—_—w-V_——Iv—w- PATHOLGGY OF THE SKIN IN EXPERIMENTAL ZINC DEFICIENCY IN SWINE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY FRANK A. VOELKER 1970 . ..‘:!r"-T.‘. " -.«. 4‘ -1 - z' ’ "‘ *1; ’9". . r a u~r ‘ f.“ ‘ . .‘ V- ‘ _ - - a '1' " . 1:11.) K .l‘i I: .L fl,'° 1-,. ‘7' l .r' F!" ‘ -~ W“ ' Avitwu .zoua‘ L1 LL; we . {n.2- .. .,‘ . {1“}. L ‘$‘ IV in 4- 01:: r ,4 I .~.p..,—.-....~- —'m‘-W S -"..'I.I_,&‘ &" 44 3f. 1" magma av III!“ & SUNS' 800K 3mm " It. . LIBRARY E. ‘ 2:222:12! air-I. ._. I ABSTRACT PATHOLOGY OF THE SKIN IN EXPERIMENTAL ZINC DEFICIENCY IN SWINE By Frank A. Voelker A total of 28 baby pigs were utilized in 2 experiments to determine the influence of infection and antibiotics on zinc metabolism. During the course of these experiments skin was made available for histopatho- logic evaluation. The morphology of the skin from different anatomical areas was evaluated histopathologically in the beginning, end, and during the course of the experiments.- In the first experiment healing of wounds was studied and, in the second, the effects of skin abrasion and of plucking hair_onfthe development of dermatitis were investigated. The hairs from the plucked areas were classified as to follicle type to determine growth activity. The skin lesions due to a deficiency of zinc in the pig were parakeratosis, hyperkeratosis, acanthosis and a dermal inflammation. The healing of open wounds and of abraded skin was delayed in pigs fed zinc-deficient diets. At the onset of the experiment there was a predominance of anagen hair follicles on the lateral thorax and of telogen hair follicles on the volar surface of the foot. By the end of the experiment the pre- dominance of the respective follicle types had diminished in both areas, except on the feet of the deficient pigs, in which there was a derma- titis. Plucking of hairs caused an increase in the percentage of Frank A. Voelker anagen follicles on the feet of pigs fed both the adequate and zinc- deficient diets; it did not cause any change in severity of the dermatitis. PATHOLOGY OF THE SKIN IN EXPERIMENTAL ZINC DEFICIENCY IN SWINE By egffi Frank AQVVoelker A THESIS Submitted to Michigan State University in partial.fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1970 ACKNOWLEDGEMENTS The author wishes to express sincere gratitude to Dr. C. K. Whitehair of the Department of Pathology, for advice and guidance during this research. Appreciation is also conveyed to Dr. D. L. Whitenack, of the Department of Pathology, and to Dr. E. R. Miller, of the Department of Animal Husbandry, for allowing the simultaneous. use of animals and research space, and for continual cheerful sympa- thetic aid in difficult portions of the research. Sincere thanks are also extended to Dr. R. F. Langham and to Dr. S. D. Sleight for teach- ing the pathology necessary in interpreting research of this type, and of the Department of Pathology for its support during the tenure of. this training. The author is deeply appreciative of the opportunity and support given him by the United States Air Force in attending Michigan State University while a member of that organization. Finally, the author wishes to express deep appreciation to his wife, Jan, for her patience and cooperation. ii TABLE OF CONTENTS INTRODUCTION 0 O O O O O O O O O O OBJECTIVES O O O O O O O O O O O 0 REVIEW OF LITERATURE. . . . . . Parakeratosis in Swine . . The Dermatosis in Other Animals. Physiopathology of Zinc in the Skin. Summary. . . . . . . . . . MATERIALS AND METHODS . .1. . . General Experimental Design. Animals. . . . . . . . . . Housing and Care . . . . . Experimental Procedure for Experimental Procedure for Histopathologic Technique. RESULTS . . .1. . . . . . . . . . Gross Pathology. . . . . . Pathology. . . . . . . . . DISCUSSION. . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . BIBLIOGRAPHY. . . . . . . . . . . VITA O O O O O O O O O O O O O O 0 Experiment 1. Experiment 2. iii Page 12 13 13 14 14 1s 16 18 20 20 22' 37 ‘ 40 41 45 Table LIST OF TABLES Page Summary of hair activity of the foot and lateral thorax of pigs at different ages and the effect of zinc deficiency, antibiotics and transmissible gastroenteritis virus (TGE) . s . . . . . . . . . . . . 35 Summary of the effect of plucking, zinc deficiency, antibiotics and transmissible gastroenteritis virus (TGE) on hair activity of the feet of pigs. . . . . . . 36 iv Figure 10 LIST OF FIGURES Page Gross appearance of the skin lesions of the ventral aspect of a pig fed a zinc-deficient diet . . . . . . . 24 Telogen (a), pigmented anagen (b), and nonpigmented anagen (c) follicles of plucked hairs of a pig. . . . . 24 Microscopic appearance of abdominal skin of a pig fed a zinc-adequate diet. . . . . . . . . . . . . . . . 25 Microscopic appearance of abdominal skin of a pig fed a zinc-deficient diet.‘ Parakeratosis and hyper- keratosis of stratum corneum (a); elongated and anastomosed rete pegs (b); and hyperplastic dermal capillaries (c) . . .-. . . . . . . . . . . . . . . . . 25 Apocrine sweat glands of abdominal skin of a pig fed a zinc-adequate diet. Duct (a) and coiled secretory tubule (b) O O O O O O I O O O O O O O ‘ O O O O 28 Apocrine sweat gland of abdominal skin of a pig fed a zinc-deficient diet. Thickened duct (a), and glandular epithelium (b); neutrophils in the glandu- lar lumen (c) . . . . . . . . . . . . . . . . .p. . . . 28 Telogen hair follicle of abdominal skin of a pig fed a zinc-adequate diet. . . . . . . ... . . . . . . . 30 Telogen hair follicle in an area on the forefoot affected with dermatitis of a pig fed a zinc- deficient diet. Note hyperkeratosis and parakera- tosis within the follicle . . . . . . . .'. . . . . . . 30 Epithelium at the margin of a lS-day-old wound on the lateral thorax of a pig fed a zinc-adequate diet. . . . 32 Epithelium at the margin of a 13-day-old wound on the lateral thorax of a pig fed a zinc-deficient diet. 0 O O O O O O O O O O O O O I O Q C C O O O O O O 32 INTRODUCTION Skin pathology has been a specific interest of the author for a number of years. The opportunity became available in the winter of 1970 to more fully characterize the dermatopathology of zinc deficiency in young pigs. The experiments were designed primarily for other objectives, namely, to study the influence of an enteric infection on zinc require- ments. However, the skin was available for a separate dermatopatho- logic study. While the design might have had shortcomings, it did supply material at a minimum expense. There is clinical evidence for the presence of zinc deficiency in many domestic animals, as well as man. In swine a severe dermatosis has been the important clinical sign and lesion. While much research has been devoted to the clinical, nutritional and metabolic aspects of this disease, only a meager amount of information is available on the nature of the skin lesions as produced by feeding specific amounts of zinc. In addition to establishing a histopathologic description of experimental zinc deficiency, research was also conducted to obtain information of the relationships of hair growth, wound healing, and mechanical abrasion to the skin condition during zinc deficiency. OBJECTIVES The objectives of this research were: 1. To study the gross and microsc0pic pathology of the skin in zinc deficiency in young swine. The influence of infection and anti- biotics on the deficiency were also evaluated. 2. To characterize possible pathognomonic lesions of the derma- tosis in both affected and unaffected skin of pigs fed zinc-deficient diets.- 3. To study the pathology of cutaneous wound healing in the zinc- deficient state. 4. To evaluate the relation of mechanical abrasion to the patho- genesis of the dermatosis. ‘ 5. To investigate the relationship of hair growth to the dermatosis° a. To establish an estimate of the activity of hair follicles in the pig, correlated with age and body regions. b. To discover if inducing the formation of anagen hair fol- licles by plucking hair would have an effect on the severity of the dermatosis. REVIEW OF LITERATURE The presence of zinc in living organisms has been recognized for almost a century, and zinc deficiencies have been produced experimentally or observed clinically in many species of animals throughout the world. Kernkamp and Ferrin, in 1953, described an enigmatic dermatosis of pigs which was of serious economic impact to farmers. Later Tucker and Salmon (1956) confirmed that the disease was caused primarily by a zinc deficiency. More recently, a syndrome attributed to a deficiency of. zinc has been found in man (Prasad, 1967). The presence of skin lesions has been a prominent manifestation of the syndrome in almost all species in which the disease occurs. This review is devoted primarily to aspects of zinc deficiency as they pertain to the skin of the pig and a number of other animals. Parakeratosis in Swine Gross pathology, Kernkamp and Ferrin (1953), at the Minnesota Experi- ment Station, originally reported "parakeratosis" as a specific disease of unknown etiology in young pigs 8 to 19 weeks of age, characterized as~a benign dermatosis with hard, dry, crpsted proliferations of the superficial epidermis. The first areas of the body to become involved were usually the areas where hair was sparse, such as the underside of the-abdomen and flank, or distally on the legs in the regions of the‘ pasterns, fetlock, and the hock. 4 In the earliest stages of the dermatitis, circumscribed erythema- tous areas were present which developed into elevated papule-like lesions 3 to 5 mm. in diameter. At the end of this transient period, the skin became scaly and encrusted (Kernkamp et aZ., 1955). On skin with short hair, the crusts were more compact and more granular in appearance, but if the hair were longer, it usually became entangled or matted in the crusts. The crusts, often 5 to 7 mm. thick, usually were not firmly adherent to the underlying skin. The thicker crusts were separated by fissures or cracks which contained a moist and sticky dark-colored mixture of exudate and debris (Kernkamp and Ferrin, 1953). Histopatholggy, The histopathology of dermatosis due to a zinc deficiency in pigs has not been extensively described in the literature. In the earliest description (Kernkamp and Ferrin, 1953) of the parakeratosis syndrome, the crusted proliferations observed grossly were composed of thickened layers of cornified epithelium. These layers of hyperkera— totic stratum corneum containing retained nuclei, either diffusely dis- tributed or piled together in dense aggregates, were termed "parakera- totic." At the bases of the fissures in the skin, collections of granu- locytes and amorphous material were present. .In later studies, Minnesota workers (Kernkamp et aZ., 1955) mentioned the presence of acanthosis and an increase in number and length of rete pegs. The histopathologic changes in the skin of swine with experimental zinc_deficiency (Anderson et aZ., 1967) were comparable to those described in previous studies of the clinical disease. They noted additionally that mitotic figures were numerous in the Malpighian layer, and the parakeratotic response in the stratum corneum, which tended to 5 be layered with no retained nuclei in the interposed layers, was especially concentrated around the orifices of hair follicles. The stratum granulosum was either diminished or absent in affected skin. In the dermis, capillaries were increased in number and more tortuous. The dermal papillae were increased in height, often extending above the basal margin of the stratum corneum in adjacent areas. Although a deficiency of zinc was not definitely established, Itikawa et al. (1964) have verified the presence of parakeratosis or a similar dermatologic condition in Japanese swine. They noted the disappearance of keratohyaline granules and the presence of acidophilic inclusions in cells of the stratum granulosum. Dyskeratotic cells or corpus ronds were present above dermal papillae. Rete pegs in affected skin tended to be acanthotic and elongated to the extent that reticular bifurcation of these structures occurred. There was degenerative atrophy of the sebaceous glands. Histochemically Anderson et al. (1967) detected a significant increase in both sulfated and unsulfated mucopolysaccharides in the dermis. There was more sulfhydryl and less disulfide, generally, in all layers of parakeratotic skin when compared to normal skin. Increased glycogen in the capillary endothelium of the dermal papillae and cells of the Malpighian layer was demonstrated by the periodic acid-Schiff (PAS) technic. The Dermatosis in Other Animals Rats. In rats, Follis et al. (1941) found experimentally that skin lesions began with a sparseness of hair, and then definite areas of alopecia occurred in the mid-dorsal area and the shoulders. Graying of hairs in dark-colored animals occurred, and occasionally a slight 6 reddening and encrustation about the eyes, nose and feet was observed (Stirn et al., 1935). Skin lesions may be minimal or absent in rats unless the deficiency is severe and of long duration (Hove et aZ., 1938). Alopecia developed sooner in more rapidly growing animals (Todd et al., 1934). Microscopically, Follis et a2. (1941) observed initial lesions in weanling rats after a deficiency of 33 days in the form of hyperkeratini- zation and thickening of the epidermis. The epidermis thickened from a normal of 3 to 4 cell layers to an 8- to 104ce11 thickness, and epi- thelium lining the hair follicles became keratinized. The presence of clear spaces was noted in the cytoplasm of the basal cells and the nuclei became more pyknotic. Also an increase in intercellular space was observed. Following this, the epidermis became covered with a crust which consisted of keratinized fibers, bacteria and leukocytes. There was a complete disappearance of some follicles with only a few mononuclear cells remaining to.mark their former location. In the dermis there was an increase in neutrophils and monocytes and also an increase in vascularity. More recently other workers (Alvares and Meyer, 1968) have found a reduction in keratohyaline granules in abdominal skin and oral epithelium. Skin lesions of zinc-deficient mice resemble those of rats (Day and Skidmore, 1947). Dogs. When feeding dogs a diet low in zinc and high in calcium, Robertson and Burns (1963) recognized superficial skin lesions on the posterior aspect of the abdomen and hind legs. The lesions were char— acterized by areas of superficial desquamation 10 to 20 mm. in diameter 7 which soon became covered with a crust-like formation. No parakeratosis or acanthosis was evident histologically. Lambs. The first visible signs of zinc deficiency in lambs, according to Ott et al. (1964), at Purdue University, was the loosening, rubbing and eating of fleece by the fourth week of the deficiency. Areas of alopecia which appeared became slightly red and wrinkled, and skin on the ventral surfaces became encrusted. Histopathologic changes in skin included hyperkeratosis, parakeratosis, acanthosis, accumulation of debris, and elongation of the rete pegs. Lesions seemed less pronounced in sheep fed phytic acid in the diet. Hyperkeratosis disappeared after 5 weeks of zinc repletion, but acanthosis persisted. A naturally occurring zinc deficiency has been reported in sheep in Colorado which clinically resembles its experimentally produced counterpart (Pierson, 1966). Calves. In general, the distribution of skin lesions is similar in calves during experimental zinc deficiency to that found in sheep (Ott et al., 1965). The dermatitis was especially pronounced on the head and, if severe enough, involved the entire body. Affected skin tended to be hard, dry, scaly, and easily injured upon trauma. Alopecia.was also present (Miller and Miller, 1962). Cracks and deep fissures appeared in the skin of the coronary bands around the hooves (Blackmon at aZ., 1967). The occurrence of structural distortion of the hooves and the horns was reported by Mills et a1. (1969), who attributed this to defects in the hardening of keratin. According to Miller and Miller (1962), histopathologic changes in the skin of zinc-deficient calves included parakeratosis, acanthosis, 8 elongation of rete pegs and reduction in numbers of cells in the stratum granulosum. Poultry. Studies in poultry nutrition indicate that zinc is important in normal feathering of the bird (Young at aZ., 1958). The feathers' of deficient chicks are characterized by a small, weak rachis and a lack of barbs near the feather tip (Edwards et aZ., 1958). Rahman et al. (1961) also noted poor feathering, brittle feathers, and unlaced thorny barbules by the second week of the deficiency. Scaliness was present on the dorsal midline, head, and neck. Squirrel monkeys. Skin lesions in zinc-deficient squirrel monkeys OMacapinlac et aZ., 1967) were typified by sparsity of hair over various areas of the body, especially those areas stressed by constant apposi- tion, such as the dorsal aspects of the knees. HistOpathologic evalua- tion of the skin of at least 1 squirrel monkey revealed no parakeratosis over the knees. Physiopathology of Zinc in the Skin. Nutritional requirements in swine. It has been difficult to establish a definite minimum nutritional requirement for zinc because of its propensity for interaction with other dietary and physiological factors. Skin lesions can be produced experimentally more readily if there are large amounts of calcium in the diet. At levels of about 30 to 36 ppm of zinc, a dietary calcium intake of 0.482 or greater will pro- gressively enhance signs of parakeratosis and inhibit weight gain. Under these conditions, increasing the zinc to 80 to 100 ppm will either prevent or alleviate the dermatitis (Vallee, 1959; Luecke et aZ., 1957; Tucker and Salmon, 1955; Stevenson and Earl, 1956). 9 Beardsley and Forbes (1957), however, found that 0.8%‘calcium had no effect on severity of parakeratosis, and Smith et a2. (1958) found that the severity of the parakeratosis was in inverse proportion to the zinc content of a basal ration containing 0.662 calcium.‘ Copper has recently been found to reduce the incidence and severity of parakeratosis and to maintain growth rate in zinc-deficient pigs, especially at high calcium levels (Hoefer et aZ., 1960). However, Smith et al. (1962) saw no improvement in skin condition in parakeratotic pigs fed 125 ppm copper. Oberleas et al. (1962), at Missouri, demonstrated that phytic acid in the diet decreased zinc availability and thus increased the incidence and severity of parakeratosis. In additional studies, O'Dell and Savage (1962) suggested that it is the high content of phytic acid in soybean protein which may precipitate zinc deficiency with more facility than in casein based diets. They also demonstrated that zinc in a phytic acid-supplemented casein diet is less available if fed to chicks. One nutritional factor, the amino acid, histidine, alleviates the' dermatosis of zinc-deficient pigs but has no action in correcting the reduced growth rate (Dahmer et aZ., 1967). Utilizing a casein-based diet, Shanklin et a1. (1968), at the Michigan State Experiment Station, found that a dietary zinc level for pigs of 20 ppm is sufficient to maintain body tissue levels, but that 14 ppm is inadequate. Zinc in skin and hair. Zinc has an important physiological function in maintaining the health of the skin. This is indicated by the fact that lowered zinc levels are frequently concurrent with skin lesions in animals fed zinc-deficient diets. Significant reductions of zinc content 10 have been observed in hooves of zinc-deficient calves (Miller and Miller, 1962), wool of zinc—deficient lambs (Ott et aZ., 1964), skin and hair of zinc-deficient baby pigs (Shanklin et aZ., 1968), hair of zinc-deficient human dwarfs (Strain et aZ., 1968), and in hair from human patients with thermal burns of the body surface (Pories and Strain, 1966). Lewis at al. (1957) found that increasing the calcium content of the ration of pigs from 0.52 to 0.82 or 1.22 produced a significant decrease in zinc content of hair, but not of skin. Increasing zinc content to 1028 ppm in the ration increased zinc content in hair but not in the skin. Klevay (1970), using hair from human subjects, discovered a dis- tinct correlation of zinc content with age. The zinc values were found to decrease gradually during the first decade of life, and then to increase during the second. No correlation could be made with the zinc level of hair to that.of blood plasma or of red blood cells. However, plasma-zinc concentrations in patients with psoriasis, and other dermatoses, were significantly lower than those of healthy controls according to Greaves and Boyde (1967). Klevay (1970) later acknowledged that the extent of contamination of.hair_with exogenous zinc is at present unknown and that studies which have assessed zinc concentrations of hair may have to be reevaluated. Autoradiographic studies of the distribution of zinc-65 in the dorsal skin of normal.rats have shown that it is preferentially concen- trated in the epidermis, rather than in the dermis. It is also concen— trated in the shaft of the hair follicles and, to a lesser extent, in the follicular epithelium. In mice the radionuclide is concentrated more in the proximal unkeratinized portions of the hair shaft than in the distal portion of the hair shafts (Miller et aZ., 1961). 11 When sections of normal human skin were stained histochemically for zinc, using the dithizone method, a homogeneous, moderately positive stain was obtained for the epidermis, arrectores pilorum, sweat glands and ducts, outer root sheath of hair follicles and the muscle layers. of large blood vessels. The stratum corneum stained variably in dif- ferent sections and all connective tissue components of the dermis remained unstained. When sections were incubated in vitro for 72 hours- in the presence of Mayer's albumen fixative or a.dilute zinc solution, an intense zinc-positive stain was observed in the stratum granulosum (transitional zone). This was-not observed in psoriatic skin when the transitional zone was absent, except for staining of keratohyaline granules. This specific binding of zinc, which occurred precisely at the zone of keratinization, may be suggestive of an important physio— logical phenomenon (Reaven and Cox, 1962). Later, using the diazonium coupling reaction, Reaven and Cox (1963) found that histidine, concentrated in the stratum granulosum, was probably responsible for the selective in vitro zinc-binding property. Zinc in wound healing. Pories et a1. (1967) observed an increase in the rate of healing of granulating wounds caused by excision of pilonidal sinus tracts in young men with concurrent oral administration of zinc sulfate. Earlier studies (Strain et aZ., 1960) had demonstrated an increase in the rate of wound healing in rats fed a mixture of methionine and zinc oxide, but not if either component were fed separately. The tensile strength of healing surgical incisions in the skin of zinc-deficient, pair-fed rats is significantly decreased (Sandstead and Shepard, 1968). After reaching this conclusion, they suggested that collagen formation might be impaired in wounds of the zinc-deficient animals. 12 Savlov et al. (1962) demonstrated a preferential accumulation of radiozinc-65 in wounds of rats in the acute phase of healing, coinciding with the develOpment of vascularity. No such increase was seen in normal skin of the same rats. There was no difference in the rate of healing of wounds in the paralumbar fossas of zinc-deficient calves and pair-fed control calves; however, wounds in control calves fed ad Zibitum healed at a faster rate (Miller et aZ., 1965). An important observation in this study was a zone of parakeratosis which was present around the wounds of the zinc- deficient calves and was thought to be due to trauma. These workers speculated that the distribution of skin lesions in zinc deficiency is due to external or secondary factors such as mechanical stress. In another report Miller et al. (1965) stated that the hair when it grew at the site of the healed wounds was gray in the zinc-deficient calves. Summary Experimental zinc deficiency will produce skin lesions in a variety of animals. In swine it is characterized as a benign dermatosis with hard, dry, crusted proliferations of the superficial epidermis, involv- ing especially the ventral aspects of the body.- Histologically, the presence of parakeratosis, hyperkeratosis, acanthosis and a dermal inflammatory response is apparent. The develOpment of skin lesions at specific dietary levels of zinc is complicated by interaction with other dietary factors such as calcium, phytate, capper and histidine. Zinc values are affected by experimental zinc deficiency in the hair more than-in skin. Moreover, zinc is micro- scopically concentrated in the hair follicles and, to a lesser extent, in the epidermis; but none is found in the dermis. Zinc is known to be an important factor in healing of cutaneous wounds in man, rats and cattle. MATERIALS AND METHODS General Experimental Design A total of 28 pigs were used in this research in 2 separate experi- ments. In the first experiment, which was more preliminary in nature, 12 pigs were used. The second, using 16 pigs, was conducted several months later in order to more precisely delineate and extend results observed in the first trial. The experiments were designed primarily to determine the influence of an enteric infection and antibiotics on zinc metabolism by Drs. Whitenack and Miller. The design was: Experiment 1: Group 1. 6 pigs were fed a zinc-deficient diet containing 12 ppm zinc. Group 2. 6 pigs were fed a zinc-adequate diet containing 100 ppm zinc and served as controls. Experiment 2: Group 1. 8 pigs were fed a zinc-deficient diet containing 12 ppm zinc. Group 2. 8 pigs were fed a zinc-adequate diet containing 100 ppm zinc and served as controls. One-half the pigs in each group of both experiments were infected with transmissible gastroenteritis virus (TGE). In Experiment 2, the ration fed one-half the pigs in each group was supplemented with 50 mg. 13 l4 oxytetracycline* and 400,000 units mycostatin**/kg. ration, on a dry weight basis. Animals The baby pigs used in Experiment 1 were 10 days old and those used in Experiment 2 were 7 days old at the onset of experimentation. The pigs in both experiments were of mixed breeds from each of 4 litters from the Michigan State University Swine Farm. In Experiment 1 all were males, except for 1 female which was used as a control. In Experi- ment 2 a mixture of both sexes was used. Animals were grouped randomly- with corrections for initial weight and sex. Housing and Care The pigs were placed in individual, grated-bottom, stainless steel cages and fed the experimental rations. During the periods of viral infection, the pigs were kept in separate rooms in which conditions were similar. The ambient temperature in each room was 85 F. During the experiment the ration fed the pigs was provided by Dr. E. R. Miller, Animal Husbandry Department, Michigan State University. This was basically a purified soybean-based ration and has been used in previous zinc deficiency studies OMiller et.aZ., 1968). The basal diet contained 12 ppm zinc, and the control pigs were fed the same ration supplemented to 100 ppm zinc. Early in Experiment 1 the pigs were fed twice daily and given just enough food so that it was completely consumed within 15 minutes. The amount given in this manner approximated that which would have been fed on an ad Zibitum *Terramycin, Pfizer Corp., New York, N.Y. **Nystatin, Squibb and Sons, E. R., 745 Fifth Avenue, New York, NOY. 15 basis. Initially, the pigs were fed the ration mixed in a slurry with water, but in the last several weeks the ration was fed dry ad Zibitum in a separate container to enhance consumption. In Experiment 2 the pigs were fed ad Zibitum throughout the experiment with the ration fed dry and separately from the water. Experimental Procedure for Experiment 1 Skin biopsies were taken from selected pigs in each group at 4 and 18 days after beginning the experiment in order to create wounds and to provide tissue for early histopathologic evaluation. In some cases 2 biopsies were taken from a.single pig at different times. Also some pigs were not included in the wound healing experiment. The general procedure involved, first, tranquilizing, shaving the pig over an area of the lateral thorax and then scrubbing the area several times with Phisohex,* followed by rinsing with water and.application of 70% alcohol. Approximately 2 cc. of local anesthetic** was then infiltrated subcu- taneously in an inverted "V"-shaped area immediately dorsal to the area to be biopsied.' A lune-shaped section of skin was then removed, which measured 20 mm. on its major axis and 10 mm. on its minor axis, by tracing an incision with a scalpel. The biopsy was taken from the lateral thorax 4 inches ventral to the dorsum of the spine and 4 inches anterior to the last rib. Its depth extended through the hypodermal fat and almost to the bony prominences of the ribs. *Phisohex, Winthrop Laboratories, New York. N.Y. 10016. **Epidural Solution, Haver-Lockhart Laboratories, P.O. Box 390, Shawnee Mission, Kansas 66201. ***Sparine, wyeth Laboratories, Box 8299, Philadelphia, Pa. 19101. 16 It was not possible to measure the rate of healing of the wounds directly, since a scab soon covered the wound and remained of constant I size until it was shed. Removal of it would have-caused disruptions and difficulty in microscopic interpretation. Pigs were necropsied at approximately weekly intervals in order to provide a continuous progres- sion of the healing process at comparable stages in both zinc-deficient and control animals. Therefore, there was considerable variability in the extent and severity of dermatitis present. Experimental Procedure for Experiment 2 In this experiment the pigs were all killed and necropsied at the end of the experiment. The pigs which had been infected with TGE were euthanatized and necropsied on the 29th day of the experiment, while the uninfected pigs were euthanatized and necropsied on the 33rd day of the experiment. 0n the third day of the experiment, procedures involving mechanical abrasion and hair plucking were performed. The hair was clipped short on the left lateral thorax in the same area used in Experiment 1 for the wound healing study. Following this, a No. 21 Bard-Parker scalpel blade was used to shave a 2.5 cm. square area, and then the same blade held.in a vertical position was used to quickly and uniformly abrade the area until bleeding points were seen and there was oozing of sero- sanguineous fluid. On the same day all hairs were plucked in the_same area only on the right lateral thorax. All hairs were plucked in a 2.5 cm. square area using a blunt-nosed Arista forceps.* They were plucked with a quick *Arista Surgical Co., 67 Lexington Ave., New York, N.Y. 10010. 17 forceful manner, with about 25 hairs being pulled atva time. In some cases a few very fine hairs which could not be grasped with the forceps remained, but these accounted for only a small percentage of the total. In the same manner, all the hairs in an area on the volar surface of the lateral (fourth) digit of the right rear leg were plucked. This area extended from the coronary band about 3 cm. proximally, and measured approximately 1 cm. in width. The hairs, once plucked, were tabulated (Table 1) according to follicle type, either (1) anagen (active) or (2) telogen (inactive) and- catogen (transitional) using a method similar to that described by Van Scott et a1. (1957). The telogen and catogen hairs were placed in the same group. All hairs which were plucked were placed into a 20 cm. by 1.5 cm. plastic petri dish which contained a small amount of water. Parallel lines approximately 1 cm. apart had been scored in the bottom to facilitate counting. The roots of at least 600 to 1800 hairs per area of plucking were thus counted and classified. A dissecting microscOpe was used to examine the hair roots using transmitted light and a variable magnification, depending on the depth of water in the petri dish, so that hairs were in simultaneous focus. At the end of the experiment the plucked areas were compared to surrounding skin to see if there was any grossly visible difference in severity of the dermatitis due to plucking. Sections from.the plucked area on the lateral digit of the right hind food and from the correspond- ing volar surface of the medial digit of the right hind food were com- pared by histopathologic evaluation for any difference in severity of the dermatitis. Pathologic criteria used in differentiation included the severity and extent of acanthotic and parakeratotic responses. Microscopic evaluation included the length of a line extending from the 18 coronary band to 3 cm. proximally on the volar surface of the digit, and corresponded exactly to the length of the plucked area. Hair follicles were also counted and classified microscOpically using the tissue sections to determine the percentage of anagen activity that had been induced by the plucking 1 month earlier, and to correlate this with variations in the severity of the dermatitis. It was found that classifying follicles in this manner introduced a factor of varia- bility since some follicles could not be distinguished as to type, especially if they were sectioned near the surface of the epidermis. Such follicles were then classified as "undetermined" and were considered to be nonactive. Thus, in reality, there was a higher anagen percentage than was actually recorded. Also, at the end of the experiment hair follicles were again plucked, this time on the lateral digit of the left hind foot and on the right. lateral thorax slightly anterior to the area formerly plucked. They were again counted and classified using the dissecting microscoPe. The hair follicle population was then evaluated as to the effects of age and zinc deficiency in both the foot and lateral thorax. The results obtained using the plucking technique were then correlated with the results obtained with tissue section to test for validity of the latter method. Hisgppathologic Technique At necropsy in both experiments skin samples from the_abdomen, chest, sacrum, and lateral thorax were collected. In addition, skin from selected areas of the foot.and wounds was collected. After removal and prior to fixation, the pieces of skin were affixed by staples to wooden tongue depressors to prevent shrinkage and distortion during l9 fixation. Care was taken not to stretch the skin. The reverse side of the tongue depressor was then labeled to mark the anatomic origin of the skin. The tissues were fixed in 10% formalin. Sections were prepared for examination and stained according to standard techniques described in the Manual of'HistoZogio and Special Staining Technics (1957). Sections were stained with Harris' hematoxylin and eosin and also with the periodic acid—Schiff technique (PAS). RESULTS While the primary objectives of this research were to determine the influence of an enteric infection on zinc metabolism, some interest- ing results on the skin were also obtained. These seemed to depend mainly on the level of zinc in the ration and length of time the pigs were on experiment. Only the results pertinent to the skin will be discussed, and other results are included in the thesis of Dr. D. L. Whitenack. Gross Pathology Experiment 1. Several unexpected factors during the experiment had a significant effect upon the dermatitis. First, the animals which were to remain free of transmissible gastroenteritis were infected acci- dentally 10 days after the start of the experiment. This affected the distribution of the skin lesions. Second, the dermatitis was extremely variable in its time of onset and so it was decided to terminate each' pig when it reached a stage in the severity of the dermatitis approxi- mately equivalent to that of other pigs. In addition, 2 of the pigs (091502 and 091433) died of transmissible gastroenteritis prior to attaining a severe dermatitis. Erythema was first noted in those portions of the body that were later affected with dermatitis, especially in the perineal region. Following this, the skin became scaly and encrusted with dark crusts which became more extensive as the disease progressed. Cracking of the 20 21 crusts occurred with subsequent oozing of serosanguineous exudate. Initially, the first areas to become involved were the abdomen and perineum, followed by extension to the chest, thighs and distal por— tions of the limbs. The hind limbs were characteristically more affected than the forelimbs. The distribution of the dermatitis appeared to correspond to skin which was in contact with the surfaces of the cages, and also skin contaminated with feces during the periods of diarrhea. The hair coat in the pigs fed zinc-deficient diets was sparse, greasy to the touch, and rough in appearance. Individual hairs appeared more erect. Alopecia was present to a variable degree, but only in areas affected with the dermatitis. The hair in these areas was more brittle and was easily removed with the crusts. A linear zone of dermatitis on the dorsal sacral area in several pigs extended from the base of the tail anteriorly to the last lumbar vertebra. No explanation was known for its presence, since this seemed not to be an area exposed to mechanical trauma or accumulation of bacteria-laden debris. The pigs fed the zinc-adequate ration had no dermatitis and their hair cost was shiny and smooth in appearance. They were fat and healthy in appearance. Experiment 2. The dermatitis of the pigs in Experiment 2 was similar in appearance, but different in distribution, to the dermatitis of the pigs of Experiment 1. It appeared first on the sternum and from there it spread to the contact surfaces of the forelimbs and the axillae. In more advanced cases it extended posteriorly from the chest to involve the abdomen. The perineal area was involved in those pigs infected with TGE. The dermatitis was usually severe on the forelegs and milder on the hind legs. 22 At necr0psy the skin with the most advanced lesions was leathery and resisted cutting with the scalpel. It was greatly thickened, fre- quently 3 to 4 times thicker than normal skin. The onset of the dermatitis occurred in all pigs fed zinc-deficient diets at approximately the same time, becoming visible at 10 days to 2 weeks after the start of the experiment. It became progressively worse, thereafter, until the experiment was terminated. By the end of the experiment it was noticed that, in general, the skin lesions were milder in the TGE-infected pigs than in the noninfected pigs. The difference in the duration of the zinc-deficient state (4 days) may have partially accounted for this. Within the infected and the non- infected groups there was no visible difference in severity of the lesions of pigs fed the antibiotic supplemented ration. Again, as in Experiment 1, no dermatitis was observed in any of the pigs fed the ration containing 100 ppm zinc. Pathology The microscopic lesions were similar in both experiments and therefore are summarized together. Epidermi . Hyperkeratosis was the first prominent change in affected skin, as indicated by the stratum corneum increasing in depth, especially around the orifices of the hair follicles. As the reaction became more intense, parakeratosis was observed throughout the stratum corneum, but frequently layers of retained nuclei alternated with layers of more normal appearing keratin. Kerstin appeared more eosinophilic in affected areas than in areas of the body free of the dermatitis. In the most severely affected areas the stratum corneum.was.thin and was covered by thick adherent crusts containing large numbers of neutrOphils 23 and bacteria (Figures 1 and 2). In skin of this nature there were occasional crevasses which penetrated through the epidermis into the underlying dermis. In sections of skin from the feet, large globular eosinOphilic inclusions were seen within the parakeratotic scale. These were usually found at the apices of folds of the epidermis and were thought to result from the filling of spongiform vacuoles in acanthotic regions with proteinaceous fluid and its subsequent condensation into solid masses. The globules were not birefringent to polarized light, unlike most of the parakeratotic scale. They were deeply PAS-positive. The stratum granulosum was frequently absent in the most severe lesions. Its presence in less severely affected areas was marked by a pronounced irregularity, in.which it was either greatly thickened or absent in immediately adjacent regions. Keratohyaline granules in the thickened areas were often enlarged. Also greatly enlarged keratohyaline granules could be seen surrounding the orifices and ducts of the apo- crine sweat glands. Acanthosis was a major component of the dermatitis found in thev pigs fed the zinc-deficient diets. In mildly affected areas it began as a thickening of the Malpighian layer with some elongation of the rete pegs. Increased thickening occurred as the lesions intensified and the rete pegs became progressively elongated,~but widened only to a certain width. As the rete pegs extended deeper into the dermis, they tended to anastomose with adjacent ones to form a 3-dimensional reticulated pattern resembling a honeycomb. Cellular arrangement in the acanthotic Malpighian layer became haphazard so that the stratum germinativum was irregular and was poorly defined from the more superficial stratum spinosum. Increased 24 Figure 1. Gross appearance of the skin lesions of the ventral sapect of a pig fed a zinc-deficient diet. Figure 2. Telogen (a), pigmented anagen (b), and nonpigmented anagen (c) follicles of plucked hairs of a pig. x 100. 25 Figure 3. Microscopic appearance of abdominal skin of a pig fed a zinc-adequate diet. Hematoxylin and eosin. x 190. .0" r "Q I ’ .. , . , - . . '_"- ' ’-’\‘ a-‘. a_.. t a I J I", ‘ ' I I v‘ ,,«4- I / I III... g‘ . 0‘ ’1’“! , t’ 4' Q I. ’ , . .'. ' " \ ‘ ‘ \‘ ~.‘ ‘\ 41“,- ~_~ ,.. -~ .\ ,N',.'\ \ D 1 ° ” ' - . " f a . 1‘ .‘ , . .f . ‘1 w .,__‘ ’ . . t ‘ ,‘ . -I\\ ‘ ‘ Q Figure 4. Microscopic appearance of abdominal skin of a pig fed a zinc-deficient diet. Parakeratosis and hyperkeratosis of stratum corneum (a); elongated and anastomosed rete pegs (b); and hyperplastic dermal capillaries (c). Hematoxylin and eoxin. x 190. I 26 intercellular space was present between cells so that intercellular bridges were more prominent. Epithelial cells were variable in size and shape and were densely hyperchromatic. Nuclei were often pyknotic or vacuolated, and possessed very large nucleoli. Mitotic figures were extremely numerous, especially in cells of the deeper regions. The stratum spinosum became thin over the dermal papillae so that these structures were elevated above the nearby stratum corneum. Varying degrees of spongiosis were noted, but it was most prominent in the skin of the abdomen. The spongiform spaces frequently became filled with neutrophils and other inflammatory cells to form spongi- form abscesses with resultant disruption of the epidermis._ In addi- tion, neutrophils and lymphocytes were present in the intercellular spaces between the epithelial cells. Dermis. The dermal papillae between the rete pegs became elongated and bulbous in nature. Their vascular components became greatly dilated and edema was pronounced. Extravascular erythrocytes and inflammatory cells were present. Deeper in the dermis the vascula- ture was increased in amount and was tortuous. The endothelial cells were always swollen and hyperplastic. In the perivascular regions fibroblasts appeared active and immature, and lymphocytes, monocytes and neutrOphils were increased in number. EosinOphils and mast cells were not numerous. Apgcrine sweat_glands. The presence of keratinaceous plugs in apocrine’ sweat gland duct orifices at the level of the epidermis was observed in normal skin of pigs fed both diets. They were believed not to cause retention of glandular secretion. However, the plugs were more prevalent and of greater size in regions of dermatitis and seemed to be a result 27 of the hyperkeratosis. When the dermatitis was severe, PAS-positive material was present in many of the plugs. The apocrine ducts appeared to participate in the same type of acanthotic response present in the epidermis. The walls were thickened and contained more layers of cells than normal. There was also an increase in intercellular space and of cytoplasmic basOphilia. The duct lumens were frequently dilated in direct proportion to the thick- ness of the walls. In affected skin there was a marked increase in the activity of the glandular epithelium, so that it was deeply basophilic and increased in height, often to the point of being pseudostratified columnar in appearance. In skin with obvious secondary bacterial infection the lumens of the glands were filled with neutrophils and proteinaceous material (Figures 5 and 6). In some cases this continued to the point of obliteration of the glandular structures. Dilation of the glands was infrequent. Fat cells in tissue surrounding the apocrine glands were small and contained small cytoplasmic fat globules in the pigs fed the zinc-deficient diets. ‘Hgigp Surprisingly, there was little histologic alteration of the anagen hair follicles in skin with severe dermatitis due to zinc deficiency. All cellular layers of the follicle were normal in appear- ance, although on occasion it was suspected that cellular arrangement was more haphazard. Trichohyalin granules in Henle's layer were normal in size and distribution. The outer root sheath was thicker, especially in the more distal regions near the epidermis where it seemed to participate in the acanthotic reaction. Hyperkeratosis was present at the orifice of the hair follicle and between the hair shaft and external root sheath distal to the sebaceous gland opening. 28 Figure 5. Apocrine sweat glands of abdominal skin of a pig fed a zinc-adequate diet. Duct (a), and coiled secretory tubule (b). Hematoxylin and eosin. x 180. Figure 6. Aporcine sweat gland of abdominal skin of a pig fed a zinc-deficient diet. Thickened duct (a), and glandular epithelium (b); neutrOphils in the glandular lumen (c). Hematoxylin and eosin. x 180. 29 There were increased numbers of telogen hair follicles in regions of skin affected with dermatitis. The hair shafts of telogen follicles were frequently absent and, when they were present, were of a smaller diameter. Others were not circular in cross section, but presented an irregular profile. Follicular keratosis, also present in telogen follicles, was especially pronounced (Figures 7 and 8). Masses of keratinized material extended downward from the stratum corneum through the orifice of the hair follicle and separated the club hair from the external root sheath. Retained nuclei were found often in the keratin, especially at the base of the follicle, and sometimes they were seen within the shaft of the club hair itself. The external root sheath was also greatly thickened on telogen hair follicles. The presence of dystrophic hair follicles was noted in some of the more severely affected areas of skin, especially on the abdomen of pigs in Experiment 1. These structures were represented as downward extending columns of epithelial cells which contained no hair shafts or any other structures normally present in anagen or telogen follicles. No abnormalities of the sebaceous glands were observed. Sebaceous glands in the young pigs apparently are not well developed, and were extremely small in b10psy specimens taken at the start of Experiment l.- In sections taken at the termination of the experiment they were larger and more extensive differentiation had occurred. No histOpathologic changes were observed in any hair follicles of the unaffected skin of pigs fed zinc-deficient diets. Wound healing. The control pigs, which had been fed the ration sup- plemented to 100 ppm zinc, had wounds which healed much faster than those fed the deficient diet. The scabs began falling off.in the 30 J'Ik \A 0.— Figure 7. Telogen hair follicle of abdominal skin of a pig fed a zinc-adequate diet. Hematoxylin and eosin. x 160. Figure 8. Telogen hair follicle in an area on the forefoot affected with dermatitis of a pig fed a zinc-deficient diet. Note hyperkeratosis and parakeratosis within the follicle. Hematoxylin and eosin. x 160. 31 control pigs after 2 weeks, revealing that the underlying wounds had been covered by epithelium. In the deficient animals, however, the scabs remained on the wounds for the duration of the experiment. On histological evaluation, it was apparent that healing was retarded in the zinc-deficient pigs. In none of the wounds from these pigs was granulation tissue as mature as it was in the control pigs. Fibroblasts were large, deeply basoPhilic and collagen bundles were smaller and more sparse. None of the wounds in the zinc-deficient pigs was completely covered by epithelium migrating from the periphery of the wound. In control pigs this was accomplished in 2 weeks. In deficient pigs, hyperplastic epithelium, normally found‘on the periphery of wounds, tended to have increased intercellular spaces and the epithelial cells were hyperchromatic (Figures 9 and 10). There was more inflammatory cellular infiltrate, especially of neutrophils, in the wounds of the zinc—deficient pigs. Mechanical abrasion. Shortly after the skin scrapings were accomplished, they became encrusted with scabs in all the pigs. Within 2 weeks the lesions had completely healed in the control pigs and, by the end of the experiment, it was difficult to ascertain the original site because of regrowth of hair in that area. The regrowth of hair was at a slower rate in the deficient pigs and was only approximately half as long as surrounding hair at the end of the experiment. There was no loss of pigmentation of the hair upon regrowth in the dark-colored pigs. The zinc-deficient pigs retained scabs at the site of-the scrapings for the duration of the experiment. The skin in this area appeared erythematous and felt twice as thick as surrounding skin. 32 Figure 9. Epithelium at the margin of a l3-day-old wound on the lateral thorax of a pig fed a zinc-adequate diet. Hematoxylin and eosin. x 160. Figure 10. Epithelium at the margin of a 13-day-old wound on the lateral thorax of a pig fed a zinc-deficient diet. Hematoxylin and eosin. x 160. 33 Upon histopathologic evaluation of the scraping sites, the only changes found in 6 of the zinc-deficient pigs were hyperkeratosis of the stratum corneum and a thickening of the dermis with increased density of collagen. In 1 pig (095938) there was some thickening of the Mal- pighian layer and elongation of rete pegs suggestive of acanthosis. A chronic dermatitis was observed in l pig (095940) which was isomorphic with the dermatitis seen in other areas of the body. Hair pluckipg. Complete regrowth of hair was observed on the feet of the control pigs within 2 weeks; however, in the zinc-deficient pigs the hair still had not completely regrown by the end of the experiment. Hair did not regrow on the sides of either group of pigs, even by the' end of the experiment, except for a few scattered larger hairs, and it was thought that these had been missed during the original plucking. Plucking of hair did not appear grossly to have any effect on the inten- sity of the dermatitis either on the feet or the sides of any of the pigs. Microscopically, a more severe dermatitis was observed on the digits of 2 pigs fed zinc—deficient diets from which the hair had been plucked than on the Opposite digits of the same feet. It was less severe in 2 other pigs and of the same intensity in the rest of the pigs. Criteria utilized in assessing severity were acanthosis and para- keratosis. In milder lesions there was little anastomosing of rete pegs as part of the acanthotic response. Also in mild lesions the acan- thosis and parakeratosis were confined to an area just above the hoof, while in the more severe lesions, both processes extended throughout the section. It can be concluded that, microscOpically, there was no effect of plucking of hair upon the intensity of the dermatitis. 34 The hair activity of plucked and unplucked skin as influenced by rations is given in Tables 1 and 2. Initial values for the percentage anagen of the hair population on the foot and on the lateral thorax were quite different. It was evident that the majority of hair fol- licles on the lateral thorax of the l3-day-old pig were anagen and that follicles on the foot were predominantly telogen. By the end of the experiment the percentage anagen had markedly decreased on the lateral thorax and the percentage telogen on the hind foot had done likewise in the pigs fed zinc-adequate diets. One may conclude that the dif- ferent results for the 2 time periods represent a normal physiological change in hair follicle population, which is unique for different ana- tomical areas of the skin. There was a significantly greater depression of the percentage of anagen follicles of the lateral thorax at low levels of zinc in the ration if TGE were not present (P < 0.01), and if antibiotics were not present (P < 0.05). Infection by TGE virus was more significant in preventing anagen depression than antibiotics. A state of nutritional zinc deficiency, therefore, has an effect on the hair follicle population in terms of activity. However, anagen per- centages were even more greatly decreased on the feet of the zinc- deficient pigs, and it was thought that this decrease was largely a result of the dermatitis which was present in that location. Values of percentage anagen obtained from microsc0pic evaluation of tissue sections appeared to correlate with those obtained by the use of direct observation of plucked hairs by the dissection micro- scope, at least for the hairs of the hind feet in which both methods were used simultaneously. If this is assumed, it can be observed that anagen activity was induced by plucking hairs. The greater concentra- tion of anagen hair follicles did not prevent the dermatitis due to zinc deficiency in this experiment. 35 .aofiumu .wx\afiuoumzz mafia: ooo.ooq pom aaomamuuou,.ma omaaa .Aao.o v av ones too saga museumomae annuamaamam soon so .Aao.o v av mug x moauoanauaa x can» .Aao.o v mo may x can» .Amo.o v we moauOHnHuso x odes mdoauooumuofi was Aaooo v my me umeoSu Heywood now moosmuomm«p unoowwaemfimea .moHoHHHom was: now moB dogmas some you mean maeaom mwoum>< .mufimn poxosae mo smoomouoas wsfiuoommap nufis mos mazu mHoHHHom mo cowuosam>m .udofiauoexo on» mo was on» or uoom peas umma on» no unHp.HououoH man was .uaoauumaxo onu mo meadowwon mnu um uoom pea: unwau ago no ufiwap Hosanna or» ones maoamom .mwfie N Mom soawou a mo nomads ammuaooume ea omouo>o one munommumou osam> zooms moauoanfiuq¢ <¢.m¢| mm.m¢ Hm.mm om.~m+ No.m< 00.0 new oaHN and 00H Hm.¢ maufiuouooouummw oHnHmmHSmamuu use mofiuownfiuao .NOdmaonov uses no assume we» was some usoquMHv um swam mo Nouonu HonouoH pom uoom oru mo huu>auoo has: no hudaasw .H edema 36 Table 2. Summary of the effect of plucking, zinc deficiency, anti- biotics and transmissible gastroenteritis virus (TGE) on hair activity of the feet of pigs Z Anagen* Z Anagen* Z Anagen Treatment unplucked medial digit plucked lateral digit Change** 12 ppm zinc 4.6 27.0 +24.4 12 ppm zinc*** Antibiotics 1.8 26.4 +24.7 12 ppm zinc TGE 7.9 30.0 +22.1 12 ppm zinc TGE 17.1 37.1 +20.1 Antibiotics 100 ppm zinc 39.7 50.5 +10.8 100 ppm zinc Antibiotics 41.3 54.6 + 9.8 100 ppm zinc TGE 39.8 63.8 . .' +24.l 100 ppm zinc TGE 27.5 66.7 +39.7 Antibiotics *Each value represents the average in percentage anagen of a region on the right hind foot for 2 pigs at the end of the experiment. Evaluation of follicle type was by direct microscOpic observation of tissue section. Average sample size for each region was 35 follicles. **No significant difference among treatment groups. ***50 mg. terramycin and 400,000 units Nystatin/kg. of ration. DISCUSSION HistOpathologic changes found in the skin of the pigs fed zinc- deficient diets were similar to those reported by earlier workers describing field cases of pigs with parakeratosis. MicroscOpic evalua- tions were suggestive of changes consistent with those of a chronic dermatitis. The pathogenesis of the skin lesions or the reason for their peculiar distribution could not be ascertained. Skin from unaffected areas of pigs fed deficient and adequate diets was entirely normal in every aspect, and it was thought for this reason that the distribution of the dermatitis was due to some external localized factor causing a specific type of damage to the more susceptible, zinc-deficient tissue. More changes were expected in the hair follicles, especially the architecturally complex anagen follicles, in view of the amount of zinc they are said to contain. It seemed interesting that changes believed associated with protein deficiency (Bradfield et aZ., 1967) were not observed in hair follicles plucked from regions-not affected with the dermatitis, despite the anorexia and reduced feed consumed. Plucking telogen follicles to induce anagen activity apparently does not confer resistance to deve10pment of dermatitis due to zincw deficiency in the pig, at least not at the levels of anagen achieved in this research. In contrast to these results, it is reported the degree 37 38 of damage that irritants inflict is less in the epidermis of skin with growing hairs than to skin with hairs in the resting condition (Montagna, 1956). The Koebner phenomenon, seen most frequently in the presence of human psoriasis, is the "development of isomorphic lesions in the~ traumatized uninvolved skin of patients who have cutaneous disease” (Pedace et aZ., 1969). The mechanical abrasion applied to the sides of the pigs was done in the same manner used to induce the Koebner phenomenon in man. Positive response, 12.5% in this experiment, was lower than the 24% reported by Pedace et al. The dermatitis in the skin scraping of the one pig could be interpreted to be characteristic of either the Koebner phenomenon, delayed wound healing, or merely secondary bacterial infection. Mechanical abrasion is probably not a primary cause of the localized dermatitis of zinc deficiency, but rather a secondary or contributing cause. In interpreting the results of the wound healing experiment, one should bear in mind that a slower rate of wound healing can also be caused by nutritional inadequacies. This could also have been the case in Experiment 1, since the zinc-deficient pigs were quite anorectic. Miller et a2. (1965) reported no difference in the rate of wound heal- ing in zinc-deficient and in zinc-adequate, pair-fed cattle, thus indicating that anorexia may be an important factor to consider. Microscopically, bacteria were prominent in all regions where the dermatitis was intense. Therefore, it would be informative if an experiment was conducted utilizing germfree pigs fed a diet low in zinc to determine the extent of bacterial infection as a cause of the dermatitis. 39 The role of TGE in preventing a greater decrease in percentage of anagen follicles on the lateral thorax during a state of zinc deficiency could not be explained. Further experimentation should be conducted so that this phenomenon could be investigated.' In other future research, hair plucking, tabulation of hair fol- licle types and mapping hair activity in different anatomical regions of skin would be informative and might be useful in interpreting dermato- logical conditions. This could be done with swine as well as other animals. Experiments to study specifically skin lesions would be most apprOpriate. SUMMARY A total of 28 baby pigs were utilized in 2 experiments to determine the influence of infection and antibiotics on zinc metabolism. During the course of these experiments skin was made available for histopatho- logic evaluation. The morphology of the skin from different anatomical areas was evaluated histopathologically in the beginning, end, and during the course of the experiments. In the first experiment healing of wounds was studied and, in the second, the effects of skin abrasion and of plucking hair on the development of dermatitis were investigated. The hairs from the plucked areas were classified as to follicle type to determine growth activity. The skin lesions due to a deficiency of zinc in the pig were parakeratosis, hyperkeratosis, acanthosis-and a dermal inflammation. The healing of Open wounds and of abraded skin was delayed in pigs fed zinc-deficient diets. At the onset of the experiment there was a predominance of anagen hair follicles on the lateral thorax and of.teIOgen hair follicles on the volar surface of the foot. By the end of the experiment the predominance of the respective follicle types had diminished in both areas, except on the feet of the deficient pigs, in which there was a dermatitis. Plucking of hairs caused an increase in the percentage of anagen follicles on the feet of pigs fed both the adequate and zinc-deficient diets; it did not cause any change in severity of the dermatitis. 4O BIBLIOGRAPHY BIBLIOGRAPHY Alvares, O. F., and Meyer, J. 1968. Regional differences in parakera- totic response to mild zinc differences. Arch. Derm. (Chicago), Anderson, J. W., Cooper, G. A., and Hoekstra, W. G. 1967. The histo- chemistry of the parakeratotic lesion of swine. J. Invest. Derm., 48: 521-530. Beardsley, D. W., and Forbes, R. M. 1957. Growth and chemical studies of zinc deficiency in the baby pig. J. Ani. Sci., 16: 1038. (abstract) Blackmon, D. M., Miller, W. J., and Morton, J. D. 1967. Zinc deficiency in ruminants. Vet. Med., 62: 265-270. Bradfield, R. B., Bailey, M. A., and Margen, S. 1967. Morphological changes in human scalp hair roots during deprivation of protein. Science, 157: 438-439. Dahmer, E. J., Grummer, R. H., and Hoekstra, W. G. 1967. Effect of histidine on zinc deficiency in baby pigs. J. Ani. Sci., 26: 1475. (abstract) Day, H. G., and Skidmore, B. K. 1947. Some effects of dietary zinc deficiency in the mouse. J. Nutr., 33: 27-37. Edwards, H. M., Jr., Young, R. H., and Gillis, M. B. 1958. Studies on zinc in poultry nutrition. 1. The effect of feed, water and environment on zinc deficiency in chicks. Poultry Sci., 37: 1094-1099. Follis, R. H., Jr., Day, H. G., and McCollum, E. B. 1941. Histological studies of the tissues of rats fed a diet extremely low in zinc. J. Nutr., 22: 223-2380 Greaves, M., and Boyde, T. R. C. 1967. Plasma-zinc concentrations in patients with psoriasis, other dermatoses and venous leg ulcera- tion. Lancet, 1019-1020. Hoefer, J. A., Miller, E. R., Ullrey, D. E., Ritchie, H. D., and Luecke, R. W. 1960. Interrelationships between calcium, zinc, iron and copper in swine feeding. J. Ani. Sci., 19: 249-259. IHove, E., Elvehjem, C. A., and Hart, E. B. 1938. Further studies on zinc deficiency in rats. Am. J. Physiol., 124: 750—758. 41 42 Itikawa, 0., Ishida, K., Tamate, H., Hoshino, T., Yoneya, 8., and Goto, K. 1964. Patho-histochemical studies on the swine parakeratosis. Kernkamp, H. C. H., and Ferrin, E. F. 1953. Parakeratosis in swine. JOAOVOMOAO’ 123: 217-2200 Kernkamp, H. C. H., Hoyt, H. H., Sorensen, D. K., and Sautter, J. A. 1955. Parakeratosis in swine. Proc. Book A.V.M.A., 77-81. Klevay, L. M. 1970. Hair as a biopsy material. I. Assignment of zinc nutriture. Am. J. Clin. Nutr., 23: 284-289. Klevay, L. M. 1970. Letter to the editor: Hair as a biopsy material. Am. J. Clin. Nutr., 23: 377-378. Legg, S. P., and Sears, L. 1960. Zinc sulphate treatment of para- keratosis in cattle. Nature, 186: 1061-1062._ Lewis, P. K., Jr., Hoekstra, W. G., and Grummer, R. H. 1957. Restricted calcium feeding versus zinc supplementation for the control of parakeratosis in swine. J. Ani. Sci., 16: 578-588. Luecke, R. W., Hoefer, J. A., Brammell, W. S., and Schmidt, D. A. 1957. Calcium and zinc in parakeratosis of swine. J. Ani. Sci., 16: 3-11. Macapinlac, M. P., Barney, G. H., Pearson, W. N., and Derby, W. J. 1967. Production of zinc deficiency in the squirrel monkey (saimiri soiureus). J. Nutr., 93: 499-510. Manual of’Histologic and Special Staining Technios. 1957. Armed Forces Institute of Pathology, Washington, D.C. Miller, M. J., Vincent, N. R., and Mawson, C. A. 1961. An autoradio- graphic study of-the distribution of zinc-65 in rat tissues. J. Histochem. Cytochem., 9: 111-116. Miller, E. R., Luecke, R. W., Ullrey, D. E., Baltzer, B. V., Bradley, B. L., and Hoefer, J. A.‘ 1968. Biochemical, skeletal and allometric changes due to zinc-deficiency in the baby pig. J. Nutr., 95: 278-288. Miller, J. K., and Miller, W. H.: 1962. Experimental zinc deficiency and recovery of calves. J. Nutr., 76: 467-474. Miller, W. H., Pitts, W. H., Clifton, C. M., and Morton, J. D. 1965. Effects of zinc deficiency per se on feed efficiency, serum alkaline phosphatase, zinc in skin, behavior, greying, and other measurements in the holstein calf. J. Dairy Sci., 48: 1329- 1334. Mills, C. F., Quarterman, J., Cheaters, J. J., Williams, R. B., and Dalgarno, A. C. 1969. Metabolic role of zinc. Am. J. Clin. Nutr., 22: 1240-1249. 43 Montagna, W. 1956. The Structure and Function of’Skin. Academic Press, New York, New York. Oberleas, D., Muhrer, M. E., and O'Dell, B. L. 1962. Effects of phytic acid on zinc availability and parakeratosis in swine. Jo A111. SCie, 21: 57-610 O'Dell, B. L., and Savage, J. E. 1960. Effect of phytic acid on zinc availability. Proc. Soc. Exptl. Biol. Med., 103: 304-305. Ott, E. A., Smith, W. H., Stob, M., and Beeson, W. M. 1964. Zinc deficiency syndrome in the young lamb. J. Nutr., 82: 41-50. Ott, E. A., Smith, W. H., Stob, M., Parker, H. E., and Beeson, W. M. 1965. Zinc deficiency syndrome in the young calf. J. Ani. Sci., 24: 735-741. Pedace, F. J., Muller, S. A., and Winkelman, R. K. 1969. The biology of psoriasis. Acta Derm. Venereol., 49: 390-400. Pierson, R. E. 1966. Zinc deficiency in young lambs. J.A.V.M.A., 149: 1279-1282. Pories, W. J., Henzel, J. H., Rob, C. G., and Strain, W. H. 1967. Acceleration of wound healing in man with zinc sulphate given by mouth. Lancet, 1: 121-124. Pories, W. J., and Strain, W. H. 1966. Zinc and wound healing, in A. S. Prasad, Zinc Metabolism. Springfield, 111.: Thomas, 379-391. Rahman, M. W., Davies, R. E., Deyoe, C. W., Reid, B. L., and Couch, J. R. 1961. Role of zinc in the nutrition of growing pullets. Poultry Sci., 40: 195-200. Reaven, E. P., and Cox, A. J., Jr. 1962. Binding of zinc by the transitional layer of epidermis. J. Invest. Derm., 39: 133-137. Reaven, E. P., and Cox, A. J., Jr. 1963. The histochemical localiza- tion of histidene in the human epidermis and its relationship to zinc binding. J. Histochem. Cytochem., 11: 782-790. Robertson, B. T., and Burns, M. J. 1963. Zinc metabolism and zinc deficiency syndrome in the dog. Am. J. Vet. Res., 24: 997-1002. Sandstead, H. H., and Shepard, G. H. 1968. The effect of zinc deficiency on the tensile strength of healing surgical incisions in the integument of the rat. Proc. Soc. Exptl. Biol. Med., 128: 687-689. Savlov, E. D., Strain, W. H., and Huegin, F. 1962. Radiozinc studies in experimental wound healing. J. Surg. Res., 2: 209-212. 44 Shanklin, S. J., Miller, E. R., Ullrey, D. E., Hoefer, J. A., and Luecke, R. W. 1968. Zinc requirement of baby pigs on casein diets. J. Nutr., 96: 101-108. Smith, W. H., Plumlee, M. P., and Beeson, W. M. 1962. Effect of source of protein on zinc requirement of the growing pig. J. Smith, W. H., Plumlee, M. P., and Beeson, W. M. 1958. Zinc require- ment for growing swine. Science, 128: 1280-1281. Stevenson, J. W., and Earl, I. P. 1956. Studies on parakeratosis in Stirn, F. E., Elvehjem, C. A., and Hart, E. B. 1935. The indispensa- bility of zinc in the nutrition of the rat. J. Biol. Chem., 109: 347-359. Strain, W. H., Pories, W. H., and Hinshaw, J. R. 1960. Zinc studies in skin repair. Surg. Forum, 11: 291-292. Strain, W. H., Steadmen, L. T., Lankau, C. A., Berliner, W. P., and Pories, W. J. 1966. Analysis of zinc levels in hair for the diagnosis of zinc deficiency in man. J. Lab. Clin. Med., 68: 244-249. Todd, W. R., Elvehjem, C. A., and Hart, E. B. 1934. Zinc in the nutrition of the rat. Am. J. Physiol., 107: 146-156. Tucker, H. F., and Salmon, W. D. 1956. Parakeratosis of zinc deficiency disease in the pig. Proc. Soc. Exptl. Biol. Med., 88: 613-616. Young, R. J., Edwards, H. M., Jr., and Gillis, M. B. 1958. Studies on zinc in poultry nutrition. 2. Zinc requirement and deficiency symptoms of chicks. Poultry Sci., 37: 1093-1100. Vallee, B. L. 1959. Biochemistry, pathology and physiology of zinc. Physiol. Rev., 39: 443-490. Van Scott, E. J., Reinertson, R. P., and Steinmuller, R. 1957. The growing hair roots of the human scalp and morphologic changes. therein following amethOpterin therapy.‘ J. Invest. Derm., 29: VITA Frank A. Voelker was born in Shevlin, Minnesota, on February 16, 1942. He attended Bemidji High School, Bemidji, Minnesota, and entered the University of Minnesota in 1960. He was admitted to the University of Minnesota College of Veterinary Medicine in 1963 and graduated in 1967. In July of the same year he entered active duty in the United States Air Force Veterinary Corps and subsequently served as base veterinarian for 2 years at Forbes Air Force Base, Topeka, Kansas. He was accepted as‘a graduate student in the Department of Pathology in 1969. Currently in the Air Force, he is scheduled to begin resi- dency training in pathology at the Armed Forces Institute of Pathology, Washington, D.C., in the fall of 1970. The author married Jan Marie Lindstrom of International Falls, Minnesota, in August of 1965. They have 2 children, Brian, age 2, and Michele, age 3 months. 45 ”'TIIIIIITIIIIIIIITITIIIIIJIIIIIES