NEONATAL THYMECTOMY IN CALVES THESIS FOR THE DEGREE OF M. S. MICHIGAN STATE UNIVERSITY DONALD L. BU ELKE 1966 ' LIB R A R Y Michigan State , University ABSTRACT NEONATAL THYMECTOMY IN CALVES by Donald L. Buelke During the past decade the lack of immune com- petence produced in various species of laboratory animals by neonatal thymectomy has been repeatedly demonstrated. This experiment was conducted to explore the feasibility of such an operation in calves and to study some of the basic effects upon the animals with the possibility of using this procedure for creating animals of lowered immunologic capabilities for use in certain viral or neoplastic disease transmiSsion studies. Ten newborn Holstein-Friesian bull calves were alternately selected and placed into either the thymectomy or control group. Each calf was given an initial 2 liter hand feeding of colostrum which was previously obtained from several cows, mixed and frozen. Within 24 hours after birth the calves were thymectomized or subjected to a sham operation. The surgical procedure involved resectioning of the central Donald L. Buelke portion of the third left rib and bluntly dissecting the thoracic portion from its anterior mediastinal position; then placing the calf on his back and bluntly dissecting the cervical portion through a midventral cervical incision. The sham operation was identical with the exception of the extirpation of the gland. Postsurgical care included appropriate anti— microbial and palliative treatment. The calves were fed an aseptically prepared milk replacer, a pelleted dry ration, hay, and water. During the first month of age all calves were troubled with frequent attacks of enteritis with ensuing loss of condition and anorexia. From about the fourth week until the termination of the experiment at 20 weeks of age no clinical disease entities were observed in either group. From the weekly blood samples, no significant difference between groups for erythrocyte count, hemo— _globin level, packed cell volume, total leukocyte count, and monocyte or neutrophil values of the leukocyte dif- ferential was detected. However, highly significant values (P4 22 23 26 29 31 32 35 36 38 46 47 49 58 INTRODUCTION In the past decade considerable emphasis has been placed on elucidating the function of the thymus gland, especially its relationship to immune mechanisms. In the past the function attributed to the thymus was that it had some obscure, or certainly controversial function; possibly concerned with lymphOpoiesis, immunity, or sexual maturation; and the subject was dropped at that. Today researchers in the field generally concede that the thymus is concerned with immunologic develOpment, although many details remain controversial. Extensive research programs in the past five years have demonstrated the role which the thymus plays in early immunologic develOpment in some animal Species. A wasting syndrome has been demonstrated numerous times in neonatally thymectomized mice. Other Species of laboratory animals have also been used to demonstrate various degrees of depression which neonatal thymectomy will produce on immunologic capacity. Recently there has also been an increase in the number of publications on the subject of agammaglobulinemia and various thymic disorders. To date however, the effect of neonatal thymectomy in larger domestic species has not been reported. Within the past 10 years studies have been con- ducted to demonstrate transmissability of the animal lymphomatoses. Early workers deve10ped an inbred strain of leukemia-susceptable mice in order to gain better insight into the develOpment and characteristics of the disease. However, those working with bovine and canine leukemia's are hampered in this respect because of the longer generation time and the economics involved with these Species. Thus, investigative studies have been limited to spontaneous clinical cases. Since thymectomy at birth in a number of mammals has been shown to produce various degrees of immunologic incompetence, it is possible that thymectomy in newborn calves could result in an animal more susceptable to leukemia. Thy- mectomy would then be eSpecially valuable in transmission studies on the bovine species. This study was conducted to develOp a suitable procedure for thymectomy in the newborn calf, explore the feasibility of such an operation, and study some of the basic effects upon the animal. The particular effects this study is designed to observe are changes or variations in: (l) erythrocyte count; (2) hemoglobin level; (3) packed cell volume; (4) leukocyte count; (5) leukocyte differential; (6) total serum protein; (7) serum albumin and globulin; (8) weight gains; and (9) physiologic behavior. REVIEW OF LITERATURE Introduction Review of veterinary literature reveals an absence of information on neonatal thymectomy in species other than those normally considered to be laboratory animals. The only documentation on thymectomy in calves is by Papp (1960) who performed the operation on older calves. On the other hand, there are numerous reports on the effect of thymectomy in laboratory mice, rats, hamsters, rabbits, and even dogs. It is this material which serves as a framework for the design of this study. Anatomy Sisson and Grossman (1962) describe the thymus of the calf as a pale and distinctly lobulated gland which is divided into two connected parts. The thoracic part occupies most of the anterior mediastinal Space. It is bordered by the pericardium posteriorly, molded by the .great vessels on the right surface, bordered by the chest wall and the left apical lobe of the lung on the left, and is continuous anteriorly through the thoracic inlet with the cervical part. The cervical portion forms the bulk of the gland. It is divided into a right and left lobe which extend from the thyroid gland to the thoracic inlet along the esophagus and trachea. The two lobes are larger and apposed at the base of the neck where they cover the eSOphagus, trachea, carotid arteries, and vagosym- pathetic trunks. Part way up they become separate entities and gradually taper off on the sides of the trachea. The description of Mcleod (1958), Frandson (1965), and Trautmann and Fiebiger (1957) coincides with that of Sisson and Grossman (1962). Trautmann and Fiebiger (1957), Frandson (1965), and Andrew (1959) classify the thymus as an atypical endocrine gland, but many current texts on histology including Bloom and Fawcett (1962), COpenhaver (1964), and Maximow and Bloom (1957) regard the organ as being more closely allied to the lymphatic system. Trautmann and Fiebiger (1957) describe the microscopic anatomy of the thymus of domestic animals as 5 to 13 mm. pyramidal or polygonal lobules. Each lobule, on stained section, presents a dark peripheral cortex which may be partially subdivided by extensions of the surrounding connective tissue and a lighter medulla centrally. The fine meshes of the reticulum of the cortex contain vast numbers of small, compacted lymphocytes, or thymocytes, along with a few eosinOphils, plasma cells, and small stellate reticulum cells. The medullary area differs in that the ilymphocytes are fewer in number and less compacted. The reticulum cells, too, are not as compacted and appear larger. Peculiar to the medulla of the thymus of all species are Hassall's bodies. These are acidophilic, con- centric formations of epithelial reticulum cells which appear to be partially degenerated and hyalinized. According to Bloom and Fawcett (1962) the medullary tissue extends from one lobule to another. Trautmann and Fiebiger (1957) state that the blood vessels are ensheathed in active lymphatic tissue. The arteries lie in the general area of the cortico-medullary junction and divide into a dense network of capillaries which in turn are drained by veins on the surface of the lobule. Lymphatics also encircle the medullary area and are drained by interlobular lymph channels. Nerves are almost exclusively vasomotor. Embryology Bloom and Fawcett (1962), Andrew (1959), and Trautmann and Fiebiger (1957) describe the reticulum of the thymus in further detail. One of the main reasons some scholars will not classify the thymus as a lymphatic organ is that the thymic tissue itself, the reticulum, is of entodermal origin while lymphatic tissue is of mesodermal origin. It arises by outgrowths from the third and fourth brachial pouches. During this histogenesis the surrounding mesodermal lymphoid primordium is incorporated into the structure, thus resulting in a mingling of two germ layers. The epi- thelial nature of the entodermal reticulum is more noticeable in the medullary portions of normal glands than in the cortex. It is quite pronounced in the embryo, in the irradiated thymus where the lymphocytes are depleted, and also in tumors of the thymus. Although no reference to the eXplicit embryology of the calf thymus could be found, Ruth (1964) mentions the fact that in the pig, chicken, and possibly in man and all mammals the thymus may have both an ectodermal and entodermal origin. This leaves the status of the embryology of the calf thymus in a state of conjecture. Physiology Physiologically much remains to be learned about the thymus; however, certain basic phenomena and func- tions have been described. Good (1964) and Hammar (1921) state that the thymus is largest in relation to the body at birth and gradually grows until puberty, but at a much lesser rate than the body in general. At puberty the gland begins to involute and all but disappears in some species according to Fisher (1964) and Trautmann and Fiebiger (1957), while in other species, particularly dogs and cattle, remnants of the thoracic part will remain until old age. Accidental involution is not an uncommon entity. In the early part of this century it was probably this factor more than any other which caused the great con- fusion over the relationship of the thymus to certain childhood disease entities. Hammar (1921) wrote an extensive treatise reviewing and discussing previous work and current concepts on the thymus. It was evident to him that many references to the "normal“ thymus were based upon postmortem findings in children who had died of a debilitating disease or inanition. By showing that these factors had caused the thymus to involute, he refuted the concept of "status thymicolymphaticus"--a widely held concept of the day which implicated the thymus in many unexplainable childhood deaths based upon .gross evaluation of thymus size. Possibly due to the superficial treatment given to the study of the thymus in basic courses in veterinary medicine, accidental involution in animals is a less often reported entity. Texts on anatomy such as Sisson and Grossman (1962) and Trautmann and Fiebiger (1957); or physiology, such as Dukes (1955) mention its occur- rence only in passing. Turner (1955) reported that adrenal corticosteroids, whether from exogenous or endogenous sources, as well as androgens, estrogens, _gonadotr0phins, or infections and intoxications cause involution. Trautmann and Fiebiger (1957) add that other causes of accidental involution include exhaustion, mal- nutrition, cachectic and debilitating diseases, and radiation. Fisher (1964) states that almost any "stress" will result in thymic involution. Radiation and corti- costeroids are two common means by which thymic involu- tion can be produced either accidentally in therapy, or for experimental purposes. Hammar (1921) recognized the marked reactability of the thymus during periods of stress in his conclusion that the thymus is not just a dormantnvestige. From the reports of Trautmann and Fiebiger (1957), Good (1964), and Hammar (1921) it appears that the thymus normally begins to involute at the time of puberty in all mammals studied; a phenomenon termed age involution. Again, early scholars were confused by the turmoil of establishing a "normal" thymus size and accidental involution. Fisher (1964) states that involution, whether accidental or age, can only be determined by microsc0pic and histologic means, an important criterion not considered by earlier researchers. 10 Hammar (1921) gave one of the first systematic descrip- tions of age involution of the thymus. Hammar's (1921) description was complete and accurate and has weathered the test of time. Bloom and Fawcett (1962), Copenhaver (1964), and Maximow and Bloom (1957) describe the involutionary processes much the same as Hammar had 40 years before. Briefly, the thymus is largest in relation to total body size at birth, but continues to grow until puberty, which is between 11 to 14 years in man according to Hammar (1921), or 6 to 18 months in cattle according to Roberts (1956), after which growth ceases and it begins to involute. Lymphoctes gradually disappear from the reticular net— work and adipose and fibrous connective tissue slowly replaces the parenchyma. Age involution is not a dramatic and definite process, but rather the slow disappearance of lymphatic tissue with an encroaching connective tissue proliferation. This process never becomes complete and traces of thymic tissue may still be present in aged individuals according to Hammar (1921), Dukes (1955), and Trautmann and Fiebiger (1957). The function of the thymus has been very obscure and often debatable. Good (1964) reviewed the history of the thymus and found that as early as the 17th century, literature appeared describing the thymus with some conjecture as to its function. Late in the 11 19th and early in the 20th centuries, because of the popular concept of status thymicolymphaticus, enthusi- astic research was conducted in an attempt to delineate the function of this organ. Hammar gathered informa- tion from most of the earlier investigations and develOped a format which serves as a basis for much of the current work. In one of his most comprehensive works, Hammar (1921) established 5 major premises describing the histogenesis, anatomy, physiologic behavior, pathologic variations, and postulated cell controlling hormones. However, by disproving the concept of status thymi- colymphaticus, there appeared to be no pertinent clinical need for further studies on the thymus and it became an unpopular research subject for several decades. Current interest developed simultaneously from several sources during the 1950's. Good (1964) develOped interest in the function of thymus after being confronted with a patient exhibiting both an acquired agamma- iglobulinemia and a thymoma. He considered these rare conditions occurring together as too significant to be passed off as chance, and investigated the problem by studying thymectomized adult rabbits. However, from his unfortunate choice of subject, in retrospect, he could conclude nothing and consequently discontinued 12 his experiments. More recent work has shown this relationship more clearly, such as that reported by Burnet (1962a) and Good (1964). About this time Chang (1955) discovered the immunologic role the thymus-like bursa of Fabricius plays in the chick. In preliminary studies Glick (1955) demonstrated a bursa-testes relationship and a relation- ship with other endocrine glands including the pituitary, thyroid, and adrenal glands which affected the bursa's development. He then bursectomized chicks at 2 to 26 days of age; but as others had found before, he could not demonstrate any gross effects produced by its removal. Six months later, 9 birds were borrowed by Chang, a fellow graduate student, for a class demonstration in immunology. Six of the birds injected with Salmonella typhimurium antigen died immediately, while the remaining 3 failed to produce any demonstrable antibody. Bursec— tomy and injection of S. typhimurium antigen was repeated in other breeds of chickens and a link between the bursa of Fabricius and the immune mechanism was revealed. Not dismayed by earlier failures, Good (1964) returned to studying thymectomized animals, this time utilizing newborn rabbits. By challenging them.with bovine serum albumin at 7 to 8 weeks he was able to demonstrate a significant reduction in antibody response, which varied with the type of antigen and 13 was insignificant if the thymectomy was not performed during the immediate neonatal period. Miller (1961a) is probably the first to report the phenomenal syndrome in mice following neonatal thymectomy. Prior to this time the only well-established function of the thymus was lymphopoiesis. Evidence to support endo- crine and immunologic functions was far from conclusive. He found in his experiments that if thymectomy was per- formed after the neonatal period, at 3 weeks, no overt differences from controls were detected. However, when thymectomy was performed on newborn mice, they appeared normal until 3 to 4 weeks of age, then develOped a wasting syndrome characterized by weight loss, diarrhea, lethargy, ruffled hair, and a hunched posture with death ensuing in l to 3 weeks. Parrott (1962a) and Jancovic/(l962) duplicated and confirmed this work. Miller (1964) reported that attempts to save these afflicted mice by high protein, high vitamin diets and the therapeutic use of broad spec- trum antibiotics failed; however, by reimplanting the thymus a 70 % recovery rate was achieved. On necropsy a common finding in wasted mice was depletion of small lymphocytes from the peripheral lymph nodes and spleen. When peripheral blood was studied an analogous deple- tion of lymphocytes was discovered although other blood cell types were not significantly different. 14 After the initial work of Miller (1961a) and Metcalf (1956) two theories developed regarding the function of the thymus. Metcalf believed a hormonal "plasma lymphocyte stimulating factor" was the mechanism by which the thymus functioned in immunity. Burnet (1962b) proposed that the thymus produced potential _germ cells which seed various lymphoid organs during early life, a theory which Parrott (1962b) tended to support. Although Miller (1961a, 1962a) accepted this theory he could not refute Metcalf's theory and tended to combine the two. Profound success stimulated research initiative and it appeared that the field was just beginning to Open. Archer (1961) and Glick (1956) had already demonstrated diminished antibody response in other species, and Miller (1961a, 1962b) was able to demonstrate complete survival of skin homofgrafts on thymectomized mice. He dis- covered that as the time lapse before thymectomy in- creased, a poorer degree of homograft survival occurred, up to a point of about 3 weeks, after which homograft immunity was apparently normal. This work and variations of it were soon replicated by Martinez (1962a, 1962b, 1964a, 1964b). The theory that the thymus was the seat of immunologic development became more widely accepted, as exemplified ina report by Miller (1964b), although the exact mechanisms still needed clarification. 15 An interesting sidelight now appeared unpredict- edly. For some time it had been known that thymectomy at 1 to 2 months of age prevented development of leukemia in inbred susceptable strains of mice as well as its development from usual carcinogenic methods. Miller (1961b) using mice and later Kunii (1965) using rats attempted to induce leukemia with viruses in neo- natally thymectomized animals but met with failure. Miller offered a simple explanation by attributing the resistance to leukemia to an absence of leukemia-pre— cipitating factors within the thymus. This is further supported by Siegler (1964) in view of the fact that lymphoma of mice is primarily of thymic origin. Furth (1964) has performed extensive research on the thymic lymphoma factor and its relationship to viruses known to induce leukemia. Although Hammar (1921) and Metcalf (1956) proposed that humoral factors were produced by the thymus it remained a very controversial subject until recently. Lymphopoiesis was one of the earliest established functions of the thymus, but Miller (1962b), in light of recent developments, explored the mechanisms of this function in further detail. By thymectomizing mice short— ly after birth and later reimplanting a donor thymus he found the mice did not suffer from wasting disease and 16 the thymus graft and the host's lymphopoietic system appeared to be functioning normally. However, auxiliary information from these studies opened a new realm of thymic function. Lymphocytes of the implanted thymus as well as lymphocytes multiplying in the host‘s lymphoid organs were of host rather than V donor origin. Secondly, transplanting a thymus from a young mouse into an old mouse whose thymus was deteri- orating resulted in a rapid rate of lymphOpoiesis as would be expected in the thymus of a young animal. This evidence tended to support the contention that a humoral factor may be secreted by the thymic reticulum which stimulated lymphOpoiesis in dormant lymphoid organs of a neonatally thymectomized host. Metcalf (1956) origi- nally reported a hormone produced by the thymus and found in high levels in human leukemia patients. Levey (1963) set out to further investigate the stimulating influence of the thymus. By implanting a donor thymus enclosed within a Millipore filter into a neonatally thymectomized mouse, he was able to produce results similar to implanting an intact thymus directly. Furthermore, he demonstrated the immunologic competence of these mice, which were previously incompetent; thus demonstrating conclusively the presence of a non-cellu- lar lymphocyte stimulating factor. Osoba (1963, 1965) 17 was able to confirm this reaction in mice, and Trench (1966) produced similar results in rabbits. To date much of the basic information has been obtained from mice. The obvious question is whether similar results can be obtained for all species, or is the mouse the only animal which is capable of such a dramatic reSponse. After the initial discovery of the wasting syndrome, new discoveries led to questioning the theory shared by Miller (1961a) and most others that the wasting syndrome was due to a form of self-immunity. McIntire (1964) and Wilson (1964) concluded that a latent infectious agent was reSponsible for the wasting and death of mice, because they were unable to demon- strate such a syndrome in germ-free animals. This in part would explain the relative absence of a wasting syndrome in other species. Results in other species have been variable. Sherman (1963) reported a fatal wasting syndrome in hamsters occurring only in the males; however, females would undergo the wasting syndrome if 00phorectomized and treated with testosterone. Balner (1966) recently reported a tendency for a more pronounced wasting in male mice than in females. Defendi (1964) demonstrated an early, atypical wasting syndrome in male and female hamsters in which losses and symptoms occurred only during the first 3 post-thymectomy weeks, after which 18 survivors appeared normal. One cannot help but wonder if the more extensive trauma of neonatal thymectomy as compared to the sham Operation is responsible for these results. Wasting syndrome in rats is rarely reported although the neonatally thymectomized rat will undergo some degree of immunologic depression of the delayed or cellular type, and a loss in ability to form humoral antibodies according to Arnason (1962a, 1962b, 1964) and Defendi (1964). Associated with this is a deficiency of small lymphocytes in the peripheral circulation and in the remaining lymphoid tissues. An analogous condition in the rabbit is reported by Archer (1962, 1964). Thymectomy of newborn dogs was reported by Tilney (1965) to produce a classical wasting syndrome including loss of condition, lymphoid hypoplasia and reduction in Vgamma_globulins. Although VandeWater (1964) observed no such syndrome, Tilney suggested that those who met with failure in attaining similar results perhaps did not remove the entire gland or left fragments behind which regenerated. Chickens have been used almost as extensively as mice in thymus studies, and have played an important role in first deve10ping the link between thymus-like organs and immunity. The chicken has both a cervical thymus and a thymic analog, the bursa of Fabricius. 19 This latter organ was reputed to function as everything from a storehouse for semen, secondary sex gland, or egg reservoir to an auxiliary urinary bladder, cloacal lubricator, or third cecum as Glick (1964) mentioned in his review. It was not until he and his associate Chang (1955) discovered immunologic suppression in neonatally bursectomized chickens that any plausible function of the bursa could be substantiated. Meyer (1959) performed hormonal thymectomies by injecting testosterone into the incubating egg on the fifth day and Mueller (1964) found the effect of this method comparable to postnatal surgical thymectomy. However, Warner (1962) found that by thymectomizing and bursectomizing the chick he was able to demonstrate a homograft immunity impairment which bursectomy alone did not alter. No attempt to study neonatal thymectomy in calves has been reported. Papp (1960) reported a significant lymphopenia on calves thymectomized at approximately 3 weeks of age. Coleman (1964)* performed neonatal thymectomies in calves, but to date no followup report has been published. *Coleman, Gerald L.; Personal communication. Pesticide Regulation Division, USDA, ARS, Washington, D.C. 20250 20 From data accumulated over the past few years current reviews such as Miller's (1965, 1966) and Good's (1966) favor the theory that the thymus is essential in the prenatal or neonatal period to produce small lymphocytes as precursors to form immunologic competent lymphoid centers throughout the body, and that lympho- poiesis, especially when related to immuno—capable forms, is regulated by a thymus reticular hormone throughout life. But as Good (1964) suggests there may be other functions of this long—mysterious gland. Furth (1964) reported work implicating the thymus hormone directly in leukemogenesis, but further research in this aspect is needed. A more recent development has been Rieke's (1966) work reporting that lymphocytes in neonatally thymectomized rats contain a defective ribonucleic acid (RNA). In still another aspect of thymus function Pansky (1965) has reported finding an insulin-like substance produced by the thymus which he feels is reSponsible for post-thymectomy hypoglycemia. Another particular line of research involving thymectomized animals has been in transmission studies of viral neOplasms. As previously cited there is a decrease in the incidence of leukemia following thy- mectomy; however, this decrease is attributed to a lack of thymus per §e_in mice, which is apparently necessary 21 as a primary nidus for infection. This has recently been substantiated by Haran—Ghera (1966) who found foci of potentially neOplastic cells in the thymus only one week following the direct injection of a leukemogenic virus into the gland, whereas no effect was encountered when the agent was injected into other lymphoid organs. After discovering the impairment of immunity following thymectomy Miller (1963, 1964a) indicated this tool may have other potential. Perry (1963) and Vandeputte (1963) were able to demonstrate a significant increase in tumors in thymectomized rats. Defendi (1964) observed a similar decrease in resistance to the polyoma virus in hamsters and mice. Grant (1965) reported an increase in sarcomata in mice resulting from injected carcinogens. Law (1966) found the thymus necessary in oncogenic immunity. MATERIALS AND METHODS Ten newborn, colostrum-deprived Holstein bull calves were obtained from area dairy farms and were alter- nately selected and placed into either the thymectomy or control groups. Upon arrival at the clinic each calf was fed 2 quarts of a "standardized“ colostrum and given a physical examination. The "standardized" colostrum was previously obtained from several cows, mixed, and frozen as Allen (1944), Hansen (1947), Jacobson (1951), and Morrison (1957) have recommended. The physical examination included: a rectal temperature reading, auscultation of the heart and lungs, observation for anomalies or physical defects, assessing the general condition, and examination of the mucous membranes. Following feeding and the physical examination, 10,000 units procaine penicillin G per pound of body weight was injected intramuscularly as a general prOphylactic measure . 22 23 Surgical procedure Several hours before commencing surgery, each animal was injected with a 0.01 mg./kg. intramuscular dose of 9-alpha-fluorOprednisolone acetate* as a prOphylactic measure to allay surgical shock. AtrOpine was administered at a dose rate of 0.01 mg./kg. by intravenous injection to inhibit secretory activity and to suppress vagal activity since the surgery necessitated handling this nerve trunk. The left thoracic wall was clipped from the mid-abdominal region anteriorly over the shoulder, and from the sternum to the Spinal column-- using a fine clipper head-** The ventral half of the neck, from the sternum to the intermandibular space, was also clipped. Calves were anesthetized while standing, using a mask on an open circuit gas anesthetic apparatus employing methoxyflurane*** and placed on a table in lateral recumbency. The mask was removed and the animal was intubated with a size 40, cuffed endotracheal tube. The surgical site was prepared for aseptic surgery. In * Predef 2X, Upjohn, Kalamazoo, Michigan ** Oster Small Animal Clippers, Size 40 head, Milwaukee, Wisconsin ***Metofane, Pitman—Moore, Indianapolis, Indiana 24 securing the animal suitably for the surgical procedure, the left foreleg was extended over the animal's head, thus exposing the entire left thoracic wall. The other limbs were secured in their normal extended position. The surgical procedure employed was a modification of Coleman's (1964).* The left thoracic area was covered with a sterile drape which permitted longitudinal exposure of the third rib. After controlling capillary bleeding in the incised skin and fascia, the incision was continued through all muscle layers down to the lateral surface of the rib. A 12 cm. segment of the third rib was then resected, from the costochondral junction dorsally, and entrance was made intoihe thoracic cavity. Using a Balfour retractor the opening was stretched transversly; thus, the anterior portion of the thoracic cavity was exposed from approximately the anterior aSpect of the heart to a point just posterior to the thoracic inlet. The left apical lobe of the lung was manipulated posteriorly thus exposing the thoracic portion of the thymus in the anterior mediastinum. A portion of the ,gland was picked up and by blunt dissection, freed from surrounding structures. Extreme care was necessary *Coleman, Gerald L.; Personal communication. Pesticide Regulation Division, USDA, ARS, Washington, D.C. 20250 25 throughout the tedious dissection, eSpecially when work- ing next to the heart so as not to disrupt the pericar- dium, and when removing the more dorsal portions of the Agland through which the vagosympathetic and phrenic nerve trunks coursed. Blood vessels of the gland, branches of the internal thoracic vessels and the anterior vena cava, were ligated. Blunt dissection was continued until the entire thoracic portion of the gland was removed. Closure was effected with a continuous lock-stitch pattern using No. l chromic catgut. The stitch pattern did not encircle the periosteum of the resected rib, but rather passed through the doubled thickness of the anterior and posterior halves of the periosteum thus apposing the original incised edges. The suturing was started from each end of the incision and just prior to closure the lungs were inflated and the pleural cavity was evacuated. Muscle layers were closed individually with a similar lock-stitch pattern, and the skin was closed with simple interrupted sutures employing a non-capillary, synthetic suture material*. An assistant now repositioned the animal in dorsal recumbency and prepared the ventral cervical surgical site. The area was again shrouded and a bold incision *Vetafil, 0.4 mm., Bengen, West Germany, Dr. S. Jackson, Bethesda, Maryland 20014 26 was made on the midline from the mandibular rami to the thoracic inlet. Following incision of the underlying fascia the ventral muscles of the neck were bluntly separated. This exposed the complete cervical portion of the thymus. As with the thoracic portion of the thymus, the cervical portion was removed by blunt dissection. Difficulty was experienced in removing the small portions of the gland located dorsolaterally to the trachea adjacent to the submaxillary salivary glands because of the proximity to the thyroid gland and the carotid arteries, and because Of its poorly accessible location and a strong fibrous attachment. Excised tissue from this area was later confirmed to be thymus by histologic examination. Muscles and fascia were apposed with a single continuous lock-stitch pattern and the skin was closed using material and techniques previously described. The sham Operation on control animals included all of the procedures used in thymectomy with the exception of extirpation Of the gland. Surgical procedure for removal of the entire thymus lasted from 3 to 3-1/2 hours. Postsurgical care Postsurgical care included treatment for surgical Shock and hypothermia. Two hundred fifty milliliters of 5 % dextrose in 0.85 % physiological saline warmed to 27 body temperature and administered intravenously. For severely depressed individuals amphetamine sulfate* was injected subcutaneously and intravenously. Follow- ing return to a pen, the calf was covered, warmed with hot water bottles,.and Observed until able to stand unassisted, a period of time usually not exceeding 1 hour. As a routine regimen, 10,000 units procaine penicillin G per pound Of body weight was injected intramuscularly daily for a minimum of 10 days. Diarrhea, which frequently became a problem in both groups, was controlled by the oral administration of a neomycin— anticholinergic preparation.** Fever during the first few postoperative days was controlled with the aid of aSpirin; 60 gr. b.i.d. the first day and 30_gr. b.i.d. the second day, administered per gs. After the initial 10 day period, treatment was allayed unless absolutely necessary, in which case appropriate measures were taken. * Amfetasul, Pitman-Moore, Indianapolis, Indiana **BiOSOl—M, Upjohn, Kalamazoo, Michigan 28 All calves were fed a synthetic milk diet* following their first and only meal of colostrum. As they learned to eat a pelleted calf meal the diet was .gradually changed to a pure skim milk milk-replacer. The initial feeding rate was 1/2 liter Of reconstituted milk-replacer per 10 kg. body weight and an attempt was made to increase this level to 1/2 liter per 5 kg. body weight by 1 week of age. However, the initial level of feeding was reverted to whenever the first sign Of enteric disorder appeared. The calves were maintained on a skim milk diet until they consumed the pelleted feed at a rate Of 1/2 kg. per day. Thereafter, according to the manu- facturer's directions,** the calves were gradually phased Off Of the liquid diet and placed on a complete dry ration consisting of pellets and a ground grain mixture. Fresh, second-cutting hay and water were available at all times. At approximately 3 months of age the pellets were completely deleted from the grain ration. . * V-2 Vealer, Mutual Products Inc., Minneapolis, Minnesota . **Calf Manna, Albers Milling Company, Kansas City, Missouri 29 Hematologic determinations At birth, and at weekly intervals for 20 weeks, 2 blood samples were taken from the 5 animals in each group. One 7 ml. sample was collected in a tube con- taining 0.05 ml. Of 15 % tripotassium ethylene-diamine- eteracetic acid (EDTA) anticoagulant to be used for cell studies. The other 7 ml. sample was permitted to clot and the serum was separated and frozen at -7 C. for subsequent protein analyses. Total erythrocyte and total leukocyte counts were performed on the Coulter Counter* using duplicate samples. Duplicate determinations Of the packed cell- volume (PCV) were performed using the microhematocrit method. Hemoglobin (Hb) was determined by the cyan- methemoglobin spectrophotometric method. Weight gains were assessed by biweekly weighings. Animals were authanatized and necrOpsied at 20 weeks of age. The thymectomized animals were examined carefully for remnants or regeneration of thymus tissue. Any tissue which could possibly be considered thymus was submitted for histological examination. Specimens of liver, kidney, lung, spleen and various lymph nodes were *Coulter Electronics, Model b, Hialeah, Florida 30 submitted for histologic examination along with any specific tissue showing gross lesions. Total serum protein and serum albumin were determined by the "Improved" Biuret Method, Ferro-Ham modification (1955)* as discussed by Bray (1957). The albumin value was subtracted from the total protein value to determine the globulin value. The albumin: ‘globulin (A:G) ratio was determined from these values. The data Obtained were processed employing trend analysis. *Lab—trol Manual of Clinical Methods (1963) Dade Reagents, Inc., Miami, Florida RESULTS Following the extensive procedure of surgical removal Of the thymus, a state of shock ensued as evi- denced by marked hypothermia (as low as 35 C.), rapid, weak pulse, and a comatose condition. Since Operative hemorrhage was minimal, shock was judged to be the result Of a long period Of general anesthesia (up to 5 hours) and trauma inherent to removal Of tissue from a large area. PostOperative recovery was marked by hyperthermia, Often as high as 43 C., which persisted for at least 2 weeks despite antibiotic therapy. Calves in both the thymectomy and control groups were frequently troubled with an enteritis during the initial 4 to 6 weeks during which time their diet was primarily liquid. At the first indication of an enteric disorder, the amount of liquid diet was decreased by one-half and apprOpriate antimicrobial and palliative measures were instituted. Of the bacterial organisms isolated from fecal specimens, Escherichia coli was the only one that could have been incriminated as an etiological agent, with the exception of the isolation 31 32 of Salmonella EE- from one calf. Rarely was the anti— biotic Of choice (as indicated by sensitivity tests) effective in controlling diarrhea. Between the ages of 4 to 6 weeks the calves began to gain weight rapidly and did not exhibit any further clinical illnesses. Hematologic findings The data were processed employing trend analysis. When the thymectomized calves were compared with the non-thymectomized control calves, no significant differences were noted for total erythrocyte counts (Appendix, Table l), the hemoglobin values (Appendix, Table 2),the packed cell volumes (Appendix, Table 3), or the total leukocyte counts (Appendix, Table 4). Differential leukocyte counts Lymphocytes. NO significant difference was noted between the 2 groups for absolute lymphocyte counts. However, the thymectomized group had a considerably lower relative lymphocyte count. Analysis showed that the mean values for the 2 .groups throughout the 19 week period (factor B) approached a significant difference, with an approximate probability of F statistic (i.e. the 33 probability this situation would occur by chance) of 0.06. (Appendix, Tables 5a and b.) Neutrophils. NO significant difference was noted between the 2 groups for either absolute or relative neutrOphil counts. (Appendix, Tables 6a and b.) Moncytes. NO significant difference was noted between the 2 groups for either absolute or relative monocyte counts. (Appendix, Tables 7a and b.) EosinOphils. NO significant difference was noted between the ngroups for absolute eosinophil count. However, the thymectomized group exhibited a relative eosinOphilia. The means averaged throughout the 19 week period (factor B) approached a significant difference with an approximate probability Of F statistic Of 0.08. (Appendix, Tables 8a and b.) BaSOphils. A significant baSOphilia, both absolute and relative was detected for calves in the thy- mectomized group. The average mean values through- out the 19 week period (factor B) were significantly different at the P“0.0l level with an approximate probability of F statistic of 0.00 for the absolute values and 0.01 for the relative values. (Appendix, Tables 9a and b.) 34 The following hematologic determinations were performed on the serum collected at weekly intervals: Total protein Thymectomized calves had a higher total serum protein level throughout the 19 week period (factor B) which was significant at the P“0.01 level. The approx- imate probability of F statistic was 0.00. (Appendix, Table 10.) Serum albumin Thymectomized calves had a lower serum albumin level throughout the 19 week period (factor B) which was significant at the P“0.0l level. The approximate probability of F statistic was 0.00. (Appendix, Table 11.) Serum'globulin Thymectomized calves had a higher serum globulin level throughout the 19 week period (factor B) which was significant at the P“0.01 level. The approximate probability of F statistic was 0.00. (Appendix, Table 12.) Albumin:globulin ratio Thymectomized calves had a lower albuminzglobulin ratio throughout the 19 week period (factor B) Wthh was 35 significant at the P <0.01 level. The approximate probability of F statistic was 0.00. (Appendix, Table 13.) Weight gains Weight gains were assessed by a biweekly weigh- ing. Cumulative gains, using birth weight as base zero were employed to equilibrate gains regardless of initial weight. Thymectomized calves gained significantly less weight throughout the 19 week period (factor B) which was significant at the P"0.01 level. The approximate probability of F statistic was 0.00. (Appendix, Table 14.) The control calves had a mean weight Of 27.6 kg. or 1.8 kg. more than the thymectomized calves which had a mean weight of 25.8 kg. Due to lack of foresight the calves were all weighed at two week intervals rather than weighing each calf every other week of his life starting from birth. This resulted in an alternate week arrangement Of data for the different calves so that in any given week of their lives actual weights were available for only some of the individuals. Therefore, when the data were selected for analysis they were based upon a two week weighing period and the figures presented in Table 14 36 would not be the actual average weight gains for every other week of all calves' lives. However, a sum of weights which was projected by the analysis with a mean net gain of 64.4 kg. for the thymectomized group and 69.0 kg. for the control group compared almost exactly with a mean of the actual final weights. All calves with the exception of T4 and Cl had weight losses as great as 5 kg. during the first few weeks after birth. At about 4 to 6 weeks of age all calves began a rapid growth period. Necropsy: Gross and histolOgic observations Gross pathological findings were minimal. The terminal portion of one ureter in thymectomized calf T3 was involved with a chronic inflamatory process. In 2 of the thymectomized calves, T1 and T4, small remnants of tissue were removed from the postmandibular area which were later histologically confirmed as thymus tissue. In all other thymectomized calves careful examination revealed no traces Of thymus tissue. Histologically the lungs showed the most frequent, although minor, pathologic involvement. Calves C2, C3, C4, T3, T4, and T5 had various pneumonic involvements. Renal disorders included glomerular atrOphy in calf T2, glomerulitis in calf C5, and accumulations of 37 lymphocytes in various areas of the kidneys in calves C1, C4, C5, and T1. Splenic involvement included decreased lympho- blastic activity in calf T1, and neutrOphil infiltration of the red pulp in calves C3, T1, T3, T4, and T5. The liver in calf T4 had neutrophilic infiltration in the hepatic sinusoids and lymphocytic infiltration in the portal triads. Lymph node involvement was minor and varied coin— cidental with the pathology mentioned above. However, of pertinent, general interest was the mild reactivity of the lymph nodes in calves T1 and T5; the hypoplasia of the germinal centers of the lymph nodes of calf T2; the eosinophil infiltration Of the lymph nodes of calves C2, T3, and T4; and the neutrophil infiltration of the lymph nodes of calves Cl and T3. DISCUSSION The problem of rearing calves, subjected to extensive surgery within 24 hours after birth, and deprived of colostrum except for an initial hand feed- ing is difficult. Numerous experiments have been con- ducted which point out the paramount necessity of feed- ing colostrum. Smith (1948), Morrison (1957), and Ingram (1958) state that colostrum is absolutely necessary for prOper antibody protection as well as for nutrients necessary for growth. Davis (1962) adds that the calf has practically no antibodies during the first 48 hours of life. Smith (1922) was probably one of the first to run a controlled experiment on colostrum feeding. He found that two-thirds of the group deprived of colostrum died within the first few weeks of life while all the colostrum fed control animals lived. Hansen (1947) demonstrated immediate increases in serum globulins following colostrum ingestion. Miles' (1948) work supported Smith's earlier study. He also noted the runted condition of colostrum deprived calves which survived. Recent work by Jacobson (1951) and Gay (1965), further support the importance of colostrum during the first few days Of neonatal life. 38 39 Sweat (1965) derived calves by hysterectomy and raised them in isolation without colostrum. His initial experiment failed when the animals died of a coliform septicemia after changing from a sterile diet to milk replacer on the third day. Further attempts, maintaining the animals on sterile diets, did prove more successful. DeSpite the new, clean facilities in which these experimental animals were housed and the sanitary technique used in food handling, every calf suffered repeated attacks Of enteritis and consequential systemic sequelae. Antibiotics, anticholinergic drugs, and intravenous fluids were employed as needed to maintain the calves, but these measures seemed to be of only temporary value. Enteritis would soon reappear follow— ing cessation of medication. It is the Opinion of this author that without the antibodies which a calf can apparently Obtain only from ingestion of colostrum early in life, antimicrobial and symptomatic treatment are of only palliative value. From the analyses of data it is apparent that thymectomy Of neonatal calves has no effect on their erythrOpOietic capabilities. Likewise, total leukocyte counts did not differ significantly between groups. A mean of 9,169 leuko- cytes/cmm., falls well within the normal ranges 40 established for cattle, although no specific references were available for Holstein cattle during early life. An analysis of the leukocyte differential does Show a statistically significant difference between groups. LymphOpenia has been one of the most character- istic findings of the wasting syndrome as Miller (1961a) and others have demonstrated. In this experiment, how- ever, no significant differences were noted between ‘groups although the thymectomized calves had a consider— ably lower relative percentage of lymphocytes which approached statistical significance at the P-t0.05 level. In the concept of trend analysis, had more animals or a longer period of time been employed, a significant difference between the 2 groups may have been noted. NO significant difference was noted between the 2 groups for either absolute or relative neutrOphil counts. Both groups had extreme variance which ranged from a low of 180 neutrOphilS/cmm. which constituted 5 % of the leukocytes for one calf to a high of 29,050 neutrOphils/cmm. which constituted 89 % of the leukocytes for another calf. Since individuals of both ,groups diSplayed similar variation which could be roughly correlated to attacks of enteritis, the author concludes that the variability seen is prOportional to the challenge presented to the individual. 41 The reason for a baSOphilia in the thymectomized ’group is not readily apparent. Schalm (1961) mentions that the function Of the baSOphil is very Obscure. It is believed they are related to tissue mast cells since the granules of both contain heparin which indicated an anticoagulant activity. Guyton (1962) comments that basophil numbers increase slightly during the healing phase of inflammation as well as during chronic in— flammatory processes. Benjamin (1964) states that a baSOphilia Often occurs concurrently with an eosinOphilia. The normal mean value for baSOphil count for cattle is 0.5 % or 40 cells/cmm., according to Schalm (1961). 'These values coincide with those presented by Benjamin (1964). The mean value for the control group is considerably above this at 0.9 % or 74 cells/cmm., and the thymectomized_group had a mean value significantly higher than this at 1.2 % or 101 cells/cmm. No reports were found in which other research workers found a baSOphilia following thymectomy. Serum protein values were significantly different between the 2 groups. Because of an extremely small amount of variation among calves from week to week, the thymectomized group has a significantly higher total protein level at 5.25 gm./100 ml. vs. 5.23 gm./100 ml. for the control group; a lower albumin level of 3.18 gm./ 100 ml. vs. 3.25gm./100 ml.; a higher globulin level 42 of 2.07 gm./100 ml. vs. 1.98 gm./100 ml.; and a lower A:G ratio of 1.61 vs. 1.74. The finding of hypo— globulinemia in some thymectomized Species was not Observed in this work. Hypoalbuminemia and a low A:G ratio, on the other hand, is typical of debilitated or cachechtic animals. These values for serum proteins are considerably different from those put forth by Dukes (1955). The values he gives for cattle are: 7.60_gm./100 ml. total protein, 3.63 gm./100 ml. albumin, 3.97ng./100 ml. globulin, and 0.91 A:G ratio. None of the available literature gave age, sex, or environmental differences for serum protein values in cattle. The initial fluctuations in the weight of the calves were judged to be a result of colostrum depriva— tion and the neonatal surgery. Calves in both groups experienced similar weight losses which persisted up to 3 weeks in some instances. Since no abrupt changes were made in the diet and each calf was individually phased off the liquid diet as he consumed more of the dry ration, the lack of substantial weight gains prior to 5 or 6 weeks of age was judged to be in part due to factors other than nutrition alone. Smith (1965) and Ingram (1965) State that depend— ing on the type of antigen, calves can develOp few 43 antibodies prior to 1 month of age. Prior to this age the calves in this experiment frequently had enteritis and suffered from sequential anorexia. However, at the age Of 4 to 5 weeks these calves ceased having enteric disorders, and appetites and gains increased concom- itantly. In the judgement of this author the inability of these calves to combat infectious diseases during the first month of life was reSponsible in part for the lack of good general health and weight gains. Of particular interest in necrOpsy findings was the fact that 2 of the thymectomized calves retained small amounts of thymus tissue. The amount present was judged to be minimal considering the mass of thymus tissue removed from these calves. Whether remnants Of thymus tissue can profoundly affect the results of this experiment cannot be accurately assessed. Tilney (1965) suggested that subtotal thymectomy in dogs essentially negated the entire effect; however, other workers have been unable to duplicate Tilney's result deSpite scrutinous extirpation of the thymus of dOgs. Although accurate statistical analysis is not feasible, observation of the data does not reveal any noticeable difference between calves T1 and T4 and the rest of the thymectomized group. HistOpathOlogic findings suggest no difference between the 2 groups. Both appeared to be equally 44 afflicted with various pneumonic or renal pathologic processes. However, of interest is the preponderance of reticuloendothelial disorders in the thymectomized calves. Here again the diversity of abnormalities Observed obviates drawing any specific conclusion. Since Miller (1961a) and others have Observed depletion of lymphocytes from lymphoid organs in thymectomized animals,the author feels it is pertinent to point out only that these abnormalities are almost exclusively limited to the thymectomized_group. Immunologic studies were beyond the sc0pe of this experiment and empirical Observations of the general health of the calves can give only a crude estimate of the immunologic capabilities of thymectomized calves. The lower relative lymphocyte and eosinOphil values could be of importance, however, when their place in current immunologic theories is Observed. Whether thymectomized calves will be of value in virus or neoplasm transmission studies remains to be explored. Theilen (1965) and Dungworth (1964) reported thymus involvement in the calf form of bovine leukemia. This could mean the thymus contains necessary factors in the evolution of this disease as Miller (1961b) pointed out in his work with mice. On the other hand, Siegler (1964) reported that the mouse form of leukemia 45 is primarily Of thymic origin, whereas, Dungworth (1964) and Theilen (1965) did not find thymus involve- ment in all cases of leukemia in calves. CONCLUSIONS Neonatal thymectomy in calves is a feasible surgi- cal operation. Raising calves with only a small initial feeding of colostrum is difficult and is probably the main factor resulting in frequent enteritis and general unthriftiness. Neonatal thymectomy has no observable effect on the erythrOpOietic system. While not producing an observable effect on the total leukocyte count, neonatal thymectomy altered the leukocyte differential. A highly significant baso— philia, both relative and absolute, occurred in the thymectomized animals. A tendency toward an eosino- philia and a lymphOpenia was also observed in the thymectomized_group. Neonatal thymectomy produced a highly significant difference in serum proteins. The thymectomized group had higher total proteins and serum globulin level, and a lower serum albumin level and A:G ratio. Neonatal thymectomy resulted in reduced weight _gains which were highly significant. 46 SUMMARY During the past decade the lack of immune com- petence produced in various species of laboratory animals by neonatal thymectomy has been repeatedly demonstrated. This experiment was conducted to explore the feasibility Of such an operation in calves and to study some of the basic effects upon the animals with the possibility of using this procedure for creating animals of lowered immunologic capabilities for use in certain viral or neOplastic disease transmission studies: Ten newborn Holstein—Friesian bull calves were alternately selected and placed into either the thymectomy or control group. Each calf was given an initial 2 liter hand feeding of colostrum which was previously Obtained from several cows, mixed and frozen. Within 24 hours after birth the calves were thymectomized or subjected to a sham Operation. The surgical procedure involved resectioning Of the central portion Of the third left rib and bluntly dissecting the thoracic portion from its anterior mediastinal position; then placing the calf on his back and bluntly dissecting the cervical portion through a midventral 47 mkl an IE 48 cervical incision. The sham Operation was identical with the exception of the extirpation Of the gland. Postsurgical care included appropriate anti— microbial and palliative treatment. The calves were fed an aseptically prepared milk replacer, a pelleted dry ration, hay, and water. During the first month of age all calves were troubled with frequent attacks of enteritis with ensuing loss of condition and anorexia. From about the fourth week until the termination of the experiment at 20 weeks of age no clinical disease entities were Observed in either group. From the weekly blood samples, no significant difference between groups for erythrocyte count, hemo— Iglobin level, packed cell volume, total leukocyte count, and monocyte or neutrOphil values of the leukocyte dif- ferential was detected. However, highly significant values (P4<0.01) for higher total serum protein and serum _globulin, and lower serum albumin and albumin:globulin ratio were detected for the thymectomized group, as were highly significant lower weight gains. The thymectomized _group also had lower relative eosinophil and lymphocyte values which approach significance at the P«‘0.05 level. LITERATURE CITED Allen, N.N.: The Use of Stored Colostrum to Replace Marketable Milk for Calf Feeding. J. Dairy Sci., 27, (August, 1944): 652-653. Andrew, Warren: Textbook of Comparative Histology. Oxford University Press, New York, N.Y., 1959, 349-350. Archer, O. and Pierce, J.C.: The Role of the Thymus in DeveIOpment of the Immune Response. Fed. Proc., 20, (1961): 26. Archer, 0., Pierce, J.C., Papermaster, B.W., and Good, R.A.: Reduced AB ReSponses in Thymectomized Rabbits. Nature, 195, (July, 1962): 191—193. 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Saunders Co., Philadelphia, Pa., 445. 87. 88. 89. 90. 57 Vandeputte, M., Denys, P. Jr., Leyten, R., and DeSomer, P.: The Oncogenic Activity of the Polyoma Virus in Thymectomized Rats. Life Sci., 7, (1963): 475-478. VandeWater, J.M. and Katzman, H.: Studies Of the Immune Mechanisms in Thymectomized Pups. J. Surg. Res., 4, (1964): 387-390. Warner, N.L. and Szenberg, A.: Effect of Neonatal Thymectomy on the Immune ReSponse in the Chicken. Nature, 196, (1962): 784—785. Wilson, R., Sjodin, K., and Bealmear, M.: The Absence of Wasting in Thymectomized Germfree (Axenic) Mice. Proc. Soc. Exptl. Biol. and Med., 117, (1964): 237-239. APPENDIX ,- y E 0v In: M GE TABLE 1. 59 control calves (millionS/cmm.) Overall mean Individual means for 19 weeks Weekly means for eachygroup Analysis Of variance table fo Source Sum Of of variance squares A; 0.179 Error (a) 386.015 B** 18.759 AB*** 10.246 Remaining 118.226 error Total 533.425 * ’Treated .** Trials ***Treated vs. trials week Thymectomized 8.61 T1 9.93 T2 11.10 T3 7.06 T4 7.81 T5 7.15 2 7-85 3 7.82 4 8.44 5 8.01 6 8.61 7 8.53 8 8.57 9 8.74 10 8-43 11 8.30 12 8.61 13 8.87 14 8.47 15 9.21 16 9.28 17 9.12 18 9.04 19 9.12 20 8.59 Degs. of Mean Masses 1 0.179 8 48.252 18 1.042 18 0.569 144 0.821 189 week F stat. 0.004 1.269 0.693 Erythrocyte counts for thymectomized and Control 8.55 C1 10.41 C2 9.54 C3 7.51 C4 7.79 C5 7.50 2 8.59 3 8.64 4 7.64 5 8.42 6 8.88 7 8.60 8 8.04 9 8.90 10 8.48 11 8.55 12 8.38 13 8.77 14 8.56 15 8.49 16 9.15 17 8.82 18 9.13 19 8.23 20 8.17 r erythrocyte values Approx. signif. prob. Of F stat. 0.91 0.22 0.81 60 TABLE 2. Hemoglobin values for thymectomized and control calves (gm./100 m1.) ‘Thymectomized Overall mean 10.49 Individual means Tl 11.36 for 19 weeks T2 12-70 T3 8.93 T4 10.01 T5 9.43 Weekly means for week 2 10.70 week each group 3 10.93 4 10.24 5 9.98 6 10.34 7 9.82 8 10.18 9 10.84 10 10.66 11 10.22 12 10.28 13 10.62 14 10.50 15 10.60 16 10.92 17 10.68 18 10.64 19 10.88 20 10.16 Control 10.40 C1 11.64 C2 11.63 C3 9.30 C4 9.86 C5 9.55 2 11.82 3 10.96 4 10.48 5 10.18 6 10.02 7 9.96 8 9.80 9 10.50 10 10.38 11 10.36 12 10.08 13 10.56 14 10.14 15 10.36 16 10.76 17 10.40 18 10.10 19 10.58 20 10.10 Analysis of variance table for hemoglobin values Source Sum Degs. 0f of Of Mean variance squares ‘freedom square F stat. A* 0.380 1 0.380 0.011 Error (a) 279.541 8 34.943 8“ 22.048 18 1.225 1.068 AB*** 6.001 18 0.333 0.291 Remaining 165.131 144 1.147 error' Total 473.101 189 * Treated ** Trials ***Treated vs. trials Approx. signif. prob. of F stat. 0.88 0.39 1.00 We ea TABLE 3. 61 control calves (%) Packed cell volume for thymectomized and Thymectomized Control Overall mean 31.72 30.93 Individual means T1 35.09 Cl 34.07 for 19 weeks T2 38.63 C2 33.88 T3 27.05 C3 28.09 T4 29.49 C4 29.83 T5 28.32 C5 28.79 Weekly means for week 2 34.04 week 2 36.20 each group 3 35.56 3 33.80 4 31.70 4 32.60 5 30.84 5 30.40 6 30.20 6 30.50 7 29.60 7 29.50 8 31.60 8 28.56 9 32.00 9 31.60 10 31.60 10 30.60 11 30.60 11 29.96 12 31.20 12 30.90 13 32.20 13 30.20 14 31.36 14 29.52 15 31.36 15 30.80 16 32.00 16 31.30 17 31.76 17 31.10 18 31.90 18 30.30 19 32.50 19 30.80 20 30.60 20 29.10 Analysis of variance table for packed cell volume Approx. Source Sum Degs. Signlf- 0f of of Mean prob. of variance squares freedom square F stat. F stat. A* 29.133 1 29.133 0.094 0.76 Error (a) 2468.309 8 308.539 B** 372.383 18 20.688 1.308 0.19 AB*** 61.098 18 3.394 0.214 1.00 Remaining 2278.074 144 15.820 error Total 5208.999 189 * Treated ** Trials ***Treated vs. trials TABLE 4. 62 calves (leukocytes/cmm.) Overall mean Individual means for 19 weeks Weekly means for each‘group Analysis Of variance Source Sum of of variance ‘squares A* 4184286 Error (a) 1393058899 B** 215410094 AB*** 210444677 Remaining 1873604347 error] Total 3696702302 * Treated ** Trials ***Treated vs. trials ‘Thymectomized 9317 T1 13439 T2 10979 T3 9858 T4 6574 TS 5737 week 2 7817 3 8886 4 10639 5 ’8051 6 11745 7 12659 8 6880 9 7957 10 9659 11 12156 12 8513 13 9973 14 8400 15 8079 16 7819 17 9202 18 8584 19 9724 20 10284 Degs. Of free- Mean dom square 1 4184286 8 174132362 18 11967227 18 11691371 144 13011141 189 week F stat. 0.024 0.920 0.898 Leukocytes counts for thymectomized and control Control 9020 C1 13004 C2 10798 C3 8258 C4 7163 C5 5879 2 6928 3 11026 4 7884 5 8135 6 7155 7 9649 8 12189 9 6304 10 8324 11 10956 12 8366 13 8455 14 9141 15 8663 16 9506 17 9865 18 9700 19 9790 20 9352 table for erythrocyte values Approx. signif. prob. of F stat. 0.85 0.56 .58 TABLE 5a. Overall mean 63 and control calves (%) Individual means for 19 weeks Weekly means for eachygroup Analysis of variance table for relative lymphocyte Thymectomized 47.87 T1 48.16 T2 46.32 T3 38.32 T4 49.74 T5 56.84 week 2 35.60 3 61.80 4 55.00 5 55.20 6 40.80 7 38.00 8 58.00 9 54.40 10 40.00 11 40.60 12 45.00 13 49.60 14 53.60 15 50.60 16 48.20 17 43.00 18 47.60 19 44.00 20 48.80 Source Sum Degs. of of of Mean variance squares ‘freedom square A* 2535 l 2535 Error (a) 7655 8 957 B** 5004 18 278 AB*** 4778 18 265 Remaining 28448 144 198 error. Total 48419 189 -* Treated ** Trials ***Treated vs. trials week F stat. 2.649 1.407 1.344 Relative lymphocyte values for thymectomized Control 55.18 Cl 55.00 C2 44.16 C3 54.10 C4 57.63 C5 65.00 2 63.60 3 55.60 4 63.20 5 65.00 6 67.40 7 51.80 8 48.80 9 61.20 10 54.80 11 43.20 12 45.20 13 49.40 14 48.40 15 48.20 16 54.00 17 50.60 18 53.00 19 58.40 20 66.60 values Approx. signif. prob. Of F stat. 0.14 0.14 0.17 TABLE 5b. and control calves (lymphocytes/cmm.) Absolute lymphocyte values for thymectomized ‘ Thymectomized Control Overall mean 4066 4781 Individual means Tl 5977 C1 7259 for 19 weeks T2 4812 C2 4455 T3 3369 C3 4338 T4 3054 C4 4038 TS 3119 C5 3816 Weekly means for week 2 2503 week 2 4251 each group 3 5134 3 6308 4 5737 4 4690 5 4394 5 4926 6 3079 6 4585 7 3271 7 4640 8 3645 8 5382 9 4465 9 3855 10 3639 10 4833 11 4377 11 4231 12 3690 12 3574 13 3974 13 4219 14 4215 14 4282 15 3936 15 3903 16 3860 16 5151 17 4057 17 5080 18 4200 18 5008 19 4436 19 5872 20 4645 20 6049 Analysis Of variance table for absolute lymphocyte values Degs. Approx. Sources Sum Of Slgnlf. of ‘Of free- Mean F prob. of variance squares dom “square 'Stat. 'F stat. A* 24276753 1 24276753 0.703 0.43 Error (a) 276299199 8 34537400 B** 66205228 18 3678068 1.634 0.06 AB*** 31399065 18 1744392 0.775 0.73 Remaining 324147049 144 2251021 error' Total 722327294 189 * Treated ** Trials ***Treated vs. trials 65 TABLE 6a. Relative neutrophil values for thymectomized and control calves (%) “Thymectomized Control Overall mean 43.96 41.20 Individual means T1 46.00 C1 38.53 for 19 weeks T2 45.74 C2 49.10 T3 38.32 C3 54.10 T4 54.84 C4 36.21 T5 34.89 C5 28.05 Weekly means for week 2 47.00 week 2 51.20 each group 3 46.20 3 35.80 4 40.40 4 40.00 5 38.80 5 33.00 6 42.80 6 30.20 7 48.00 7 39.20 8 39.20 8 46.60 9 50.60 9 39.20 10 40.40 10 34.60 11 48.20 11 49.80 12 43.60 12 48.00 13 48.20 13 39.40 14 34.00 14 42.60 15 44.00 15 46.60 16 38.60 16 39.40 17 43.00 17 41.00 18 44.40 18 44.00 19 48.40 19 44.80 20 49.40 20 37.40 Analysis of variance table for Sources Sum Of of variance squares A* 361 Error (a) 12800 B** 2760 AB*** 2029 Remaining 32886 error Total 50836 * Treated ** Trials ***Treated vs. trials Degs. 0f free- Mean dom square 1 361 8 1600 18 153 18 113 144 228 189 relative neutrophil values Approx. signif. F prob. of stat. F_§tat. 0.226 0.65 0.671 0.84 0.494 0.96 TABLE 6b. 66 and control calves (neutrOphils/cmm.) Absolute neutrophil values for thymectomized 'Thymectomized Control Overall mean 4589 3678 Individual means T1 6723 C1 4953 for 19 weeks T2 5342 C2 5653 T3 5810 C3 3453 T4 2992 C4 2674 TS 2076 C5 1657 Weekly means for week 2 4773 week 2 2500 each group 3 3202 3 4161 4 4001 4 2476 5 3005 5 2863 6 8009 6 2185 7 8915 7 4536 8 2656 8 6259 9 3070 9 1971 10 5350 10 3387 11 7084 11 6103 12 4249 12 4139 13 4972 13 3730 14 3634 14 4067 15 3526 15 4133 16 3342 l6 3651 17 4378 17 3984 18 3585 18 3785 19 4527 19 3221 20 4908 20 2681 Analysis Of variance table for absolute neutrophil values Degs. Approx. Sources Sum Of Slgnlf. 0f of free— Mean F prob. of Variance squares dom __square 'Stat.‘ F_stat. A* 39375711 1 39375711 0.634 0.45 Error (a) 496712418 8 62089052 B** 201517588 18 11195422 1.169 0.29 AB*** 184808612 18 10267145 1.072 0.39 Remaining error' 1378697497 144 9574288 Total 2301111826 189 * Treated ** Trials ***Treated vs. trials TABLE 7a. Overall mean Individual means for 19 weeks Weekly means for each_group Analysis of variance table Sources Sum of of variance 'squares A* 0.047 Error (a) 109.432 B** 85.916 AB*** 50.253 Remaining 690.568 error Total 936.216 * Treated ** Trials ***Treated vs. trials week Degs. of free- 'dom 1 8 18 18 144 189 Thymectomized 5.10 Tl 4.21 T2 7.05 T3 4.74 T4 4.26 T5 5.26 2 4.40 3 4.60 4 6.60 5 6.00 6 4.80 7 3.20 8 5.80 9 4.40 10 5.20 11 5.60 12 4.40 13 4.60 14 5.00 15 5.00 16 5.20 17 6.40 18 5.60 19 5.20 20 5.00 week Relative monocyte values for thymectomized and control calves (%) Control 5.14 Cl 5.21 C2 5.32 C3 4.74 C4 5.42 C5 5.00 2 4.40 3 5.00 4 5.60 5 3.60 6 3.60 7 4.20 8 4.40 9 5.60 10 5.00 11 4.80 12 5.80 13 4.80 14 6.40 15 6.20 16 5.80 17 7.00 18 6.00 19 5.00 20 4.40 for relativenmnocyte values Mean sg‘uare 0.047 13.679 4.773 2.792 4.796 Approx. signif. prob. of F stat. 0.91 0.47 O. 91 TABLE 7b. 68 and control calves (monocytes/cmm.) Overall mean Individual means for 19 weeks Weekly means for each'group Analysis of Sources Sum of of variance ‘squares A* 813 Error (a) 4277701 B** 1043819 AB*** '851372 Remaining 7329869 error‘ Total 13503574 .* Treated ** Trials ***Treated vs. trials Thymectomized 465.0 T1 526.2 T2 724.5 T3 468.0 T4 277.9 T5 328.4 week 2 371.2 3 ‘408.4 4 684.6 5 536.0 6 595.6 7 368.0 8 427.8 9 339.6 10 478.4 11 580.0 12 359.4 13 384.0 14 415.0 15 439.6 16 425.2 17 569.8 18 499.6 19 522.6 20 .430.6 Degs. of free- 'dom 1 8 18 18 144 189 square Mean 813 534713 57990 47298 50902 week variance table for absolute monocyte EEEE; 0.002 1.139 0.929 Absolute monocyte values for thymectomized 991291. 460.9 Cl 702.5 C2 527.5 C3 393.6 C4 385.2 C5 295.6 2 332.2 3 525.0 4 498.6 5 302.6 6 280.2 7 403.8 8 470.2 9 336.2 10 370.4 11 416.6 12 500.4 13 380.2 14 545.6 15 535.8 16 592.4 17 668.8 18 604.4 19 543.0 20 450.4 values Approx. signif. prob. of 'F‘stat. 0.92 0.32 0.55 69 TABLE 8a. Relative eosinOphil values for thymectomized and control calves (%) ThymeCtomized Control Overall mean 8.30 5.20 Individual means T1 1.32 Cl 1.25 for 19 weeks T2 0.37 C2 0.58 T3 0.47 C3 0.16 T4 1.10 C4 0.32 T5 1.10 C5 0.42 Weekly means for week 2 0.60 week 2 0.00 each group 3 0.80 3 0.40 4 0.60 4 0.80 5 0.40 5 0.20 6 0.20 6 0.60 7 0.40 7 0.20 8 0.20 8 0.80 9 0.60 9 1.20 10 0.80 10 0.40 11 1.00 11 0.80 12 1.20 12 0.60 13 0.60 13 0.80 14 0.60 14 0.60 15 0.40 15 0.00 16 1.40 16 0.20 17 1.00 17 0.20 18 2.60 18 1.60 19 1.80 19 0.20 20 1.40 20 0.80 Analysis of variance table for relative eosinophil values Degs. Approx. Sources Sum of Slgnlf. of of free- Mean F prob. of variance sguares dom __square stat. F stat. A* 5.058 1 5.058 1.466 0.12 Error (a) 27.600 8 3.450 B** 32.379 18 1.799 1.556 0.08 AB*** 15.642 18 0.869 0.752 0.74 Remaining 166.400 144 1.156 error‘ Total 247.079 189 * Treated ** Trials ***Treated vs. trials TABLE 8b. 70 and control calves (eosinophils/cmm.) Overall mean Individual means for 19 weeks Weekly means for each‘group Analysis of variance tabl Sources Sum of of variance sguares A* 58048 Error (a) 460900 B** 305359 AB*** 147792 Remaining 2370644 error Total 3342752 * Treated ** Trials ***Treated vs. trials Thymectomized 87.31 T1 167.84 T2 90.89 T3 44-16 T4 81.79 T5 74.84 week 2 46.80 3 118.80 4 64.40 5 49.20 6 24.20 7 88.00 8 19.00 9 235.20 10 87.40 11 101.60 12 113.60 13 79.80 14 38.20 15 26.60 16 98.00 17 86.40 18 214.60 19 148.40 20 106.00 Degs. of free- dom 1 8 18 18 144 189 Mean square 58048 57613 16964 8211 16463 week F i355; 1.008 1.030 0.499 Absolute eosinophil values for thymectomized Control 54.10 C1 164.79 C2 60.00 C3 12.95 C4 22.00 C5 25.00 2 0.00 3 32.20 4 71.60 5 21.40 6 42.00 7 34.20 8 100.20 9 94.00 10 45.40 11 128.20 12 77.60 13 81.40 14 83.20 15 0.00 16 30.40 17 22.40 18 125.40 "19 13.40 20 79.00 e for absolute eosinophil values Approx. signif. prob. of F stat. 0.45 .137 F :10» 0.8: 1082' for WEI ea TABLE 9a. Relative baSOphil values for thymectomized and control calves (%) Thymectomized Control Overall mean 1.23 0.93 Individual means T1 0.37 Cl 0.58 for 19 weeks T2 0.84 C2 0.79 T3 1.95 C3 0.84 T4 1.10 C4 0.89 T5 1.89 C5 1.53 Weekly means for week 2 0.60 week 2 0.20 each group 3 0.40 3 0.00 4 1.60 4 0.80 5 0.80 5 0.00 6 0.60 6 1.00 7 0.60 7 1.20 8 1.80 8 0-80 9 0.40 9 1.20 10 1.20 10 1.20 11 0.80 11 0-80 12 1.60 12 1.00 13 2.00 13 0.80 14 1.40 14 0-80 15 2.20 15 0-60 16 1.80 16 1-00 17 1.20 17 1-4O 18 1.00 18 2-00 19 1.20 19 1.80 20 2.20 20 1.00 Analysis of variance table for relative basophil values Degs. Approx. Sources Sum 0f Slgnlf. Of of free“ Mean F prob. of "Variance “sguares dom “'Sguare ' stat. F stat. A* 4.426 1 4.426 0.786 0.40 Error (a) 45.074 8 5.634 B** 30.716 18 1.706 1.980 0.01 AB*** 25.474 18 1.415 1.642 0.06 Remaining 124.126 144 0.862 error Total 229.816 189 * Treated ** Trials .***Treated vs. trials TABLE 9b. 72 and control calves (baSOphils/cmm.) Overall mean Individual means for 19 weeks Weekly means for each_group Analysis of variance Sources Sum of of variance sguares A* 34063 Error (a) 165015 B** 315486 AB*** 206365 Remaining 1068763 error' Total 1789692 * Treated ** Trials ***Treated vs. trials week ThymectomiZed 100.62 T1 46.79 T2 94.89 T3 166.74 T4 77.26 T5 117.42 2 43.40 3 22.00 4 175.80 5 67.20 6 38.20 7 35.40 8 137.20 9 25.20' 10 103.80 11 52.00 12 121.00 13 178.60 14 129.60 15 153.00 16 129.20 17 110.20 18 85.20 19 111.20 20 193.60 week Absolute baSOphil values for thymectomized Control 73.84 C1 87.84 C2 68.58 C3 64.10 C4 63.47 C5 85.21 2 17.60 3 0.00 4 60.80 5 0.00 6 50.80 7 88.60 8 62.80 9 63.40 10 86.60 11 46.00 12 76.00 13 56.60 14 63.80 15 37.00 16 80.80 17 132.20 18 206.60 19 179.80 20 93.60 table for absolute basophil values Degs. 0f free- Mean dom square 1 34063 8 20627 18 17527 18 11465 144 7422 189 F EEEEL. 1.652 2.362 1.545 Approx. signif. prob. of 0 0 0 F stat. .23 .00 .08 73 TABLE 10. Total protein values for thymectomized and control calves (gm./100 m1.) Thymectomized Control Overall mean 5.25 5.23 Individual means T1 5.73 Cl 5.80 for 19 weeks T2 5.63 C2 5.31 T3 5.24 C3 4.96 T4 5.12 C4 5.26 T5 4.54 C5 4.82 Weekly means for week 2 4.24 week 2 4.56 each group 3 3.74 3 4.47 4 4.66 4 4.49 5 4.69 5 4.64 6 5.01 6 4.84 7 5.06 7 5.10 8 5002 8 4.77 9 5.60 9 5.39 10 5.40 10 5.25 11 5.36 11 5.25 12 5.41 12 5.27 13 5.50 13 5.39 14 5.53 14 5.32 15 5.48 15 5.75 16 5.68 16 5.57 17 5.73 17 5.71 18 5.78 18 5.73 19 5.99 19 5.83 20 5.88 20 6.04 Analysis of variance for total protein values Degs. Approx. Sources Sum of S1gn1f. of of free- .Mean - F prob. of variance sguares ' dom ' square stat. F stat. A* 0.020 1 0.020 0.006 0.90 Error (a) 27.991 8 3.499 B** 49.071 18 2.726 10.618 0.00 AB*** 2.647 18 0.147 0.573 0.91 Remaining 36.972 144 0.257 error Total 116.701 189 * Treated ** Trials ***Treated vs. trials 74 TABLE 11. Albumin values for thymeCtomized and control calves (gm./100 m1.) Overall mean Individual means for 19 weeks Weekly means for each.group week "Thymectomized 3.18 Tl 3.15 T2 3.12 T3 3.20 T4 3.52 T5 2.89 2 2.89 3 2.39 4 2.96 5 3.03 6 3.12 7 3.06 8 3.05 9 3.42 10 3.15 11 3.14 12 3.18 13 3.26 14 3.24 15 3.29 16 3.35 17 3.41 18 3.43 19 3.51 20 3.48 Analysis of variance table Sources Sum of of variance sgggggg A* 0.268 Error (a) 6.068 B** 8.656 AB*** 1.332 Remaining 17.251 error' Total 33.576 * Treated ** Trials ***Treated VS . Trials Degs of free- dom l 8 18 18 144 189 Mean sguare 0.268 0.758 0.481 0.074 0.120 week F EEEE; 0.354 4.014 0.618 Control 3.25 Cl 3.15 C2 3.26 C3 3.26 C4 3.52 C5 3.07 2 3.16 3 2.95 4 2.99 5 2.85 6 3.15 7 3.22 8 2.95 9 3.35 10 3.21 11 3.25 12 3.27 13 3.29 14 3.37 15 3.64 16 3.41 17 3.45 18 3.33 19 3.48 20 3.48 for albumin values Approx. signif. prob. of F statt__ 0.57 0.00 0.88 75 TABLE 12. Globulin values for thymectomized and control calves (gm./100 m1.) 'Thymectomized Control Overall mean 2.07 1.98 Individual means T1 2.57 Cl 2.65 for 19 weeks T2 2.50 C2 2.05 T3 2.03 C3 1.70 T4 1.60 C4 1.74 T5 1.64 C5 1.74 Weekly means for week 2 1.35 week 2 1.40 each group 3 1.35 3 1.52 4 1.70 4 1.50 5 1.66 5 1.79 6 1.87 6 1.70 7 2.00 7 1.89 8 1.97 8 1.82 9 2.18 9 2.04 10 2.24 10 2.03 11 2.22 11 1.99 12 2.23 12 2.00 13 2.24 13 2.10 14 2.29 14 1.95 15 2.19 15 2.12 16 2.33 16 2.16 17 2.32 17 2.26 18 2.35 18 2.40 19 2.48 19 2.35 20 2.40 20 2.56 Analysis of variance table for globulin values Degs. Approx. Sources Sum cf signif. of of free— Mean F prob. of variance squares dom square ' stat. F stat. A* 0.422 1 0.422 0.119 0.73 Error (a) 28.369 8 3.546 B** 18.101 18 1.006 10.031 0.00 AB*** 0.921 18 0.051 0.510 0.95 Remaining 14.436 144 0.100 error. Total 62.250 189 * Treated ** Trials ***Treated vs. trials TABLE 13. 76 Albumin;globu1in ratio for thymectomized and Overall mean Individual means for 19 weeks Weekly means for . each group Analysis of variance Sources of variance A* Error (a) B** AB*** Remaining error Total * Treated ** Trials ***Treated Sum of squares 0.902 19.794 5.507 1.009 13.603 40.814 vs. trials control calves week Control 1.75 Cl 1.24 C2 1.65 C3 1.96 C4 2.08 C5 1.79 2 2.27 3 1.94 4 2.03 5 1.71 6 1.71 7 1.86 8 1.76 9 1.80 10 1.73 11 1.83 12 1.75 13 1.66 14 1.75 15 1.79 16 1.66 17 1.62 18 1.41 19 1.53 20 1.37 for a1bumin;globu1in ratio Thymectomized 1.61 T1 1.21 T2 1.31 T3 1.63 T4 2.21 T5 1.68 week 2 2.15 3 1.52 4 1.82 5 1.86 6 1.71 7 1.64 8 1.66 9 1.53 10 1.49 11 1.52 12 1.52 13 1.57' 14 1.52 15 1.63 16 1.49 17 1.48 18 1.48 19 1.45 20 1.51 table Degs. of free- Mean dom square 1 0.902 8 2.474 18 0.306 18 0.056 144 0.094 189 Approx. signif. F prob. of stat. F stat. 0.364 0.57 3.238 0.00 0.593 0.90 77 TABLE 14. Cumulative weight gains for thymectomized and control calves (kg.) Thymectomized Control Mean weights 25.8 27.6 Biweekly cumulative week 0 0.0 week 0 0.0 weights 2 -1.0 2 -1.4 4 0.9 4 3.3 6 6.8 6 6.9 8 10.6 8 14.4 10 17.5 10 22.8 12 27.9 12 27.8 14 33.6 14 36.9 16 44.4 16 46.7 18 53.9 18 55.0 20 63.0 20 63.8 Actual final cumulative weights 64.4 69.0 Analysis of variance table for cumulative weight_gains**** Degs. Approx. Sources Sum of s1gn1f. of of free- Mean F prob. of variance squares dom square ‘ stat. F stat. A* 412 1 412 1.375 0.25 Error (a) 11992 40 300 B** 221269 9 24585 178.917 0.00 AB*** 391 9 43 0.732 0.68 Remaining 2372 40 59 error Total 236437 99 * Treated ** Trials *** Treated vs. trials _ . 1 ****These figures are presented as they were or1g1na1 y analyzed in pounds. MICHIGAN STATE UNIVERSITY 0 3 1293 3082 26 LIBRARIES 66 hi -_‘ —