by the low zinc intake and infection. TGE infection lowered the serum zinc of pigs fed 100 ppm zinc but not of pigs fed 12 ppm. Gross lesions. Infected pigs that died had empty stomachs that were moderately hyperemic with hemorrhagic erosions of the fundic mucosa. The small intestine was distended with gas and a yellowish fluid. The mucosa and mesenteric vessels were moderately to severely congested. The mesenteric lymph nodes were hemorrhagic and edematous. In 1 pig fed the zinc-deficient diet there was bilateral hemorrhage and necrosis of the testes. All pigs necropsied 10 days or longer after virus inoculation had no gross lesions of TGE. 34 MHO. v mfifi IHOI “Ame. v my mosam> osu umooH mono Monmouw haucmofiuaowfimn v moo ooame unmoa one» noumsuw hauoooamwawwmm “Ame. v my mooao> manna nosed omen uuuomuw maunmowmaowfimo mooamoqwficwam ozN "HO. V mflfl «Ame. v mv momma man no uouuu pudenduma mo.v .m.n mo.v a.HH o.ae uo.soa o.ao o.ma .Ha ooa\.wi_.au aspen no.v .m.a .m.a o.“ e.- o.o~ mo.Hm n.0H mausooeeasa mo.v .m.c .m.G N.h mo.mh mm.mo o.m¢ mo.mu mflflnmouudmn vmudflamom mo.v Ho.v .m.fi m.H mo.ma ©.MH m.h cow-MN MEE\mOH ammuhuoxafld m.a Ho.v .m.n m.H o.o~ snm.mm o.a~ nao.mm N .uauoouoaom .m.a Ho.v .m.a ~.o a.» ~.ma ~.a H.ma .Ha ooa\.am .aaaonwoaom momhamqm oooam o.oe o.ma c.5e- o.mH u .aoaucmumu onus no.v Ho.v Ho.v ¢.o ano.o oom.m 0.0: «.0 .wa .soaucouou anew .m.a .m.n .m.a H.o m.o m.o m.o m.o .wa .onHu madman: .m.: .m.: Ho.v s.o na.m m.m o.a ~.o .ma .oaau Hague o.HH n.ma m.H ~.H .ms .mxuuan onus mundane scan m.o- m.o a.ou m.o voou\as«u o.oHH o.mmH o.mOH o.ooa .am .oaeuas soon suing .m.e Ho.v .s.a o.s~ o.m~- o.ma o.osu mo.Hm .aw .eaum Asian .WUW o0.” Nomog Noo Nam 90.” he" NoN owx aunwfiM3NHQHUWQH nu- nu- -u- m m m m a a so oz oocmauowuum wok muH UGHN I. Ema .umHv ca OGAN x seam Hmm+ mmw con mwa NH eonuuuuaH moomowwfimwmw. H unuawuumxm .Hoauu.ooamaon mauln m mswuov doauoomow moy one uses assuage he voodooawoa ms momhacnm pooao one mundane omen .ooomauowuom .s manna 35 All pigs fed the low zinc diet had depletion of body fat and serous atrophy of fat in the coronary groove of the heart and renal pelvis. There was extreme atrophy of the thymus (Figure 2). In con— trast, in pigs fed adequate zinc the thymus gland was prominent along the common carotid artery from the parotid gland to the anterior thorax (Figure 3). In all of the pigs the dorsal surface of the tongue was coated with a white material. The esophageal mucosa appeared thickened and dull white. The cardia of the stomach was roughened and had raised, yellowish areas . Histopathologic changes. Significant changes were observed in the stomach, small intestine, tongue, esOphagus, thymus, skeletal muscle, pancreas, and tastes. Other tissues, including brain, pituitary gland, lung, liver, adrenal gland, and spleen, were examined; but changes in these tissues were minimal or absent. Samples of skin were taken by Dr. Frank Voelker for a separate study evaluating in detail the skin changes due to zinc deficiency alone and in combination with stress factors. Stomach. The microsc0pic changes in the stomach of pigs fed the zinc-deficient diet and exposed to infection, that were necropsied 4 days after inoculation, varied from congestion to hemorrhage and necrosis of the epithelium. There was karyorrhexis and pyknosis of epithelial cells. Foci of hemorrhage and necrosis began at the surface of.the epi- thelium and in areas extended to the muscularis mucosa. Polymorphonuclear leukocytes were numerous in the regions with extensive necrosis in the mucosa. There was inflammatory edema in the mucosa and submucosa. The blood vessels of the submucosa were engorged with blood and vessels con- tained thrombi (Figures 4 and 5). These changes were not observed in non- infected pigs or in pigs infected and fed zinc-adequate diets. 36 Figure 2. Atrophic thymus from a pig fed the 12 ppm zinc diet for 3 weeks (arrow). Figure 3. Normal thymus from a pig fed the 100 ppm zinc diet for 3 weeks (arrow). 37 Figure 4. Hemorrhage and necrosis of gastric mucosa from in— fected pig fed the zinc-deficient diet (1). H S E stain. x 75. Figure 5. Submucosa of stomach from infected pig showing a focus of neutrophils (left arrow) and a thrombus in a blood vessel (right arrow). H & E stain. x 187.5. 38 Small intestine. The lesions were limited to infected pigs. The villi of the jejunumand ileum were shortened, fused and blunt (Figure 6). The epithelium at the tips of the villi had a morphologic alteration from simple columnar to cuboidal. The fusion of 2 or more villi was observed in the more severely affected areas of the small intestine. The brush border was shortened or absent (Figure 7). The blood vessels of the mucosa and submucosa were congested. There were small accumula- tions of neutrophils at the tips of many villi. Tppgpg, esophagus, and cardia of the stomach. The white coating of the tongue and the yellowish pseudomembrane in the distal esOphagus observed grossly in all pigs was characterized by degeneration, hyper- keratosis, and parakeratosis of the epithelium and had a profuse fungal and bacterial growth. The mycotic organism appeared as hyphae and budding yeast forms interspersed between keratotic layers of epithelium. When stained with Gomori's methenamine silver nitrate, they were morpho- logically similar to Candida albicans. The bacteria in the layers of keratotic epithelium were Gram—positive mediumrsized bacilli and cocci (Figures 8, 9, 10 and 11). Infiltrations of neutrophils were observed in the squamous epithelium of the cardia of the stomach. The stratum corneum of the esophagus and tongue of pigs fed the zinc—deficient diet was thickened by partially keratinized cells. Cell borders were discernible and cell size was irregular. The cytoplasm was homogeneous and the cells had persistent pyknotic nuclei. The cells in the stratum spinosum were increased in number, contained cytoplasmic vacuoles, with pyknotic nuclei. Fungal hyphae and bacteria were in the thickened epithelial layers (Figures 12 and 13). In contrast, pigs fed 100 ppm zinc had esophageal epithelium which consisted of cornified cells devoid of nuclei, and there were no vacuoles in the cells of the stratum spinosum. 39 Figure 6. Ileum from pig fed the zinc—deficient diet and infected with TGE virus. Note shortened, fused and blunted villi. H & E stain. x 75. Figure 7. Mucosa of the ileum from TGE-infected pig fed the zinc- deficient diet. Note the cuboidal appearance of the epithelium and loss of the striated border (arrow). H & E stain. x 187.5. Ill All]: J‘l 40 Figure 8. Squamous epithelium on the dorsal surface of the tongue. Dark staining material in the cornified interpapillary epi- thelium is fungal and bacterial growth (arrow). H & E stain. x 75. Figure 9. Cornified epithelium from dorsal surface of the tongue from a pig fed the zinc-adequate diet for 51 days. Note larger dark- staining yeast forms of Candida aZbioans (arrow) and numerous Gram- positive bacteria (arrow). Gram stain. x 750. 41 Figure 10. Distal esophagus from a pig fed 100 ppm zinc diet with keratotic layers of epithelium containing fungi (arrows). Gomori's methenamine silver stain. x 75. Figure 11. High magnification of fungi in cornified epithelium of distal esophagus. Note budding yeast forms and hyphae. Gomori's methenamine silver stain. x 750. 42 Figure 12. Parakeratosis of zinc deficiency in the esophagus of a pig that had been fed the 12 ppm zinc diet for 21 days. Note the pyknotic nuclei (arrow). H & E stain. x 187.5. Figure 13. Parakeratosis of the dorsal surface of the tongue from a pig that had been on the low zinc diet for 37 days (arrow). B & E, stain. x 187.5. 43 Thygus. Alterations were a depletion of thymocytes in the cortex of the thymus of pigs fed zinc—deficient diets (Figure 14). The remain- ing cells were less closely packed in the cortex, and there was a decrease in size of the medulla. Reticular cells and fibers appeared more prominent in the medulla. In contrast, pigs fed zinc-adequate diets had thymic tissue with a deeply staining cortex and a less intensely staining medulla. The cortex was crowded with thymocytes in a background of reticular cells and fibers. The medulla contained fewer thymocytes than the surrounding cortex (Figure 15). Skeletal muscle. Typical vitamin E-selenium deficiency lesions were observed in 2 of the 3 infected pigs fed the-zinc-deficient diet. These were characterized by swelling, fragmentation, loss of cross stria- tions, and increased numbers of mononuclear cells in affected fibers. These latter cells were probably phagocytes of the reticuloendothelial system which had infiltrated in response to the degeneration of the fibers. The most severely affected fibers had an increased affinity for eosin (Figures l6, l7 and 18). Skeletal muscle from pigs fed 100 ppm and infected with a TGE virus appeared normal (Figure 19). Testes. Bilateral necrosis and hemorrhage were observed in 2 infected pigs fed the 12 ppm zinc diet. The spermatogenic and Sertoli cells in seminiferous tubules were pyknotic and were exfoliated into the 1umens.of tubules. There was hemorrhage into the interstitial tissue and the interstitial cells were degenerated. The nuclei were pyknotic and the cytoplasm was excessively eosinophilic (Figures 20 and 21). Pancreas. The pyramid-shaped secretory cells in acini in all pigs fed the zinc-deficient diet contained decreased numbers or absence of 44 -. . . I \sfizfli“; 1;‘ me» 1‘3 ‘1‘ Figure 14. Lesions of zinc deficiency in the thymus after 51 days on the zinc-deficient diet. Cortex reduced in size (arrow). H & E stain. x 75. ~«.-t’ .r , . (‘ _ Figure 15. Portion of thymus of pig fed the zinc-adequate diet. Note wide cortex containing crowded thymocytes (arrow). H & E stain. x 75. 45 Figure 16. Nutritional muscular dystrophy in a pig fed the low level of zinc and infected with TGE virus. H 6 E stain. x 75. Figure 17. Fragmentation (1), loss of striation (2) and mineral deposits (3) in skeletal muscle from an infected pig fed the zinc- deficient diet. H & E stain. x 750. 46 Figure 18. Skeletal muscle fibers partially replaced by cellular exudate. Mononuclear cells (arrow) predominate in the exudate, and probably are macrOphages or related cells of reticuloendothelial origin. H & E stain. x 750. Figure 19. Normal skeletal muscle from infected pig fed the zinc-adequate diet. H 8 E stain. x 375. Figure 20. Portion of testis from infected pig fed the 12 ppm zinc diet. Normal juvenile seminiferous tubule (l), degenerative seminiferous tubules with pyknosis of germinal epithelium and Sertoli cells (arrows). H & E stain. x 375. Figure 21. Necrosis of germinal epithelium (l) and interstitial cells (2) in the testis of an infected pig fed 12 ppm zinc. H & E stain. x 750. 48 strongly refractive, acidOphilic zymogen granules. In contrast, pigs fed the zinc-adequate diet had pancreatic acinar cells containing numer- ous zymogen granules. Prominent changes.were not observed in any of the islands of Langerhans. Experiment 2 Clinical siggg, The first signs of zinc deficiency were decreased weight gain and decreased food consumption. Results of growth and food utiliza- tion are given in Table 5. The average daily weight gains in the pigs fed the zinc-deficient diet were approximately half that of the pigs fed the zinc-adequate diet during the first 2 weeks. During the third week, weight gains essentially stopped in the pigs fed 12 ppm.zinc, while pigs fed 100 ppm zinc gained weight at a normal rate. Weight gains were slightly better in the pigs fed the antimicrobial drugs. Skin lesions, as described in Experiment 1, appeared in pigs on the low level of zinc at the end of the second week. Of the 8 pigs that were orally inoculated with TGE virus, 2 fed the zinc-deficient diet and 3 fed the zinc-adequate diet developed signs of infection. One pig fed the zinc-deficient diet died. Pigs fed the adequate diet continued to eat well throughout the infection. One pig that was fed the zinc-deficient diet plus the antimicrobial drugs developed a cough. On examination of the oral cavity a diffuse white coating on the dorsal surface of the tongue was observed resembling the fungus infection that was seen in the pigs of Experiment 1.- Zinc balance and hematology, The results of the zinc balance studies and blood analyses are summarized in Table 6. 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