ABSTRACT STUDIES ON GNUPOBIOTIC PIGS PART ONE. A TECHNIQUE FCE HEARING GNOTOBIOTIC PIGS PART TWO. A CQIPARISON OF BODY WEIGHTS, ORGAN WEIGHTS, AND SGIE HISTOLOGICAL FEATUES OF GNO‘I‘OBIO'I'IC AND FARM-RAISED PIPE by Glenn 1.. laxler Part One describes the technique used in h experiments to ob- tain and rear young pigs to the age of approximately 3 weeks in the ab- sence of bacteria or in the presence of known species of bacteria. A hysterectonw was performed on the anesthetized saw, and the pigs were removed from the uterus inside a sterile, vinyl-film isolator. The diet, sterilized with steam under pressure, consisted of pasteurized, homogenized milk with mineral and vitamin supplements. The technique was found to be successful in that 2 pigs were raised in the absence of any demonstrable bacteria. Four animals were raised in the presence of gmphylocmcus aureus, 3 in the presence of Bacillus sp. , and h in the presence of Achranobacteriaceae and an un- . identified organism. ‘ Part Two describes the comparison of body weights, organ nights, and the histology of selected tissues of the 13 pigs from Part One, 5 pigs raised in open cages on the sterilized diet, and 11 animals raised by the saw to the age of 3 weeks under fam conditions. Glenn L.‘Wax1er The farm-raised pigs were significantly heavier at the end of the 3-week period than were the artificially-raised animals. When the organ weights were expressed as relative weights, the mandibular lymph nodes of the farm-raised pigs were significantly heavier than those of the gemfree pigs. This did not hold true for the other groups of lymph nodes studied. The kidneys of all artificially-reared animals were relatively heavier than those of the farm-raised animals. The relative adrenal weights of the germfree pigs and those animals raised in the presence of Staphylococcus aureus were greater than those of the farm-raised pigs. Light-centered nodules, corresponding to the usual germinal or reaction centers were found in the lymph nodes of the germfree ani- mals, but these did not appear as large or numerous as in the farm- raised pigs. The germfree pigs usually displayed more solid-centered primary nodules than did those animals raised under farm conditions. Numerous immature eosinophils, neutrophils, and small, intensely- staining cells resembling hematOpoietic cells were found in the lymph nodes of all groups of animals. The histological picture of the lymph nodes seemed to be influenced more by the diet and rearing conditions under which the animals were kept than by the presence or absence of bacteria in the environment. No significant differences were found in the histolog of the spleens of the various groups of animals, except for the fact that tin artificially-reared animals appeared to have more iron-containing pig- ment in this organ. in. "—._‘— - I Glenn L. Waxler The hepatic interlobular septa of the artificially-reared ani- mals were less well-develOped than in the farm-raised pigs, and in many cases the only indication of the borders of the lobules was a lining up of the hepatic cells and reticular fibers in this area. Two germfree pigs left in the germfree environment 3 and 5 days, respectively, after death showed no gross evidence of post-marten degeneration. However, dehydration was noted. Histological examina- tion revealed less degenerative changes than would be expected under conditions in which the animals were in contact with bacteria. 0f the ' tissues examined, the avocardium and erythrocytes showed the least de- generation, the lymphatic tissue and the adrenals the most. STUDIES ON GNOTOBIOTIC PIGS PART ONE. A TECHNIQUE FOR HEARING GNOTOBIOTIC PIGS PART TWO. A COMPARISON OF BODY WEIG-ITS, ORGAN WEIGHTS, AND SOME HISTOLOGICAL FEATURES OF GNOTOBIOTIC AND FARM-RAISED PI (‘6 ' By Q’Q. Glenn 1.. Healer A THESIS Submitted to Hichigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Veterinary Pathology 1961 'L, ”I‘m . . («we .57 ,. 'u . .3 ”R .‘Y- . a,q..r-.. 5; 2.0370 (I. ‘ :5 J / , '. .9 V I, c" L, / '1? c, ’ , . ACKNOWIMIENTS ‘ The author wishes to express his gratitude for the invaluable advice and assistance offered by Dr. C. K. Whitshair during the course of this work. The help of Dr. C. C. Morrill in reviewing the manuscript and that of Dr. R. F. Langham in interpreting the histological findings is also appreciated. Prafessor P. C. Trexler and associates at the Lobund Institute , University of Notre Dame , were very helpful with suggestions, advice, and the loan of equipment in de- veloping the gnotobiotic rearing techniques . The author also acknowledges the patience and en- couragement of his wife and her many hours of work in typing and preparing the manuscript. ii . ‘ .. sang,— when) ya?“ I. II. III . INTRODUCTION . . PART ONE. TABLE OF CONTENTS REVIEWOFLITERATURE. .. . . . . A. B. Gnotobiotic Techniques . . . . 1. 2. 3. h. 5. Terminology . . . . . . . Chronological Survey . . Techniques and.Equipment Diets O O O O O O O O O 0 Use of Germfree Animals . Artificial .Rearing of Pigs . . . mmWTAL O O O O O O O O O O C A. Procedures . . . . . . . . . . 1. 2. 3. h. Equipment . . . . . . . . Sterilization of Equipment Surgical Procedure . . . . Diets and Rearing Procedures a. EXperiment I . . . . . b. Experiment II . . . . c. Experiment III . . . . d. Experiment IV . . . . Bacteriological Procedures 111 A TECHNIQUE FOR HEARING GNOTOBIOTIC PIGS F5? \OVIF’J—“P' 16 20 23 26 26 26 1:8 53 53 57 S9 61 A... w -..~_._..m.. vi.— . gar: Wuuurui {3" _ I! x I}, 1" I .1 '.. . —.,”4—‘— “s.‘.q- - - _. _ - I“ -- fl 7 *V ,7 ..'."'_""..7 . a... ”" -- — ' ' _*1‘==3'J.t.3—§ - any-- nun ~ suntan-“5|”- Ear-u Page B.Results................ ..... ..63 l.Experimentl...................63 2.ExperimentII..................6h 3. Experiment III . . . . . . . . . . . . . . . . . . 67 h.ExperimentIV..................70 IV. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 75 V.SUWY............ ....... 00000081 PART TWO. A COIPARISON OF BODY WEIGHTS, ORGAN mums, AND SOME HISTOLOGIOAL FEATURES OF GNOTOBIOTIchND EARN-RAISED PIGS I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . 82 II.REVIENOFLITERATURE...................8h A. GrowthRatesofGermfreeAnimals........... 81; B. Organ weights of Germfree Animals . . . . . . . . . . 85 C. Histology of the Lymphatic System . . . . . . . . . . 87 D. Histology of Germfree Animals . .. . . . . . .. . . 92 E. Histology of the Lymphatic System and the Liver of the Pige‘eeeeeeeeeeeeeeeeeeeeeeee9h l. Histology of the Lymphatic System . . . . . . . . 9h 2. HistologyoftheLiver.............. 96 III. EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . 99 A.Procedures......................99 1. Animals Used . . . . . . . . . . . . . . . . . . . 99 2. BodyandOrganWeights.............. 99 3. Histological Procedures . . . . . . . . . . . . . lOl iv B . maflts O I O O O O O O O O O O O O O ‘0 O O O 1. 2. 3. h. S. BodyandOrganIeights . . . . . . Gross Observations . . . . . . . . . . Histolog of the Lymph Nodes and Spleen Histology of the Liver . . . . . . . . 0 Microscopic Evidence of Post-Morten Degeneration inGermfreePigs................ IV.DISCUSSION.................. A. BodyandOrganleights. . . . . . .. . . B. Histology of the Lymph Nodes and Spleen . C. Histology of the Liver . . . . . . . . . . D. licroscopic Evidence of Post-Horton Degeneration 0 in Gemree P188 0 O O C O O O O O O O O O O O O O O 0 SUMMARY 0 O O O O O O O O O O O O O O O O O O O 0 O LISTOFREFERENCES................ APPENDIX..‘....... 0 O C O O O O O O O O O 102 102 102 127 11.7 155 159 159 161 163 161. 165 167 179 '- - -.;: LT _- I“ _-_ .— 7:— - '1 1": w a “ mm :7!" image LIST OF TABLES Table Page I. B vitamin mixture 103, concentrated . . . . . . . . . 55 II. ‘SaltmixturelS,solutionA.....'........ 55 III. Salt mixture 15, solution B . . . . . . . . . . . . . 56 IV. Volume of diet fed in Experiment I . . . . . . . . . 56 V. Vitamin solution used in EXperiments II, III, and IV 58 VI. Volume of diet fed in Experiment II . . . . . . . . . 59 VII. Volume of diet fed in Experiment III . . . . . . . . 60 VIII. Volume of diet fed inExperiment IV . . . . . . . . . 61 IX. Totalbodyweights.,...............6S X. Sourceofgroupsofpigs..............6S 70 XI. Body weights of pigs in Experiment III III. Body weights of pigs in Experiment IV . . . . . . . . 72 ' XIII. Totalbodyweights.................103 XIV. Weights of mandibular lymph nodes . . . . . . . . . . 1014 IV. Relative weights of mandibular lymph nodes . . . . . 105 XVI. Heights of external inguinal lymph nodes . . . . . . 106 XVII. Relative weights of external inguinal lymph nodes . . 107 XVIII. Heights of prefemoral lymph nodes . . . . . . . . . . 108 III. Relative weights of prefemoral lymph nodes . . . . . 109 XX. Ieightsofhearts..................110 XXI. Relative weights of hearts . . . . . . . . . . . . . 111 XXII. Weightsoflungs..................112 XXIII. Relative weights of lungs . . . . . . . . . . . . . . 113 um. Weightsof’livers..................llh vi Table XIV. XXVI. XXVII. XXVIII. XXIX. m. XXII. XXIII. XXIIII. mIV. XXIV. xmi. Relative weights of livers . Heights of Spleens . . . . . Relative weights of spleens Weights of kidneys . . . . Relative weights of kidneys Heights of pancreases Relative weights of pancreases Heights of thyroids Relative weights of thyroids Heights of adrenals Relative weights of adrenals Hematological data . . . . . O O I 0 vii 128 U . -1 "5:11;. A i 4' sense \0 (DR) O‘W‘t’w 10 11 12 13 1h 15 16 17 18. 19 20 21 22 LIST OF FIGURES Surgical isolator . . . . . . . . . . . . Rearing isolator viewed from filter side Rearing isolator viewed from glove side . Air filter attached to rearing isolator . Air-outlet trap attached to rearing isolator Air-filter core . . . . . . . . . . . . . . ‘lrapped air-filter core . . . . . Assembled air filter . . . . . . Air-outlet trap, disassembled . . Air-outlet trap, assembled . . . Stainless-steel rearing cage Interior of rearing cage . . . . SquareéPak Flask, disassembled . Square-Pak Flask, assembled . . . . . Surgical isolator with tank in place Surgical and rearing isolators connected Gram-stained fecal smear from germfree pig Grampstained fecal smear from farm-raised pig Gnotobiotic pigs from.Experiment IV . . . . . Gram-stained fecal smear from gnotobiotic pig hmriment IV 0 O O O O O 0 O O O O O I O O O O O in Solid primary nodules in prefemoral lymph node of gamma pig L1 0 O O O O O O O O O. O O O O 0 0 Solid primary nodule in mandibular'lynph node of gamma pig 3-2 G O O O O O O O O O O O O O O O Viii 27 3o 31 32 33 35 36 37 39 ho be 1.3 145 so 51 68 69 71 73‘ 130 131 - - ‘. a .‘C*-L_'-’-—‘:— ~ ..‘4 I- ._ ‘1' “a: 'a'r i Finis 23 2h 25 26 27 28 29 30 31 32 33' 3h 35 36 37 ‘38 39 ho 1.1 hz Light-centered nodules in prefemoral lymph node of farm-raisedpigG-l................. Light-centered nodules in mesenteric lymph node of gamma pig E‘l O O I O O O O O O O O O O O O O O O Light-centered nodule in mandibular lymph node of pigc-3000eoeeeeeeeeeeeeeeweee Li ght-centered nodule in mandibular lymph node of gemreo pig E'l O O O O C O O O O O O O O O 0 O O 0 Cells in light-centered nodule from mesenteric lymph nOdBOffam-PEISOdpigG—IO..o......... EosinOphils and neutrophils in medulla of mandibular lymphnode offarm-raisedpigG—Z . . . . . . . . . . Hematopoietic cells in medulla of superficial inguinal lymph node of farm-raised pig G-l . . . . . Hematopoietic cells in trabecular sinus of super- ficial inguinal lymph node of farm-raised pig G—l . . Iron-containing pigment in medulla of lymph node of farm-raisedpigG-ll ................ Reticular framework of prefemoral lymph node of fam’raised p18 M O O O O O O O O O O O O O O O O O SpleenofgermfreeanimalE—l.. .. . Iron-containing pigment in spleen of gnotobiotic pigA.leeeeeeeeeeeeeeeeeeeeeeo Liver Of fM‘tgjused p18 6.9 e e e e e e e e e e e e LiverofgermfreepigE-2. .. .. .. . . . . Liver of farm-raised pig G-8 LiverofgermfreepigE-2.... .......... Hematopoietic cells in hepatic sinusoid of germfree pig 3.1 O O O 0‘ O O O O O O O O O O O O O O O O O O O Spleen of germfree pig 3 days after death . .. . . . . Liver of germfree pig 3 days after death . . . . . . Atrium of germfree pig 5 days after death . . . . . . ix Page 132 _ 133 DA 136 137 Ibo lhz 11.2 1113 1145 1116 lbs 151 151 153 153 151. 156 157 158 PART ONE A TECHNIQUE FOR HEARING GNOI'OBIOTIC PI$ I. INTRODUCTION Gnotobiotics, the science of rearing organisms by themselves or in the presence of specifically known associates, has received much . attention during the past several years. Most investigations have in- volved the rearing and use of laboratory animals. However, germfree chickens have been produced quite extensively, and a few attempts have been made to utilise larger domestic male in this type of research. The size of the equipment necessary to house the larger animals and the volume of diet required have been among the factors limiting the usefulness of these species in gnotobiotic research. The specific fields of research in which germfree animals have been used include, among others, the areas of nutrition and in- fectious diseases. Investigative work in the area of nutrition is made diffioalt by the various interrelationships which exist among the merous nutritional factors. In like manner, the infectious agents often exert an influence on one another, and it has been demonstrated that interrelationships exist between nutrition and infectious agents. By using gerafree animals it is possible to control the environmental conditions of the experiment more rigidly and thus more thoroughly delineate sons of the above-described interrelationships. In the field of swine diseases it is recognised that there is a need for much basic research. Diagnosis of many diseases is made 1 .. ,. . . A y.. .._. . * fir. fish n» . ._.o’- (fr/H, .. . .. . . -. a. .. ...0u..a.l.:.t ,s . . «.45 . o ... . . n v.- .,, c . .1— . . . .. ..g .c... f. .z; a. g .‘ . .. . . . y . O. l o. e . i ..2 . quite difficult by the vast array of microorganisms which may be iso- lated from affected individuals. This is especially true in some of the enterio disturbances of young animals. It is felt that adequate prevention and control of these conditions in swine will be accom- plished only when it has been found possible to clearly define the roles played by the various mic roorganisns involved. The research on these problems will be greatly enhanced and the results nade ’nore reliable if a dependable, practical technique for obtaining and rearing young pigs free from microorganisns can be developed. It will then be possible to introduce known species of bacteria and study their ability to produce pathological changes in the animal. In preliainary work at this institution, using pigs derived by Caesarean section as a source of experinental animals for various studies, it was found that certain factors often complicated the re- sults. One of the nest semen of these was the appearance of bac- terial infections, usually nanifested by enteritis, in the young colostrum-deprived animals. In an attupt to control these cmpli- cating factors, work was begun in 1959 to investigate the possibili- ties of adapting gnotobiotic techniques to the roaring of young pigs. It was hoped that this would result in the obtaining of more reliable experimntal data in the field of research utilising young pigs. The attempts to obtain germfree pigs as reported in this manuscript Iere made from the period of Hay, 1960, to January, 1961. Part One of this muscript includes a description of the equip-ant, sterilisation techniques, surgical procedures, diets, rearing methods, and bacteriological techniques used in a series of infill-1hr? an», ill! I tf‘ 3 1; experiments designed to investigate the feasibility of obtaining and raising gnotobiotic pigs to an age of approximately 3 weeks. Part Two includes some phases of a morphological characterisation of the animals obtained in these I; esperiments. This includes a study of the body weights, weights of various organs, and the histology of selected lymph nodes, the spleen, and the liver of these animals as compared to the same observations made on pigs of comparable age raised under ordinary farm conditions. II. WOFLITERATURE A. Gnotobiotic Techniques 1. Terminology Reyniers gt a_l.. (l9h9b) have reviewed the various toms used to describe the technique of working with animals in an enviroment free from detectable microorganisms and the terms applied to these animals themselves. They point out that such designations as "pure", "gens-free”, "sterile", 'aseptic", and nbacteriaa-f‘reeu have appeared in the literature frmn time to time. llore recently the term "germ- free” has by most authors been changed to one word and now appears as 'germfree'. Baker and Ferguson (19%) suggested the term "axenic" which is derived from the Greek and means "free from strangers". According to these workers, 'uenic' organisms are individuals of a species free from an demonstrable life apart from that produced by their own protoplasm. Just (1959) suggested the term 'bion" which refers to an organism that is morphologically and pivsiologically in- dependent, a condition satisfactorily met by germfree animals. Under this systu of nomenclature 'biontology” refers to the field of re- search dealing with germfree forms. A 'holobiont" is an organism free from microorganisms, a "monobiont'I is raised in contact with a single species and a "dibiont'I with 2 species of microorganisms. Reyniers gt. _s_1_. (l9h9b) and Reyniers (1956) have suggested the term 'gnotobiotics" to describe the conditions and concepts in- volved in this type research. This term is derived from 2 Greek words, one meaning "know" and the other "life“, and it may be used to h indicate the field of investigation concerned with growing living things by themselves or in an association with other canpletely known kinds of organisms. The animal grown under these conditions may then be referred to as a "gnotobiote", and the environment may be referred to as a 'gnotobiotope". Treader (1960b) has suggested the grouping of 'gnotobiotes' upon the basis of the certitude with which their specific content can be defined. He described 'slpha-gnotobiotes" as those maintained in a secure environment and subject to extensive examinations requiring re- production to furnish the necessary specimens. The "beta gnotobiotes” include animals maintained under the same rigid security but unable to reproduce under germfree conditions. The “game gmtobiotes" are gnotobiotic animals as used at the present time in which the status of each isolator is determined by rather extensive analysis by competent microbiologists. He also described 'delta gnotobiotes'l as those re- quiring the least reliable control procedures, such as simple micro- scopic observations of fecal smears combined with a few culture media. Jenkins (1960) has suggested a similar classification of laboratory animals on the basis of associated organisms. 2. Chronolgical m Credit for the first attempts to cultivate complex organisms in the absence of bacteria is generally given to Duclaux (1885) who was able to grow peas andbeans in an environment free of bacteria. The seeds were sown in a sterile soil moistened with milk, and Duclaux observed that after one or 2 months the milk was still intact with no coagulation. Pasteur (1885) in a preface to Duclaux's publications, expressed an interest in the question of whether or not it would be possible to raise animals in an environment and on a diet both free of the ordinary microbes. It was Pasteur's firm belief, however, that life tinder these conditions would become impossible. Schottelius (1899) . who studied under Pasteur, expressed the opinion that it is inconceivable that the relationship between the in- testinal mucosa and the intestinal flora should not have developed into some sort of symbiosis. If the normal flora is not allowed to establish itself at birth, as would be the case in the gemfree ex- periment, he concluded that life would not be possible. The interest of Schottelius in the possibility of germfree life was followed by several attempts to obtain germfree chickens (Schottelius, 1899, 1902, 1908, 1913), and the failure of these birds to grow seemed to strengthen the contentions of both Pasteur and his student. These chickens were below their initial weight at the end of the 30-day ex- perimental period. Control animals on the sterilised diet developed normally. Ihen the gemfree chickens were infected with pure cultures of Bacteria 3213 from hen feces or milk, they developed almost as well as the control chickens. A study of the diet used by Schottelius suggests that it was probably poor in accessory nutritive substances as compared to diets used for chickens in later successful trials (Gustafsson, 19h8). Nencki (1886) free Switserland was apposed to the theories presented by Pasteur and Schottelius and belonged to the group of those believing that the bacterial flora has the ability to produce more or less poisonous substances which would impair the health of the animal host. A few years later the German workers Nuttal and Thierfelder (1895-1896, 1896-1897) demonstrated that guinea pigs could live in the absence of bacteria. letchnikoff (1903), who, like Pasteur, never actually per- fumed a gemfree experiment, clearly lined up with the Nencki-Nuttal— Thierfelder forces in believing that the bacterial flora can assume the role of the fighting antagonist against the host. Cohendy (1912a, 1912b), a student oleetchnikoff, was quite successful in rearing germfree chickens and raised 17 such birds, some of them.to the age of to days. In some cases the physical condition.and growth of these germfree chickens compared favorably to control animals . Cohendy con- cluded that (1) life is possible fer vertebrates (chickens) in the germfree state and (2) germfree life does not cause an breakdown in the organism. Cohendy and Hellman (1911;) reared germfree guinea pigs to ages varying from 16 to 29 days and also used these animals in studying cholera infection ((30th and Iollman, 1922). Kfister (1912, 1913, 1915a, 1915b) a student of’Schottelius, 'was the first to work with larger'mammals under germfree conditions. He raised 2 germfree goats, and one of these lived to the age of 35 days. Excellent reviews of these early efforts in the field of germfree research have been presented.hy Gustafsson (l9h8), Reyniers gt g_1_. (19149:), Rowers (1956). and Gordon (1960). Balsam (1937) conducted 3 experiments, using germfree chickens, to study the correlation between the digestibility of food and vitamin requirements of normal chickens. From the results of these experiments, Balsam.coneluded that (l) the intestinal flora of the chicken does not exert an appreciable influence on the digestibility of food and (2) the lack of an intestinal flora has no effect on the need for vitains by germfree animals. kuch of the recent research in gnotobiotic techniques has been conducted at the Lohund Institute, University of Notre Dame, Notre Dame, Indiana. No attempt will be made at this time to review extensively the may reports published by this group. However, the following references may be cited as covering, in a general way, the research work conducted: Gordon (1959), Phillips 25’. 31. (1959), Beyniers (1959a, 1959b), Reyniers and Trexler (191:3), Reyniers at 91. (19h6, 19h9a), Trexler (1959) , and Truler and Reynolds (1957). The Lobund group has successfully reared germfree rats, mice, guinea pigs, chickens, rabbits, monkeys, turkeys, dogs, and other animals (Reyniers, 1959b). The Swedish workers (Glimstedt, 1936, 1959; Gustafsson, l9h6-19h7, 191:8, 1959a, 1959b) have utilised germfree rats and guinea pigs in their research. In Japan, liyakawa (1959a, 1959b) and Hiyakawa _e_t_ 2.1.3 (1957) have worked with the germfree guinea pig. In addition numerous other groups have conducted research involving germ- free laboratory animals (Lev and Forbes, 19593 Horowitz 9:. $1., 1960; Phillips st 31., 1960; Hickey, 1960; Snow and Hickey, 1960). The interest in obtaining and rearing germfree farm animals, first evidenced by Kiister (1912 , 1913, 1915a, 1915b) has been dormant until quite recently. saith and l'rexler (1960) have obtained a gem- free lamb and 2 germfree goats. The lamb was reared under gemfree conditions for 1; months. Luckey ( 1960) also reported the successful rearing of germfree lambs. In addition to the present work, at least one attempt has been made to rear gemfm pigs (Trader, l960d). ”-— ‘fl—‘A‘M , .a_ — Aw _ 1 .4— A A 4-4.... N“. 3. Techniques and m According to Reyniers (1956) the ideal germfree animal would be one resulting from the removal of all contaminants from a conven- tional animal. This animal, left with the ”experience but not the cause of this experience'I , would then be comparable to the conventional. This goal of couplets decontamination has never been accomplished, al- though much work has been done in this direction. is a result, it has been necessary to rely on techniques designed to obtain young animals before they become associated with microorganisms. The probability of obtaining germfree manuals and birds is closely associated with the contention that these animals are free from microorganisms during their ubryonic development. Royniers and Trexler (l9h3) have expressed it in this manner: "That a placental animal may be obtained germ-free is based on the theory of its being free from contaminating microorganisms while in 113933 . This, of course, is the case with normal parent animals. The reasons for this theory are partially based on anatomical and mubrans protection, germicidal activity of the amniotic fluid, self-sterilisation of the developing embryo, and chance factors.” Reyniers g_t_ g. (l9h9a) have also shown that only a mall percentage of the eggs available to them were contaminated before incubation, and they suggested that the de- fensive mechanism of the developing chick is effective in preventing the growth of the few microorganisms which find their way through the shell. In the case of both manuals and birds advantage is taken of this freedom frat microorganisms during embryonal developent to pro- cure the germfree young. 10 The equipment used by Nuttal and Thierfelder (1895-1896, 1896-1897 , 1897) for rearing germfree guinea pigs and chickens con- sisted of a cylindrical glass Jar with a volume of 6 liters. This Jar was supplied with air filtered through cotton at both the intake and outlet. L rubber glove for handling the animals was attached to an opening in the side of the jar, and a rubber nipple was attached to a second opening. The lower portion of this apparatus was placed in a water bath to help maintain the proper environmental temperature . This equipment was sterilised with a combination of sublimate solu- tion, alcohol, ether, flaming, and autoclaving. Using a protective tent, surgery was perfomed on the pregnant guinea pig with the onset of milk secretion. The young were transferred into the rearing apparatus by opening the cylindrical jar quickly at its ground-glass surface. 8 Schottelius (1899, 1902, 1908, 1913) used a sterile roan in his efforts to rear germfree chickens. This roan, constructed of metal and glass, was entered through an anteroom provided with a germicide tray containing sublimate solution. The caretaker, wearing steam-sterilized cap, mask, gown, rubber gloves, and shoes, entered through this anteroas. A small hatching caspartment, built into the room, was separated from the room by a sliding door and gauze curtain. The temperature of this hatching compartment was regulated by a water bath. Air circulation to the sterile room took place by gravity through cotton filters . The room was prepared by washing with subli- mate and lysol solution. This was followed by treatment with formal- dehyde gas. All utensils used were sterilized by autoclaving. Eggs which had been incubated for 20 days were cleaned with mercuric 11 chloride solution, rinsed, and transferred into the hatching compart- ment, where they were left to couplets incubation and hatching. Cohendy (1912a, 1912b), in his efforts to rear germfree chickens, used a glass cylinder with metal end plates. Air entering and leaving the cylinder was forced through cotton filters . A coil of cold water was used for drying and cooling the air in the rearing unit, and at the same time the water condensing on this cold coil served as a source of drinking water for the birds. A hatching cae- partment was constructed in one end of the glass cylinder. The entire apparatus was sterilised with steam under pressure. The surface of the egg shell was sterilised with mercuric chloride before it was placed in the hatching capartment. The apparatus used by Kiister (1912, 1913, 1915a, 1915b) in rearing germfree goats consisted of a rearing room and a sterile lock. Air from a compressor was passed through a cotton filter and was then dried by being passed successively through concentrated sulfuric acid and potassium hydroxide. An electrical heating unit was inserted in the air flow before it entered the unit. The rearing room was sup- plied with rubber gloves for handling the animals. The sterile lock was equipped with 2 heavy doors. Before being sterilised, the rearing row was painted with a_ ”germicidal“ paint. It was then washed with lysol solution and treated with formaldehyde gas. The lock was sterilized with steam under pressure or' with formaldehyde gas. Preg- nant goats near term were subjected to surgery inside a protective tent. The young goat was then transferred by way of the sterile lock into the rearing roan. 12 The equipnent used by Balsam (1937) in rearing germfree chickens consisted of a double be: equipped with cotton filters for sterilising the air. The apparatus was sterilised with formaldehyde vapor for 3 days, with the exception of the filters which were steri- lised separately by autoclaving . Food and other materials were steri- lised in a metal box which was then placed in the outer compartment of the unit, and the surface of the box was sterilised with a combination of condensing steam and mercuric chloride solution. The fertile eggs from which chickens were to be hatched within the isolator were intro- duced after the shells were sterilised with mercuric chloride. The equipent used by Reyniers gt _a_1_. (l9h9a) in their early work on rearing germfree chickens consisted of a simple glass churn Jar fitted with a cotton filter and attached to a flask of sterile water. This unit was sterilised with dry heat at 180° C. for one hour. The fertile egg, whose surface had been previously sterilised with mercuric chloride, was placed in the glass jar inside a sterile hood. At the same time, a supply of steam-sterilised food was added. The Notre Dame group also described a germfree apparatus of the bell-Jar type with the bottom of the glass Jar being supported in a germicidal trap. A rubber glove attached to this trap allowed manipulation of the animals and materials within the unit. Air on- tering the unit was sterilised by passing through glass wool or cotton filters. The entire unit was sterilised before use by completely filling it with germicide. A modification of this unit was also con- structed of metal. The more elaborate isolation equipment designed by the Notre Dame group was first described by Reyniers (1910) and Reyniers and 13 Trexler (19113). Since these reports, numerous descriptions of this equipment and its use in germfree studies have appeared in the litera- ture. Among these the following may be listed: Reyniers (1956, 1957, 1959b), Reyniers _e_t 9;. (191.6, 19h9a), Horowitz gt 5;. (1960), and Hickey (1960). The basic Reyniers germfree isolator has been de- scribed as a heavy metal cylinder, usually constructed of stainless steel andng fras 28t036 in. indiameterandupto 12 ft. in length. To this isolator were attached a window, one or more pairs of shoulder-length rubber gloves, a supply lock (which was essentially an autoclave opening into the isolator), inlet and outlet filters con- taining glass fibers, and outlets for attaching a steam supply. The units were sterilized by steam under pressure, each isolator simply acting as its own autoclave. Four specialised isolators were described by Reyniers (1959b). These were rearing isolator, mining isolator, transport unit, and surgical isolator. These units could be attached to each other for transferring animals, food, etc. The surgical unit was sepa- rated into upper and lower units by a metal shelf with an opening in it. This opening was covered with sterile collaphane. At the time of surgery, the abdosen of the anesthetised full-term mammal (rat, mouse, guinea pig, or rabbit) was brought into contact with this cellophane from below. Using cautery, the operator was then able to cut through both the cellophane and abdominal skin, bring the uterus inside the isolator, and remove the young. The animals were then passed to the rearing unit. A special attactnent has been described for bringing fertile eggs into the rearing isolator through a germicidal trap (Teah, 1960) . Reyniers (1956, 1957) has also described a large, colony- type isolator suitable for rearing large mnbers of animals. The attendant, wearing a plastic suit, entered this unit through a germi- cidal trap. The Japanese workers (Iiyakawa, 1959a) have also utilised the heavy steel cylinder in designing an isolator patterned somewhat after the Reyniers unit. However, the animals, diet, cages, etc., inside . the isolator were manipulated with a remote control device. This allowed the isolator to be made larger since the arm's reach of an in- dividual was not the limiting factor. The air entering the isolator was sterilised by filtration and was also heated to hoo° c. and cooled. The humidity of the air was also carefully controlled. Gustafsson (19h6-19h7) has described an isolator constructed of a metal cylinder on end with a large petri dish on top as the lid. A sluice tank containing iodine was used to transfer the young rats taken by Caesarean section from the surgical isolator to the rearing unit. In a later publication (Gustafsson, 1959a) he described a larger unit constructed of thin stainless steel in a square, angled design. The tap of the isolator was covered with thick glass, and heavy duty surgical gloves were fastened to rubber sleeves which were, in turn, secured to Openings in the side of the isolator. The young animals were passed into this unit through the sterile food autoclave or through a germicide trap. Both these isolators were sterilised within an autoclave which was evacuated to 0.05 atmospheres before steam was introduced. Air to be passed into the isolator was first cooled and dehuaidified. It was then preheated and passed through a carborundm column heated by an electrical element to 300° 0., after which it was 15 filtered through glass-wool filter material. A heat exchanger was used to cool the air after sterilisation. The outgoing air was also sterilized by heat. The construction of an isolator from rigid transparent plas- tic has been described by workers at Syracuse University (Phillips gt 91., 1960). The walls were made of 3/8-in. Plexiglas type R (Rohm and Bass Company, Philadelphia, Pennsylvania), and were cemented to- gether. The air entering and leaving the isolator was filtered through couercially-available filters attached to the unit. Neoprene gloves were attached to openings in the walls of the isolator. Fertile eggs, sterilised diet , water, etc., were passed into the unit through a germicidal lock containing mercuric chloride. A series of chemicals was used in the sterilization of this equipment. Included were a de- tergent, an iodine compound, a quaternary ammonium solution, formalde- hyde, and etmlene oxide. Trexler (1959, 1960.) and Trexler and Reynolds (1957) have described isolators constructed of flexible plastic film. The air for these isolators was sterilised by passing through glass-wool mat, and the flow of air leaving the unit was controlled by an outlet trap which was a variable orifice relief valve with a liquid seal. This outlet trap maintained a back pressure equivalent to 1/h to 3/8 in. of water in the isolator. Shoulder-length rubber gloves , attached to the wall of the unit by means of aluminum rings, allowed access to the in- terior. The flexible plastic isolators were ordinarily sterilised with an aerosol of peracetic acid. laterials could be passed into the isolator by connecting it directly to an autoclave. Steam-sterilised materials in containers could also be passed through a lock in which 16 the outside of the container was sterilised with an aerosol of pera- cetic acid. It was also possible to use a germicidal dip bath in in- troducing materials into the isolator. lork has been conducted (Trexler, 1959, l960c) on the use of a large plastic-lined roar for rearing germfree animals. This roar was chemically sterilized and was serviced by an attendant wearing a plastic garment which was also chemically sterilised. Hickey (1960) has made a comparison of the apparatus cur- rently in use for obtaining and caring germfree animals. h. Diets According to Iostmann (1959a) the main difficulty facing the nutritionist when working with germfree animals is that, while much may be known about the requiraents of the host-flora complex, virtually no quantitative knowledge is available about the nutritional requirements of the host per se. It is therefore necessary to use the known require- ments of the normal stock animal as a base line. In using these, cor- rection must be made for the absence of possible intestinal synthesis (mostly of vitamins) and for the effects of the necessary sterilisa- tion, which results in loss of vitamins and of nutritional value of protein. Nuttal and Thierfelder (1895-1896, 1896-1897) fed their germfree guinea pigs a diet consisting of undiluted milk which had been obtained by milking cows under precautions of surgical sterility. This milk was boiled and bottled in a steam bath for 1/2 hour on 3 conp secutive days. l7 Schottelius (1899, 1902, 1908 , 1913) fed his chickens 'a diet consisting of millet seed, chopped egg white, and egg shells. This diet was sterilised by autoclaving. Cohendy (1912a) used different combinations of a number of ingredients in preparing diets for germfree chickens. These ingre- dients included egg albumin, bread crumbs, cracked corn, potatoes, rice, barley, lettuce, milk, dead flies, millet, chicory, sand, and Sprat's flour. These ingredients were sterilised with steam under pressure. luster (1912, 1913, 1915a, 1915b) , after repairing the mother following the Caesarean section, used the animal as a source of milk for his germfree goats. The milk was stored in bottles and sterilised by steam under pressure within the sterile lock attached to the rearing unit. In addition, sterilised oatmeal was fed. The diet fed to chickens by Balsam (1937) consisted of corn meal, corn, meat and bone flour, fish meal, cod-liver oil, brewer's yeast, sodium chloride, and calcium carbonate. This diet was also steam sterilised. This is the first recorded investigation in which vitamins were purposely added as part of the diet. In the early work on rearing germfree chickens at Lobund Institute (Reyniers _e_t_ 31. , l9h9a) , comercial starting rations, sup- plemented in some cases with cod-liver oil, brewer's yeast, and milk, were used. Somewhat later this same group used a number of synthetic- type diets. These diets varied quite widely in their component parts. Steam sterilisation at 120° C. for 25 minutes was used for liquid and solid diets, dry heat at 170° C. for one hour was used for oils, and heat-labile substances were passed through a Seits bacteriological 18 filter. These workers also investigated the destruction of vitamins by steam sterilisation and reported that the loss of thiamin could be held to 30 per cent if the proper pH was maintained. About 25 to 30 per cent of the pantothenic acid was destroyed by autoclaving. Luckey (1959) has reported further work on the preparation of diets for gemfree chickens and described a number of practical and synthetic-type diets. He also investigated the nutrient loss in syn- thetic-type diets and found an 11 per cent loss of both fat and protein due to steam sterilisation at 17 lbs. per sq. in. for 15 minutes and storage at room temperature for one week. Under the same conditions, the loss of vitamins ranged from 3 per cent for choline up to 92 per cent for thiamin. Lackey also found considerable loss of some nu- trients due to the mixing procedure. In addition to sterilisation by steam under pressure, Luckey stated that filtration, intermittent. heating, gamma rays, and ethylene oxide may all be used for sterilisa- tion of various diets. A description of some of the early diets used by the Lobund group in attempting to rear germfree rats was given by Reyniers _e_t_ 91. (191.6) . The liquid diets included such raw materials as rat milk, stomach contents of suckling rats , and milk from cows, dogs, rabbits, and sows. Various synthetic-type diets were also formulated. The most successful liquid diet contained casein, lactalbumin, a salt mixture, raw cream, sodium hydroxide, and distilled water. This was sterilized with steam at 15 lbs. per sq. in. for 20 minutes. A vitamin supplement was also fed to the animals. The solid diet consisted of a commercial rat diet or a synthetic-type diet , both supplemented with vitamins. These workers described the steam sterilisation of dry food. Their 19 procedure consisted of evacuating the supply lock to 29 in. of mercury, allowing steam to flow freely for 10 minutes, building up the steam pressure to 15 to 20 lbs. per sq. in., holding this pressure for 20 to 145 minutes, and finally evacuating the lock to 29 in. of mercury for about 1:5 minutes to help dry the contents . The initial evacuation be— fone the introduction of steam helped to insure the circulation and penetration of steam into the solid food. Pleasants (1959) has recently described various synthetic- type diets used at 1.0de Institute for rearing germfree rats , mice, and rabbits through weaning. He also described the rubber nipples and forced-feeding techniques used for the young animals. lostmam(1959a) has listed both liquid and solid diets successfully used in rearing germfree laboratory mammals. He stated that steam sterilisation (123° 0., 17 lbs. per sq. in. for 25 minutes) is the most satisfactory method for sterilizing diets. soatmm also stated that steam sterili- sation may cause loss of dietary thiamin of 80 to 90 per cent. Losses of other vitamins do not usually exceed ho to 50 per cent. The nutri- tional value of protein is also affected by steam sterilisation, with the availability of lysine especially being affected. Phillips 23.: g. (1959) have investigated a number of liquid, dry, and semisolid diets suitable for rearing germfree guinea pigs. Iiyakawa (1959b) reported the use of a liquid synthetic-type diet for rearing guinea pigs for the first few days of life. The diet was then gradually changed to a synthetic-type solid diet so that only the solid diet was fed after the first month. The liquid diet used by Glimstedt (1936) for his germfree guinea pigs consisted of cows' milk to which was added either blood 20 plasma or cream and plasma. This diet was sterilized by steam at 110° C. for 3/h of an hour on 3 successive days. The various solid diets used were sterilized by steam at 110° C. for 2 hours. The vita- min supplements used were sterilised by filtration. Gustafsson (19h8) reported the use of a milk mixture made with ensymatically digested casein as a source of protein. Gustafsson (1959b) used a stock diet composed of casein, wheat starch, arachis oil, a salt mixture, and a vitamin supplement. This diet was steri- lised by steam at 121° c. for 30 minutes. Phillips _e_t_ 21. (1960) sterilised solid diets for chickens by electron beams, using radiation doses of h to 5.5 megarads without visible alterations in the diet. Luckey (1960) used a diet containing casein, glucose, corn oil, salts, roughage, acetate, and vitamins in his attempts to rear germfree lambs. 5. Egg 9; Germfree Animals Rush of the early work with germfree animals was concerned with the develorment of techniques suitable for rearing the various species of animals free from contamination. “later (1912) , however, realised the promising scientific field of research opened by these germfree rearing attempts and suggested that animals raised under such conditions might be suitable for studying such things as digestion and absorption, development of natural and artificial immunity, wound healing, and destruction of pharmaceutical and therapeutic prepara- tions. 21 One of the areas of particular interest in germfree research has been the anatomical comparison of gemfree and conventional animals. This has involved primarily the intestinal tract (Gordon, 1959 ; Gordon and Bruckner—Kardoss, 1958-1959) and the lymphatic system (Glimstedt, 1936; Thorbecke, 1959; Thorbecke and Benacerraf, 1959; Thorbecke e_t g. 1957; "imam, 1959b; Hiyakan 93 g, 1957). Gemfree animals have also been usedquite extensively in nutritional studies, especially in attempts to define the role of the intestinal synthesis of certain vitamins. The following may be men- tioned as contributions of the germfree techniques in the study of the rurtrition of monogastric animals: Gustafsson (1959b), lostmann (1959a), Luckey (1959), Reyniers (19116, 1956), and Luckey _e_t_. g. (1955a, 1955b). In addition, Smith and Trexler (1960) and Luckey (1960) have introduced the use of germfree animals into the study of the process of ruminant digestion. The serologic aspects of germfree life have also been in- vestigated. Ioshann (1959b) has studied the serum proteins in gem- free vertebrates as have Iostmann and Gordon (1958). The formation of antibodies in germfree animals has been investigated by Springer g a}. (1959), Noam (1959b), and Wagner (1955, 1959). Gemfree and gnotobiotic animals have proved to be of value in a study of the mode of action of antibiotics in growth promotion. Most evidence (Luckey at 51:, 1956; Wagner and Iostmann, 1958-1959; Forbes and Park, 1959; Lev and Forbes, 1959; Forbes e_t 3., 1959; Gordon 32 51;, 1957-1958) points to the fact that conventional chickens fed antibiotics show an increased growth rate because of the inhibiting action of the antibiotics on certain undesirable bacterial 22 species. These same workers in general found no growth promoting action when antibiotics were fed to germfree chickens. Luckey gt a}. (1956) , however, found that low levels of certain antibiotics produced growth increments in germfree chickens. Phillips and wens (1959) and Phillips at 5;. (1955) have studied the relationship between bacteria and infection with Entsmoeba histolytica. They were unable to produce lesions in the germfree guinea pig when the parasite alone was given. When certain species of bacteria were also given, lesions were produced. Newton e_t .a_l_. (1959) and Phillips (1960) have also studied the development of helminths in germfree animals. Germfree animals have also been used in studying diseases caused by bacteria. Orland e_t a}. (1951.) were unable to demonstrate dental caries in germfree rats fed a diet which consistently led to the production of caries in conventional rats. Later work (Orland gt e_l. , 1955; Orland, 1959) demonstrated that Enterococcus in pure culture, combined with the cariogenic diet produced caries in germfree rats. Taylor (1959) has discussed the use of germfree animals in virology. Among the miscellaneous areas of research utilising germfree animals may be listed studies involving tuners produced in chickens by the injection of methylcholanthracene (Taylor at 51. , 1959; Reyniers and Sacksteder, 1959), liver necrosis (Luckey e_t a_1_. , 1951;), honor- rhagic shock (Zwiefach, 1959) , and otolaryngic experimentation (Kelemen, 1960) . 23 B. Artificial Rearing of. 23:52. Early attempts to rear baby pigs without the aid of colostrum often met with failure. Bustad gt _s_1;. (19118) reported that pigs re- moved frm the mother at birth and placed on a synthetic milk con- taining all known vitamins and with plasma or serum as colostrmn sub- stitutes failed to survive longer than 22 days. Ihen no colostrum substitute was fed, the pigs died shortly after birth. These workers concluded that colostrum and serum or plasma contain something which is necessary for the sm'vival of the pig. A severe diarrhea developed in all animals fed the synthetic milk. The apparently critical need for colostrum in the diet of the young pig was further uphasised by the numerous reports of successful artificial rearing of pigs weaned at an early age. Iintrobe (1939) successfully reared pigs weaned at 2 to 23 days of age on a diet of casein, lard, sucrose, minerals, and vitamins. Becker gt :1. (19511) , however, working with pigs allowed access to colostrum for 211 hours, concluded that the baby pig was un- able to utilise sucrose as a source of carbolvdrate. A synthetic-type diet with glucose as the carbohydrate did produce satisfactory results. Johnson _e_t_ 31. (19118) reported the rearing of pigs weaned at a minimum 01’ 211 hours on a diet of casein, cerelose, lard, salts, and vitamins. Other successful attempts to rear pigs weaned at one to 2 days of age have been reported by Reber gt :1. (1953), Schendel and Johnson (1953), and lcCrea and Tribe (19511, 1956). It is now recognised that young pigs, along with the young °f other farm manuals are incapable of producing antibodies themselves for some considerable time after birth and that they derive their in- mnity from the mother through the colostrum (Brambell 3.2 21., 1951). 211 Rutqvist (1958) was unable to detect gamma globulin in the serum from pig fetuses and newborn pigs. Large amounts of game globulin appeared in the sense shortly after nursing. It has been shown that game globulins form the chief component of cireulating antibodies (Iostmann, 1959b). This definite correlation between the ingestion of colostrum and the level of antibody in the young pig has also been noted by Hoerlein (1957) , Straub and Boguth (1956) , and Iellman and Heuner (1953). Work by Hoerlein (1957), Asplund (1960), and Speer (1957) in. dicated that the ability to absorb gamma globulins from the colostrum is lost in the young pig after approximately the first 21) hours. Young and Underdahl (1951), in discussing the rearing of colostrum-deprived pigs, stated that it is of vital importance to pro- tect the young and vulnerable pig from infections that may be ensootic within the herd, even though such infections are not apparent in older swine or in pigs that have had colostrua. These workers caught pigs in sterile bags at the time of birth and raised them in an isolated roan heated to 80 to 90° F., using a diet based on milk. This same group has developed a technique for moving the pigs from the sow by means of hysterectav (Ioung e_t 31;, 1955). The full-term 80' '38 anesthetised with carbon dioxide , and the entire gravid uterus was re- moved and passed into a hood previously treated with formaldetwde (Underdahl and Ioung, 1957a). Here the pigs were removed from the uterus, the umbilicus was ligated and cut, and the stimp was treated with tincture of iodine. The young animals were then transported in a Previously sterilised wooden box to the rearing room. Here the pigs were placed in individual isolation units in a room previously treated with formaldehyde (Young and Underdahl, 1953; Young _e_t_ 3., 1955). 25 The isolation units were kept at 90 to 100° F. for the first few days, and then the temperature was gradually lowered. The diet described by Underdahl and Young (1957b) for starting the young pigs consisted of one quart of hanogenised milk, one whole egg, and 5 ml. of a mineral mixture containing ferrous sul- fate, copper sulfate, manganese chloride, and potassium iodide. The pigs were transferred at one week of age to an isolation broader (Underdahl and Young, 1957b) large enough to hold 12 pigs. They were continued on the same diet and gradually changed to a com- mercial pig starter feed. The pigs at 11 weeks of age were transferred to a clean area. The use of these ”disease-free" pigs to repopulate herds affected with atrophic rhinitis and virus pneumonia of pigs has been described by Young e_t g. (1959). lhitehair and Thompson (1956) reported the rearing of Caesarean-derived pigs in individual metabolism cages on a diet of casein, fat, lactose, minerals, and vitamins. Others successfully rearing colostrum-deprived pigs have included Haelterman (1956), Hoerlein e_t a}. (1956), and Shuman 33 31. (1956). , Catron e_t 51. (1953) described a practical synthetic milk formula for baby pigs receiving no colostrum. In attempts to improve the diets for rearing young pigs, various workers have studied the composition of sows' milk. work has been reported by Heidebrecht 91'. 51. (1951) and Davis 11". 21. (1951). The British workers (Brands 23 _a_l_., 191:7) have found that sows' milk contains a higher percentage of total solids, solids-not-fat, protein, 38h, calcium, and phosphorus than the milk of cows and goats and about the same percentage of lactose. Bellis (1957) also compared the com- POSition of the milk of various mammals. III . EXPERIMENTAL A. Procedures 1. Equiaent The equipment used in 1; experiments on collecting and rearing gnotobiotic pigs was similar to that described by Trexler (1959, 19608) and Trexler and Reynolds (1957) for rearing laboratory animals. Two principal pieces of equipment were used, one being the surgical isolator and the other the rearing unit. Both these isola- tors were constructed of vinyl film which was 8 mils (0.008 in.) in thickness. The surgical isolator (Figure 1) measured 36 x 36 x 72 in. and was mounted on a sheet of plywood slightly wider and longer than the unit. The top of the plywood was covered with one layer of paper- packing material to help cushion and protect the bottom of the isola- tor. At one end of the sheet of plywood was fastened a fiber-glass ring 16 in. in diameter and approximately 12 in. in length. This ring served as an opening into the isolator, and it extended about 2 in. above the top of the plywood. A circular piece of the vinyl film about 12 in. in diameter was cut out of the bottom of the isolator, and the resulting opening was stretched over the top of the fiber- glass ring. The film and fiber glass were fastened together by 3 com- plete turns of Scotch Brand Tape No. 1171 (Minnesota lining and llanufacturing Comparw, St. Paul, Minnesota). At the opposite end of the isolator were attached the air filter and air-outlet trap to be described in more detail later. 26 I (I'llvllf. Ii I‘lllil III-I'll ls l Figure 1. Surgical isolator A. B. C. D. 16-in. fiber-glass ring Air filter Air-outlet trap 30-in. dryabox gloves lZ-in. fiber-glass ring Outer door 28 Three pairs of 30-in. dry-box gloves (Charleston Rubber Compamr, Charleston, South Carolina) were attached to the sides of this surgical unit. Two pairs, both 25 mils thick, were attached opposite each other on the end nearest the fiberglass ring in the bottom of the isolator. The third pair, 15 mils in thickness, was attached on one side near the opposite end. The gloves were attached to the isolator by a method differing somewhat from that described by Treader and Reynolds (1957). A circular Opening approximately 7 in. in diameter was cut in the viml film at the place where the glove was to be attached. The cuff of the glove was cemented to the film and both were held in place in the outer, concave surface of a spun- alminms ring approximately 9 in. in diameter. Directly opposite the third pair of gloves was attached another fiber glass ring which was 12 in. in diameter and 6 in. in length. This ring was placed vertically. Both this and the larger ring were provided with flanges for securing the rings to the plywood base. The vinyl film was attached to the smaller ring in the pre- viously described manner. Inner and outer doors or covers for this ring were constructed of 20-mil viml film with the door being made slightly smaller in diameter than the fiber-glass ring. The inner door was stretched over the interior opening of the ring and held in place with a large rubber band cut from a truck inner tube. The outer door was held in place with 3 turns of Scotch Brand Tape No. 1:71. This outer door was provided with 2 openings constructed of Tygon flexible tubing (U. S. Stoneware Ganpany, Akron, Ohio) 1 in. in diameter and 3 1/2 in. long. These were either sealed to the vinyl film by use of a Callahan Dielectric Sealer (J. A. Callahan Comparw, Chicago, 29 Illinois) or were taped in place in much the same manner as the fiber- glass rings. Three such openings were provided in the isolator itself, one each for receiving the air filter and air-outlet trap, and the third for use during sterilisation of the isolator. These openings were closed with rubber stoppers when not in use. The rearing isolator (Figures 2, 3) measured 21.; x 2); x 72 in., and it was attached to a plywood base with the ends and part of the sides built up to give added support. An air filter (Figure 3;) and air-outlet trap (Figure 5) were attached to one side of the unit, along with a lZ-in. fiber-glass ring fitted with inner and outer doors as previously described. On the apposite side one pair of gloves was attached. The floor of both the surgical and rearing units was covered with a sheet of ribbed polyethylene to prevent cages and sharp objects frmn perforating the vinyl film. In the first 2 experiments no satisfactory method of weighing the pigs inside the isolator was found. In Experiments III and IV a book was installed in the top of the roaring unit by cutting a small hole in the vim'l film. A piece of Tygon tubing l/2 in. in diameter and approximately 3 in. long was taped into this hole as has been pre- viously described. A length of small stainless steel rod was then forced through a hole drilled in a small rubber stopper. A hook was bent on either end of the rod, and the stopper was taped securely into the upper end of the tubing. This allowed the upper end of the hook to be attached to a spring-type milk scale. This scale, in turn, was sus- pended from the ceiling of the room. The pig was suspended from the lower hook inasmall clothbagorbymeans ofaloop <3f 8'“sz the heck joint. 30 Figure 2. Rearing isolator viewed from filter side A. B. G. D. Air filter Air-outlet trap Sterile lock Rearing cage .. If- -‘ Figure 3. Rearing isolator viewed from glove side A. 30-1n. dry-hem gloves B. Spencer Turbo Compressor Figure 1;. Air filter attached to rearing isolator A. Air filter B. Sterile lock Air-outlet trap attached to rearing isolator Figure 5. Air-outlet trap A. 3h The sterile lock (Figure h) for the introduction of liquid diet, vitamins, minerals, etc. , into the isolator consisted of a tubu- lar piece of 20-mil viml film 12 in. in diameter and 21; in. in length. This lock had 2 Tygon-tubing Openings, one near either end. The outer door was rmoved from the rearing isolator and one end of the lock was fastened to the fiber glass ring with 3 turns of Scotch Brand Tape No. 1571. A fiber-glass ring 12 in. in diameter and 2 B/h in. in length was taped on the other end of the lock, and the outer door was also taped to this ring. This famed a chamber which could be loaded from the outside and whose contents could be brought into the rearing isolator through the inner door. The air filters were constructed with a core of wire mesh or empanded metal having solid ends (Figure 6). On the tap end, a metal tube, 1 in. in outside diameter, was welded. This core was. wrapped with four l/2-in. layers of F0 50 glass-wool mat (American Air Filter Company, Louisville, Kentucky) (Figure 7) which were held in place by several turns of small stainless-steel wire. This amount of filter material has been found sufficient to retain a dried aerosol of washed bacterial spores at velocities encountered during use (Trexler, 1959). A cover of 8-mil vinyl film was held in place over the filter material by 2 radiator hose clamps (Figure 8). In actual use the air entered through a short piece of Tygon tubing attached to the cover, went through the h thicknesses of filter material, and left through the metal tubing attached to the filter core. This tubing was secured in- side one of the Tygon-tubing outlets of the isolator by means of a small radiator hose clamp. - 35 Air-filter core Figure 6. 36 (.51 ‘ h ‘9‘. V5_ I - , , w ““ ‘ I ‘ $ ( \ ;B x "fl ," 1 ..J ‘ l ‘ . Figure 7. wrapped air—filter core A. Air-filter core B. Glass-wool mat Figure 8. Assembled air filter Vinyl cover Radiator-hose clamp Adjustable-clamp Tygon tubing for attachment to air source Metal tube for connecting to isolator 38 Air filters of similar construction have been shown by Trexler and Reynolds (1957) to pass 2 to 3 cu. ft. of air per minute at an air pressure of 5 lbs. per sq. in. The air-outlet trap (Figures 9, 10) was made of fiber glass. The trap was attached to the isolator by slipping the small side an inside one of the short pieces of Tygon tubing fanning one opening into the isolator and attaching it with a small radiator hose clamp. The lower end of the mall fiber-glass tubing, covered with a short length of Tygon tubing sealed on one end, acted as a reservoir to prevent fluid spilled from the trap from running into the isolator. The upper end of this tube rose above the level of the liquid in the trap. The end of this tube was covered by an inverted plastic tumbler with 2 openings in its well. These openings were at such a level that they were approximately l/h in. below the surface of the liquid when the tumbler was at its lowest position. - A wire ring was mounted inside the large opening of the tumbler, and a short piece of wire was cemented to the upper surface. These both acted as guides. A wire screen covered the top of the trap to shut out insects. The air- . Outlet trap was filled to the proper level with Cellulube (Celenese corporation of America, New York, N. L). The air leaving the isolator entered the side arm of the tap, traveled into the upper end of the fiber-glass tube, raised the 1”Verna tumbler, and escaped through the holes in the wall of the l‘ttOr. Aw decrease in'pressure within the isolator, caused, for example, by withdrawing one's arms from the gloves, caused the tumbler t° d-‘Pep so that the outlet openings were below the surface of the x. Figure 9. Air-outlet trap, disassembled Side arm Reservoir Upper and of fiber-glass tubing Fluid level Plastic tumbler Opening in wall of tumbler lire-screen cover ho Figure 10. Air-outlet trap, assembled i. . , a. v }.-..h..u.n Ii‘rwlufadfiafiw thrurvfifi ‘ .. ... hl liquid. This prevented a back-flow of contaminated air into the~ iso- hWe In Experiment I the air-outlet trap of the rearing isolator consisted of a 6-in. length of vinyl film tubing 1 in. in diameter. One ad of this tubing was attached to the Tygon tubing opening of the The other end, with a small metal weight attached, floated The isolator. on the surface of a l-liter beaker 3],; filled with mineral oil. air flow caused this tubing to float on the surface of the liquid. .A docrease in pressure caused the end of the tubing to sink, and the back-flow was prevented. Outlet traps of this design have been shown by Truler (1959) to maintain a pressure equivalent to l/h to 3/8 in. of water within the iBOhtOre Air flow was provided either by small individual electric blowers of the squirrel-cage type (Universal Electric Company, Owosso, Hichigen) or by a Spencer Turbo Compressor (Spencer Turbine Company, Hartford, Connecticut). The latter provided enough air flow for Sweral isolators at one time and was fitted with a ”rough" filter for removing dust and other extraneous material from the air. The air 'Ource and the filter on the isolators were connected by lengths of Va<=‘Iluln-sweeper hose. The flow of air was regulated by an adjustable “flap on the Tygon tubing of the air filter so that the inverted plas- tic t‘uabler of the outlet trap rode at the preper level. The cages (Figures 11, 12) were constructed of stainless "5.01 and measured 11 x 11 1 181a. They were provided with a door, ‘ movable floor of heavy hardware cloth, and a removable tray for °°n§cting urine, feces, and spilled food material. The door contained J— "‘ " ,J h2 Figure 11. Stainless-steel rearing cage A. Feeding pan B. Metal clip C. Removable tray D. Door catch \ . u ,. . F .A Figure 12. A. B. C. AL, 143 W -.7 minim" 0"! a '0' 3...... i oeeeeee. V umeeeeee eeeeeeea ‘ Interior of rearing cage Feeding pan Removable floor Removable tray . ,. - — Motowne- e _.__—.——\__-fi — I} a" ,r 7; e . uh an opening to receive the feeding pan which measured 5 3/1‘ x h in. and was 1 5/8 in. deep. lletal clips held the feed pan and the floor in place. The containers for liquid diet (Figures 13, 1h) were 2-liter Square-Pal: Flasks (American Sterilizer Company, Erie, Pennsylvania). These contdiners were sealed by 2-piece lids. A rubber collar slipped over the neck of the container, and a hard plastic cap covered this collar. The collar and cap were self-venting so that liquid diet could be autoclaved within them. Ihen the containers cooled, the collar and cap formed an air-tight seal. 2. Sterilisatigg 9}; M All equipnent to be sterilized was first washed with water and a detergent, rinsed, and dried. The lower end of the l6-in. fiber-glass ring on the surgical unit was covered with a sheet of filer (E. 1. dqut de Nemours and Company, Inc., Wilmington, Delaware) 1.5 mils in thickness. This was held in place by 3 complete turns of Scotch Brand Tape No. 853. The air filter was detached from the isolator, and the vinyl cover was removed. A small piece of llylar was placed over the metal tube carrying air from the filter and was taped in place with Scotch Brand Tape No. 853. The filter was then wrapped in paper and sterilized in an oven at 150° C. for 1 1/2 hours. The filter was then attached to the isolator, and the cover was re- placed. The surgical isolator was next inflaud with a mixture of Freon (10 per cent) and air (90 per cent). A Halogen Leak Detector Type 8—2 (General Electric Cmpany, Schenectady, New York) was used Figure 13. Square-Pak Flask, disassembled A. Rubber collar B. Plastic cap Figure 11;. Square-Pal: Flask, assembled h? to check the unit for leaks, covering the entire surface of the vinyl film and gloves. Any leaks found were patched with 8-mil vinyl film using the Callahan Dielectric Sealer. The interior of the surgical unit was sterilized with 2 per cent peracetic acid. Trexler (1959, l960a), Trexler and Reynolds (1957), and Kline and Hull (1960) have found this material to be a very effective sterilisation agent when it is properly used. Trexler and Reynolds (1957) have emphasized that peracetic acid is very corro- sive, and many substances such as heavy metals catalyze its decomposi- tion. They therefore recommended plastics, stainless steel, and glass as the construction materials of choice in cases where sterilization was to be accomplished with peracetic acid. Nacconal NRSF (National Aniline Division, Allied Chemical Corp., New York, N. Y.) in the amount of approximately 0.1 per cent was added as a detergent and wetting agent. This solution was made with distilled water. The in- terior surfaces of the gloves, isolator, and floor mat were thoroughly wet with this solution which was atomized with a small paint spray unit modified by inserting a small plastic tube inside the spray nozzle. After the surfaces were thoroughly wet, the isolator was in- flated with air and all openings sealed with rubber stoppers. The in- terior surface of the outer door was sprayed. The door was taped in place, and the area between the inner and outer doors was fogged through the 2 openings in the outer door. The surgical unit was allowed to remain closed for at least 2 hours. Then the Mylar film covering the opening of the air filter was punctured from inside the isolator with a small length of .."mmhlfuer .. ,. ._ . - ‘qm 148 stainless steel wire, the isolator was connected to the air supply, and the interior of the isolator was allowed to dry. Before the interior of the rearing isolator was sterilized, the cages were wrapped in paper and sterilized with steam at 250° F. for 30 minutes. Since the cages were too large to go through the 12- in. fiber-glass ring, it was necessary to cut an opening in the vinyl film, insert the wrapped cages, and reseal the opening with the Callahan Dielectric Sealer. The cages were then unwrapped and sprayed and the rearing unit was sterilized in the same manner as was described for the surgical isolator. After the rearing unit was dry, the remaining supplies were sterilized and transferred into it. These supplies included surgical instruments, towels, cloth gloves, 3 length of cotton cord, vials of minerals and vitamins, screw-capped test tubes, a h—oz. rubber bulb, a glass graduate, and the initial swply of milk in 2-liter flasks. This material was placed in a modified Reyniers unit, and a vacuum of approximately 25 in. of mercury was drawn in the unit to remove air pockets and insure better penetration of steam. The vacuum was then broken with steam, and the contents were sterilized at 252° F. for 28 minutes. A sterile lock attached to the roaring unit then per- mitted these supplies to be transferred from the Reyniers unit to the vinyl rearing unit. 3. Surgical Procedure A small, round—bottomed, galvanized tank of approximately ZS-gal. capacity was placed at the end of the surgical unit so that the l6-in. fiber—glass tubing extended into one end of the tank —— w.—m3.fl 'Xb‘ 9 ‘- a 149 (Figure 15). A piece of large-size polyetl'wlene tubing, sealed at either end, was used to line this tank, and an opening was cut in this tubing to receive the lé-in. fiber-glass ring. Two Tygon openings were also attached to the polyethylene, and 20 gal. of water were added to the lined tank through one of these openings . The water covered ap- proximately 1; in. of the outside of the fiber-glass ring. Persoetic acid was added to provide 50 parts per million, and the interior of the polyetmrlene was fogged with 2 per cent peracetic acid solution. Shortly before the unit was to be used, 1 gal. of 5.25 per cent sodium hypochlorite bleach was added to the solution in the lined tank. The outer doors were moved from both isolators, and the 2 fiber-glass tubes were connected by a short length of 20-mil vinyl tubing 12 in. in diameter with 2 Tygon openings in the vinyl (Figure 16). This connecting tubing was taped in place with 3 turns of Scotch Brand Tape No. h7l on each end. The interior surface of this comes- tion was then fogged with peracetic acid solution, and the Tygon openings were closed with rubber stoppers. After 30 minutes both inner doors on the isolators were opened, and the necessary towels, surgical instruments, and cotton cord were passed into the surgical unit. The pregnant Yorkshire sow on the 112th day of gestation was thoroughly scrubbed with a detergent solution. She was then suspended by the rear limbs, head down, using a chain hoist and cables attached above the fetlock Joints, and lowered into a large tile, 2).; in. in diameter and 36 in. deep, sunken in the floor with the upper surface level with the floor. Seven pounds of dry ice had previously been pulverized and placed in this tile. After one minute surgical rive—m . _. VT saw-u :wr Figure 15. Surgical isolator with tank in place A. Galvanized tank 42‘. Figure 16. Surgical and rearing isolators connected A. B. C. D. E. F. Surgical isolator Rearing isolator Connecting tube Galvanised tank Polyethylene lining tank Plywood incline 52 anesthesia was complete, and the sow was raised from the pit. The ab- domen was then thoroughly scrubbed with Weladol Shampoo (Allied Laboratories, Inc., Indianapolis, Indiana) and dried. In Experiments II, III, and IV the ventral abdominal wall was then sprayed with Vi-Hesive Surgical Adherent (Aeroplast Corporation, Dayton, Ohio). A sterile piece of Mylar approximately 2 ft. by 3 ft. was then cemented to the ventral abdominal wall. The abdominal cavity was then quickly opened in the ventral midline, cutting through the Mylar when it was used. The incision ex- tended frcn Just anterior to the brim of the pelvis to a point Just posterior to the xiphoid cartilage. A heavy cotton ligature was then tied around the uterus just anterior to the cervix. In Experiment III, 2 heavy forceps of the intestinal type were applied instead of the ligature. The uterus was then removed by cutting just posterior to the ligature and also Just anterior to the ovaries. In Experiment I the uterus was passed directly into the germicidal tank after the top of the polyethylene bag lining the tank had been incised. In the other 3 experiments a piece of plywood ap- proximately 15 in. square was set at the end of the tank at a slight incline (Figure 16). This was covered with sterile Mylar and the uterus was allowed to slide down this surface into the germicidal tank. The uterus was guided into the tank by an assistant wearing sterile, shoulder-length gloves. This assistant then kept the uterus in continuous motion in the germicide for 30 seconds. In the meantime, the ”lar covering the bottom of the l6-in. fiber-glass ring was punctured with scissors by the operators working through the gloves of the isolator. The uterus was then pulled into 53 the surgical isolator and torn open as rapidly as possible. Blunt scissors or forceps were used to help puncture the uterine wall. The fetal membranes were ruoved from each pig, and a small alligator-type electrical clip or a Kelly forceps was placed on the umbilical cord approximately 1 in. from the body. The pigs were then given artificial respiration by alternately pressing and releasing the chest until all were breathing. The umbilical cords were then ligated close to the body wall with heavy cotton cord, and the cords were out between the ligature and the forceps. The pigs were dried with cotton towels and were then nabbed with a towel saturated with a solution of Hyamine (Rohn and Haas Compaw, Philadelphia, Pennsylvania) in Experiment I. In Experi- ment IV they were swabbed with a solution of Quart-Sept (Fort Dodge Laboratories, Fort Dodge, Iowa). The Operator using the gloves oppo- site the lZ-in. fiber-glass ring then passed the animals into the rearing unit. The inner doors were replaced on both isolators , and the connecting tube was removed. The inner surfaces of the outer doors were sprayed with 2 per cent peracetic acid, the doors were taped in place, and the space between the doors was tagged with the solution through the Tygon openings. The rearing unit was then removed to the roan in which the aninals were to be kept. h. Diets and m Procedures a. kperimsnt I The diet used in Experiment I consisted of pasteurized, hauogenised milk containing h.S per cent butterfat and fortified with Sh hoo International Units of irradiated ergosterol per quart. Two quarts of this milk were placed in each 2-liter Square-Pak Flask, and the initial supply was sterilized with steam as described previously. The initial supply of milk was passed directly into the rearing unit from the autoclave. During the course of the experiment, milk was sterilized by steam at 250° F. for 30 minutes. The flasks were then removed from the autoclave, and the exterior surfaces of the flasks were cleaned and dried. The sterile lock (Figure 1;) was then attached to the rearing unit, and the outer door was removed. The interior surface of the lock was sprayed with peracetic acid solution, and the exterior surfaces of the Square-Pal: Flasks were likewise sprayed before being placed in the lock. The exterior door was then replaced, and the space within the lock was fogged with the solution. After a waiting period of at least 30 minutes, the inner door of the look was removed, the flasks were taken into the isolator, and the inner door was replaced. The vitamin mixture used in this experiment to compensate for vitamin loss in sterilisation was essentially B vitamin mixture 103 de- scribed by Pleasants (1959), except that it was twice as concentrated. The vitmins used are listed in Table I. This solution was made up and dispensed in 15-ml. quantities into 20-ml. glass ampules. The ampules were then sealed and sterilized with the initial milk supply, being passed directly into the rearing unit from the autoclave. The mineral supplemnt used was salt solution 15 also de- 5 cribed by Pleasants (1959). It consisted of solution A and solution B 1'3xich were made up and sterilised separately in lS-ml. quantities 55 TABLE I. B vitamin mixture 103, concentrated Vitamin Mg. per 1.5 ml. water Thiamine hydrochloride 3 Riboflavin l Pyridoxine hydrochloride 1 Niacinamide 2.5 Calcium pantothenate 12.5 Choline dihydrogen citrate 100 Biotin 0.025 Folio acid 0.25 Vitamin 312 0.025 Inositol 100 as described for the vitamin mixture. The composition of the solu- tions are listed in Tables II and III. TABLE II. Salt mixture 15, solution A IMent E. Er ml. water NaZHPOh 180 KI 0.1. Before the milk was fed to the pigs within the isolator, 30 ml. of B vitamin mixture 103, concentrated, and 15 ml. each of anineral solutions A and B were added to each Square-Pal: Flask 56 TABLE III. Salt mixture 15, solution B Iggedient g. per ml. water “8801; 11s mole-M120 h Ferric amonium citrate 21; 011012 2.1; ZnSOh°H20 5.2 00012-6320 0.8 containing 2 qts. of milk. The ampules were opened by filing a small notch in the neck of the ampule with a small 3-cornered file and then breaking the ampule at this notch. Cloth gloves were usually worn over the rubber gloves during manipulations within the unit to prevent the gloves being punctured by slivers of glass and other sharp objects. The pigs were fed 3 times each day, at approximately 8 a.m., l p.m., and 8 p.m. The milk, vitamin, and mineral diet was placed in the feed pans, and the pans were left in the cages until the next feeding. The volume of diet fed each day is shown in Table IV. TABLE IV. Volume of diet fed in Experiment I Age of igs Volume per pig per day (days; (1:11.) o-h 300 5-6 360 7-17 160 57 Waste material in the cages was aspirated from the removable trays by means of the h-os. rubber bulb and placed in the empty Square- Pak Flasks from which the diet had been fed. This material was then passed outside the isolator as new supplies of milk were passed in. The temperature in the room in which the pigs were maintained was kept at 90° F. during the course of the experiment. A total of 7 pigs was obtained in Experiment I. Since only I. cages were available in the rearing unit, the remaining 3 pigs were removed from the unit at 2 days of age and placed in open metabolism- type cages. These cages were kept in the same room as the rearing unit, and these animals were fed the same diet as the gnotobiotic pigs. b. Experiment I; The diet used in Experiment II was the same as that used in kperiment I except that the vitamin and mineral supplements were changed. The vitamin supplement used (Table V) was a modification of that described by Rsber gt 5}. (1953). As modified, this supplement was calculated to allow for destruction of 90 per cent of thiamine and 50 per cent of the other vitamins as suggested by lostmann (1959a) and still meet the minimum requirements. The fat-soluble vitamins were dissolved in absolute ethyl alcohol, the remaining vitamins were dis- solved in distilled water, and the volume of the final solution was adjusted to contain 20 per cent alcohol. This solution was dispensed in lO-ml. ampules, sealed, sterilized at 250° F. for 15 minutes, and passed into the rearing unit. It was added to the milk at the rate of 5 ml. per Quart. The mineral solution added in this experiment consisted of §olution B used in Experiment I with the addition of potassium iodide 58 TABLE V. Vitamin solution used in Experiments II, III, and IV Vitamin Amount gr 5 ml. A‘ 1600 LU. Db 2&0 LU. 3° 2.0 mg. K 0.5 mg. Thiamine hydrochloride 3.0 mg. Riboflavin 1.2 mg. Pyridoxine h.o mg. Calcium pantothenate h.0 mg. Inositol 20.0 mg. p—Aminobensoic acid 5.2 mg. Biotin h7.2 meg. Nicotinic acid 5.2 mg. Choline 260.0 mg. Folic acid 0.1 mcg. 312 5.2 mcg. a As FEB-250 dry vitamin A (Distillation Products Industries) b is irradiated ergosterol c As alpha tocopherol acetate in the same amount as it has been in solution A. The resulting modi- Iisd solution B was added to the milk before feeding at the rate of 3.5 ml. for each 2 qts. of milk. The pigs in this experiment were fed 1; times each day for ‘he first 11 days. The feedings were made at approximately 8 a.m. , 59 l p.m., 5 p.m., and 10 p.m. During the remaining time the 10 p.m. feeding was eliminated. The volume of diet fed per day is indicated in Table VI. TABLE VI. Volume of diet fed in Meriment II 0 . Age of pigs Volume per pig per day (152) (n1 ) 0-6 hso 7-17 600 The temperature in the rearing room was held at 90° F. for the first a. days, after which it was reduced to 85° F. for the re- mainder of the experiment. Of the 9 animals in the litter used in this experiment, 3 died while in the surgical isolator. Four of the remaining animals were transferred to the rearing unit, and 2 were transferred to indi- vidual isolation units, similar to those described by Ioung 33 El. (1955). These units were designed to operate under positive pressure. it the end of 7 days these 2 animals were rasoved from the individual isolation units and placed in open metabolism-type cages in the same roan where the vinyl rearing unit was maintained. These pigs were fed the same diet as the gnotobiotic animals. c. garment £I_I The diet fed in this experiment was identical to that used in kperiment II. The pigs were fed 1. times a day for the first 6 days and 3 times a day thereafter. The approximate amounts of diet fed are indicated in Table VII. TABLE VII. Volume of diet fed in Experiment III Age of pigs Volume pe(r pig per day ml (5813) . o - 2 300 3 - 7 too 8 ~ 9 500 10 - 11 600 12 - 16 1150 17 - 21 525 For the first 9 days of this experiment the room temerature was maintained at 90° 1". After this it was reduced to 85° F. for the remainder of the experiment. 01‘ the 13 pigs in this litter, only I; lived. Since the num- ber of animals in this group was so small, it was thought advisable to leave all the pigs in the plastic isolator and not place any in open cages. d. kperiment I! The diet fed in Experiment IV was identical to that used in Experiment III. The milk was sterilised in 1 1/2 liter amounts in the 2-liter Square-Pak Flasks to reduce the amount of boiling over during the sterilisation process. After the flasks were transferred into the rearing unit, they were brought to the usual 2qt. volume before addition of vitamins and minerals. The pigs were fed 6 times a day, with the feedings being made at 8 a.m., ll a.m., 2 11.31., S p.m., 8 p.m., and 11 pm. The amounts of diet fed are in- dicated in Table VIII. 61 TABLE VIII. Volume of diet fed in Experiment IV Age of pigs Volume per pig per day .1291)._ ( ) o - 6 360 7 - 9 1:50 10 - 12 51.0 13 - 16 630 17 - 21 720 Twelve pigs were obtained in this litter. Six of these were placed in the rearing unit, with 2 pigs being placed in each of 2 cages. Five of the remaining animals were allowed to nurse a sow which had fan-owed the day these pigs were delivered. These pigs were given 2 m1. of Amidsman (Armour and Compamr, Kankakee, Illinois) at 2 to 3 days of age. At the termination of these 1.; experiments, the animals were removed from the rearing units, weighed, bled, and killed. Various organs were weighed and blocks of tissue were saved for use in Part Two. Experiments I and II were each terminated at the end of 17 days, and kperiments III and IV were terminated at the end of 21 days. 5. Bacteriological Procedures Bacteriological determinations on the pigs in the rearing unit were made twice during each experiment, once at about 10 days of age, and again at the termination of the experiment. Samples for these determinations were obtained from 2 sources. Sterile cotton swabs in screw-capped test tubes, which had been placed in the rearing 62 unit at the time of initial sterilization, were used in collecting rectal swabs from the pigs. The second source of material for culture consisted of a mixture of urine, feces, and milk dipped from the bottom of the tray in each cage. This was placed in a sterile, screw-capped test tube within the isolator. These samples, one rectal swab from each pig and one liquid sample from each cage, were then passed out through the sterile lock. In Experiment I wet mounts of the samples were made and ex- amined, along with smears which were stained with Gram's stain. ‘ Eugonagar (Baltimore Biological Laboratories, Baltimore, Maryland) I (with 20 per cent bovine blood) and thioglycollate broth were also inoculated. In Experiments II, III, and IV, a method which was essen- tially that of Larson and Hill (1955) was used to determine the flora of the animals within the isolator. Vet mounts and Gram's stains were made as in Experiment I, and Eugonagar (with 20 per cent bovine blood) was inoculated and incubated both aerobically and anaerobically at 37° c. The liquid samples from the cages were used in inoculating the remaining media. Violet red bile agar (Difco Laboratories, Detroit, lichigan) was used for coliform determinations. ”SF" medium (Difco) was inoculated for detemination of fecal streptococci. Lactobacillus Selection Medium (B.B.L.) was used to detect the presence of lacto- bacilli. Glostridia were determined by the use of a modified Iilson- Blair medium, and potato dextrose agar (Difco) was utilised in checking for yeasts. 63 lhen growth was obtained on any of the media used, attempts were made to identify the bacteria involved, using standard bacterio- logical procedures. A quantitative determination of the bacteria in the feces was made, utilising a composite weighed sample of feces from the 2 ani. mals raised in open cages in Experiment II and using the techniques of Larson and Hill (1955). B. Results 1. gamut I Some difficulty was encountered with the surgical procedure - in this experiment. An attempt was made to support the gravid uterus msxmslly while it was being excised and removed. This resulted in a tear in the uterine wall, and one pig dropped from the uterus. before it could be placed in the germicidal lock. No problems were met in getting the young pigs to drink the liquid diet from a pan, and they soon were consuming the entire amount shortly after it was offered to them. Although the milk was slightly changed in color, becoming a very faint tan, and had a slight ”cooked" odor, this did not appear to affect its palatability for the young pigs. The odor from the air-outlet trap at the beginning of the ex- periment was that of the autoclaved milk. Later, however, the exhaust air took on a rather "sour" odor. At no time did the residual milk in the feed pans of the gnotobiotic pigs appear coagulated. ' Although it was not possible to obtain birth weights on these animals, they did not appear to grow very rapidly. The hair cost was not as smooth as is normally seen in 3-week-old pigs. It was also noted that the animals did not appear to possess as much sub- cutaneous fat as do animals nursing the sow. The feces of the pigs in the isolator were normal for the first few days, except for the fact that they were dark in color. By the tenth day the fecal material had become rather soft and semi-fluid in consistency. This continued until the termination of the experiment, with the feces becoming slightly more fluid as time went by. This dark, semi-fluid fecal material covered the rear quarters of the animals in the isolator. Bacteriological cultures revealed the presence of Sta 10- w m in the feces and the material from the cages. An occa- sional culture showed the presence of a mold, suggesting the possi- bility of a mold contamination. The littermate pigs maintained in open cages also grew quite slowly, although they were sanewhat smaller at the beginning of the trial than those left in the isolator. The fecal material was more nearly normal in consistency for 2 of the 3 animals (pigs B-l, 8-2, Table II. The third animal (8—3, Table 3) had diarrhea during almost all of the experiment. The body weights of the pigs in this experiment are shown in Table 11. The gnotobiotic animals are represented by group A, their littermates raised in open cages by group B. Table X sumariaes the sources of the various groups of pigs in these experiments. 2- ME 2 The use of the Mylar-covered incline leading to the germicid- al trap provided more support for the uterus as it was excised and .33 one no condenses: o .3333 moauwpnevflnu dds flaws. an. 3...e.el.poua..lnn. Ids Mo 0330.3 23 ca renegade: m .maefimemnoohoaa manganese—me has no commune on» 5. manage: a .eowao mono 5 egeaeaee .o msoum uo 303.333 9 and. a no 02.3th on» 5. venues—fie: o .moweo some 5 Redefines .4 93.5 no moaeaoauaq m .393.» msoooooHHmmeu. .m. we 00:3er on» 5 condense: < negolm a awed no museum no sou—pom .M 3m: omen." mwd mm}. oo.m 0%: om.m $0.: awakened o.» a H.” .3 a 3 «w ~.~H : m 0.2 a m ~.~H h h m.ma h 0 mg: a m H4: b m6 .m w.n : a 19“ M 9m 3 m5 2 m.m a «fin ..H m o.~.n .— m6 3 H4. 3 o.m .m 0.: a «4. a m3 3 m 0.: m 06 m 4.: .— o.m : N.m . .m ed a fin m H .mm mam .mm mm .mm mm .mm film. and mum ..fl. mum .mm mem .sz a macho ‘ .m $8.6 m mmoao a macho u macho m 95.5 < macaw a vflGHHhomm HHH fighgfifl HH anon—Huang H éfiafidhmfl “.23 3&3: has ”38 .n Ema 66 placed in the tank, and no problem was encountered with tearing of the uterus as in Experiment I. The air leaving the isolator developed a "sour“ odor when the pigs were about 12 days of age, although there was no coagulation of the residual milk in the feeding pm. One pig within the vinyl rearing unit died a few hours after delivery. Death was due to hemorrhage from the umbilical cord. It ap- peared that the stump of the cord had been torn in some way above the ligature. The pigs did not differ markedly in appearance from those in Experiment I. The consistency of the fecal matter was again semi-fluid, and it was of a dark color. Bacteriological cultures revealed the presence of Bacillus 32. The organism was not further identified. The littermate pigs in Open cages grew at about the same rate as did the gnotobiotic animals. Pig D—l deve10ped some diarrhea toward the end of the experiment, but pig D—2 had well-formed feces. The Quantitative determinations of the intestinal flora of this group of animals, using a composite sample frm the 2 animals, revealed that the bacterial count was essentially that of animals raised on the sow. The total numbers of aerobic and anaerobic organisms, along with the embers of colifoms, streptococci, and clostridia, were nearly identi- cal to that of a series of pigs the same age nursing the sow. The lactobacilli were reduced by a factor of about 10,000 in the pigs raised in open cages on the sterilised diet. The body weights of the animals in this experiment are shown in Table II. The gnotobiotic animals are represented by group C, while group D represents the 2 littermate animals raised in open cages. 6? 3. Experiment 2;; The viability of the litter used in this experiment was quite low, and only h of 13 pigs lived. Three of the remaining 9 were mummi- fiexi, and attempts to initiate respiration in the remaining 6 animals *were unsuccessful. The animals started eating quite slowly, and they ‘were much weaker than the animals in previous experiments. One of the h pigs placed in the rearing unit died on the third day and another on the fifth day after delivery. Both these animals were left in the rearing unit at 90° F. until the eighth day ‘ of the experiment. There was no evidence of bacterial decomposition ’ as evidenced by bloating, discoloration of the skin in the abdominal region, or odor. The carcasses did become dehydrated, and the eyes were quite sunken at the time of removal from the rearing unit. The odor from the air-outlet trap was that of the sterilized milk throughout the experiment, and the pigs themselves had no dis- agreeable odor at the time they were removed from the rearing unit. The faces of the 2 remaining pigs were well-formed at the start of the experiment but began to be rather soft after about one week. At the end of the 21-day experiment the fecal material was dark in color and semi-fluid to pasty in consistency. Bacteriological examination failed to reveal the presence of any demonstrable bacteria in rectal swabs or waste material from the cages. A Gram-stained smear of fecal material from one pig in this group is shown in Figure 17. For comparison, a smear of fecal material from a 3-week-old pig raised on a saw is shown in Figure 18. Since facilities for weighing the pigs had been installed in the rearing unit before the start of this experiment, it was possible 68 Figure 17. Gram-stained fecal smear from germfree pig Note absence of bacteria. 3 1600 69 _’ Vu- I C Q E. " g - ‘ .1 ; ' I , x . " a. / . 1‘ h, . z , -— (:1... F \- ‘ . a ‘ . . , w ‘ \ ' , ‘ ‘ 3 \l g ‘t‘\‘ .~ ' " I; x . I a \ I" ’ 3". . ’\ I.) ' ,.¥\ -"', _ I -— , ' f "x‘ w . ' g 0‘ ‘ ‘f' ‘5 x a" ‘2‘ \ 'l t , - - .- 0‘ .I K. a, . q .. '\ ‘ '1 .0 ’ I ‘u ( f1 ’ ‘_ f ., o ' J. ' . ' * a . ~ ~ , . .' - . . 1 o I. l .. h V I x ‘ I ‘ ,5, ' " ‘ ' e .. \ . \ T . 1 ' , _1 ,# _.__,_h v _ ..A w, , LE *‘1 Figure 18. Gram-stained fecal smear from farm-raised pig Note numerous bacteria of various shapes and sizes. x1600 we! . Fw~‘_.__‘ 70 to follow the growth rate of these pigs. The body weights at different ages are shown in Table II, and the final body weights are shown as group B in Table 11. TABLE XI. Body weight of pigs in Experiment III (IbSo) Age (m) ..a__.P1 No. 2 1 11 a 3.1 1.1; 2.0 3.0 11.1: 5.2 1.7 1.8 2.7 h.1 1;. Experiment 11 Shortly after the pigs were transferred to the rearing unit, the unbilical ligature on pig F-l loosened, and the animal lost enough blood to become rather weak. It soon regained its strength, however. On the second day of the experiment, one of the pigs was lost because of hemorrhage from the umbilical cord. It had been placed in a cage with another pig, and apparently the ligature had been sucked or bitten from the umbilical cord. The remaining pigs did quite well in compari- son to earlier groups. Two of the pigs (Figure 19) from this group are shown after their removal from the rearing unit. Sane difficulty was encountered in the one remaining cage containing 2 pigs. One of these was somewhat larger than the other, and it became necessary to hold each animal while the other was being fed to prevent the larger animal from getting more than its share of the milk. The outlet air from the rearing unit did not develop a signi- ficant odor, although it was thought possible at times to detect 71 Figure 19. Gnotobiotic pigs from Experiment IV 72 somewhat the same "sour" odor that had been encountered in previous groups contaminated with bacteria. The fecal matter was noted to be somewhat soft on day 9 of the experiment, and it became somewhat more liquid as time progressed. The 2 pigs in the same cage became covered with this dark, semi-fluid fecal material. Bacteriological determinations revealed the presence of a bac- terial strain belonging to the Achromobacteriaceae and a small Gram- negative, anaerobic rod which was lost before it could be further iden- tified. A Gram-stained smear of fecal material from one of the pigs in this litter is shown in Figure 20. Examination of the isolator at the termination of the experiment revealed a small hole in the vinyl film which may have accounted for the contamination. The body weights of the h gnotobiotic animals killed at the termination of this experiment and 3 of their littermates raised on a sow are shown in Table III. The remaining animals of this litter were TABLE XII. Body weights of pigs in Experiment IV lbs. ._...____Ass_idsxsl_.._.___ ZiaJha- §22££2 9 1 1k 2; F-l Gnotobiotic 2.h 3.3 h.7 6.0 F—2 " 2.7 3.7 5.3 6.9 F-3 " 2.8 3.2 h.s 5.6 F-h " 2.6 3.5 h.7 6.5 G-lo On sow . 2.h h.3 6.0 8.h 0-11 " 2.h 3.5 5.7 7.0 0.12 n 2.8 h.8 7.5 9.5 II a .. is}... 73 Figure 20. Gram-stained fecal smear from gnotobiotic pig in Experiment IV Note several rod-shaped bacteria in center of field. 3: 1600, 7h lost. The final body weights of the gnotobiotic pigs are shown as group F in Table I1, and 2 of the sow-raised littermates are shown as pigs G—lo and G—ll. The remaining animal raised in the vinyl isolator was trans- ferred to a sterile rearim unit when the other animals were killed. It was kept in this unit for an additional 2 weeks, and it was then re- moved from the isolator and placed in a room where pigs had been pre- viously kept. It was gradually changed to a conventional pelleted diet and pasteurized milk. While the animal was in the isolator, it was noted that the tongue protruded from the mouth and could not be com— pletely retracted. This condition gradually improved after changing to the conventional diet, and the feces also soon became normal in consis- tency. The final body weights of all pigs in these )4 experiments are shown in Table II. Included also, as group G, are 11 pigs raised on sows to the age of 21 days. These data, along with various organ weights, will be compared in Part Two. .. .... 5...... .... ....I ......1.” .b.l .. . . ‘mlu‘L-wu m. .0. ’e 1 5.4 -. n. V ' n." . D. . l ..- I I a ... gap... _ IV. DISCUSSION The fact that young pigs were raised to the age of apprenti- mtely 3 weeks in the presence of not more than 2 species of bacteria indicates that this technique and equipment can be successfully used in producing gnotobiotic pigs for use in research. It is thought that the bacteria encountered in Experiment I probably entered as a result of the tear in the uterine wall during the Waterectomy. The exact reason for the contamination in Experiment II is not known, but it is seemed that the most likely cause was improper sterilization of the diet since the contaminant was a spore former. The bacteria encountered in Experi- ment IV probably entered through the tear in the vinyl film of the iso- later. The percentage of live pigs obtained in mperinents II and III was rather disappointing. The small size of the animals obtained in the latter case suggests that the breeding date of the sow used may have been in error. Postponement of the hysterectomr for another 2 or 3 days or perhaps even longer might have resulted in heavier and stronger ani- mals. It is extremely important to be able to predict the expected farmwing date of the sow or gilt to be used in this procedure. in error of a few days in either direction can result in the failure of an attupt to obtain viable pigs by hysterectomy. Glimstedt (1936) used radiographs of the pelvis in determining the end of the gestation period in guinea pigs, depending on the amount of separation of the synphysis pubis. Phillips 91'. _a_:_l_. (1959) also relied on the diastasis of the pubic 75 76 bones of the pregnant guinea pig in determining the proper time to per- form surgery. They found a gradual increase in the separation of the pubic bones until the distance between the bones reached 17 to 18 mm. Following this they found a rapid increase during the next 21: to 36 hours, at which time the separation was 21 or 22 mm. This latter period of rapid diastasis appeared to be the most favorable time for Caesarean section, but germfree guinea pigs derived from dams with a pubic spread of 16 mm. or more could usually be reared successfully. These workers determined the amount of diastasis either radiologically or manually by palpation. Gustafseon (19b6-19b7) utilised 2 female rats bred on the same day to the same male. One female was allowed to give birth spontaneously. The other animal was then checked every half hour and surgery was started as soon as labor began. The Lobund group (Pleasants, 1959) has reported delaying surgery in rate until one of the young was delivered spontaneously. Since none of these procedures seem practical in the sow, emphasis should be placed on accurate breeding dates. If these are available, the best plan is to perform surgery on the 112th day of gestation. If no accurate breeding date is available, one must rely on the appearance of milk in the nipples as a sign of approaching parturition, but this is not highly accurate, as milk may be present for only a short time or for several days prior to farming. Another factor that no doubt affected the viability of the young pigs was the fact that the time elapsing between the attainment of surgical anesthesia in the sow and the initiation of respiration in the pigs was longer than ordinarily encountered in the procurement of specific-pathogen-free pigs. This was due to the need for cmpletely P ; p‘IIfiJ 77 aseptic procedures in the current work. It is felt that the develop- ment of equipment and techniques for the utilization of a Caesarean section in obtaining gnotobiotic pigs would result in a higher degree of viability. The placental attachment of each animal could be inter- rupted on an individual basis, each animal could be handled individu- ally, and more time could be devoted to the initiation of respiration in each pig. It might also be possible to salvage the sow for market or perhaps for raising subsequent litters. A need for larger cages was evidenced in these experiments. The present cages appeared adequate for the length of time the animals were kept in them, but it is possible that the animals might consume more food and grow more rapidly if more space for exercise were pro- vided. If attempts to rear gnotobiotic pigs to an older age were to be attempted, larger cages would no doubt be required. Another modification which would be of value would be the pro- vision of larger fiber-glass rings for the introduction of material into the rearing unit. This would allow the cages to be placed within the isolator without cutting and rescaling the vinyl film itself. It would also allow the construction of a larger sterile lock and permit the in- troduction of more diet at a given time. The diet itself seemed adequate to support life in the young pig, but it would be desirable to have a diet which would, if possible, produce growth rates and weight gains more nearly comparable to those seen in pigs raised on the sow. This suggests that much more work needs to be done on formulating such a diet. In order to study this problem thoroughly, the sterilized diet should be fed to both gnotobiotic ani- mals and those possessing a normal flora. Additional information as to ‘1 slashes.“ wfll.m.sn..._..fll..wa.wa, I I m . ’l‘J n4 78 the adequacy of the diet could also be gained by feeding the sterilized diet to one group of animals and the same diet before sterilization to a comparable group. In formulating a ration more suitable for rearing young pigs, it may be necessary to resort to something other than cows' milk as a base. It is known (Bellis, 1957) that sows' milk contains more total solids, fat, protein, and ash than does sows‘ milk. It is also quite high in energy. Sows' colostrum is even higher in total solids and protein than is sows‘ milk. It may be necessary to formulate a new synthetic-type diet to provide opthmum nutritional conditions for the young pigs. The appearance of semi-fluid feces in the animals raised in the isolator suggests that a deficiency of some nutritional factor may have existed. Whitehair (1958) has listed diarrhea as a symptom seen in some of the B—vitamin deficiencies. The fact that some of the ani- mals raised in open cages on the same diet had feces which were rather well-formed points to a possible influence of the bacterial flora on the nutritional adequacy of the diet. It is conceivable that one or more of the vitamins were destroyed during the sterilization process to such an extent that there was not sufficient quantity left in the diet to meet the minimum need. It is likewise conceivable that certain bac- teria in the intestinal flora of the pigs raised in open cages were able to synthesize the vitamin or vitamins in question and thus counteract this deficiency and prevent the formation of semi-fluid feces. It is possible that something in the diet, either as an in- gredient in the original diet or as a result of the sterilization pro- cess, may have produced the fluid feces by interfering with absorption 79 from the intestinal tract or by increasing peristalsis. This seems un- likely, however, since the same thing was not seen in the pigs in open cages. The possibility exists that the bacteria, by their bulk, in- fluence the physical characteristics of the fecal material. It is hoped that further work will result in the clarification of this prob- lem. Glimstedt (1936) reported that the stools of his germfree guinea pigs were not formed but were rather viscous. He did not con- sider this as being truly diarrheal in nature. The improved growth rate seen in the pigs in Experiment IV may be attributed to the greater amount of diet fed per day, the imp creased number of feedings per day, or a combination of both. The bacteriological procedures were apparently adequate for picking up bacterial contamination, but they were quite time-consuming and could, no doubt, be simplified. For detecting bacteria during the course of the experiment, the use of wet mounts and Gram's stains of fecal smears along with the inoculation of thioglycollate broth would probably be adequate. It would be necessary, of course, to incubate this medium at different temperatures, for example, 25, 37, and 55° C. lore elaborate cultural procedures could then be used, if desired, at the end of the experiment, or if examination of Grampstained fecal smears indicated the presence of organisms in sufficient quantities to suggest that the thioglycollate broth was not supporting growth. The lack of the usual post-marten decomposition in the 2 pigs in Experiment III left in the isolator for 3 and 5 days, respectively, after death must be attributed to the absence of bacteria. This .. .1: . . . .O.‘ 80 suggests that autolytic changes are not, in themselves, responsible for a.marked change in the external appearance of animals after death. ‘ Very careful planning is required to collect young pigs by this technique, and the success of the procedure depends upon everything being in order and ready for use at the time the sow approaches the end of the gestation period. The synchronization of all the different factors involved in a procedure of this type, including the estimation of the farrowing date of the sow, the preparation and sterilization of equipment, the preparation and sterilization of the diet, and the pro- vision of necessary media and equipment for bacteriological procedures, poses a very real problem. Each of these factors needs to be the sub- Ject of careful study, and much time and effbrt could be devoted to the improvement of each. ‘ r all use. ”is. V. SUMMARY A series of h experiments was conducted in an attempt to db- tain and rear young pigs to the age of approximately'B weeks in the ab- sence of bacteria or in the presence of known species of bacteria. The pregnant saw was anesthetized with carbon dioxide, and the uterus was excised and passed into a sterile vinyl-film.isolator by means of a germicidal trap. The pigs were removed from the uterus and passed into another vinyl-film isolator'where they were kept for the remainder of the experiment. The isolators were sterilised with peracetic acid, and air entering the units was sterilized by passing it through h layers of glassawool met. The diet used consisted of pasteurized, homogenized milk with mineral and vitamin supplements. This diet was sterilized with steam under pressure. Two pigs were raised in the absence of any demonstrable bac- teria, h were raised in the presence of’Staghylococcus'gggggg, 3 in the presence of Bacillus sp., and h in the presence of échromdbactgriaceae and an unidentified organism. In addition, 5 animals were raised in open cages on the same diet, and 2 were raised on.a saw. The pigs raised on the experimental diet grew rather slowly, and.those raised in the isolators developed semi-fluid feces during the latter part of the growing period. Pigs kept in open cages but mainp tained on the same diet generally had feces which more nearly approached normal. Two pigs left in the sterile isolator for 3 and 5 days, re- spectively, after death showed no external post-mortem changes. They did, however, show evidence of dehydration. 81 - A- III as“ ”In... PART TWO A common or sour wanna-rs, own name, AND sow: HISTOLOGICAL mm or snowstorm AND FARM-RAISED PIGS I. INTRODUCTION The morphological and physiolOgical characteristics possessed by gnotobiotic pigs must be studied sufficiently to determine whether or not they deviate significantly from those seen in animals raised on the saw. The possible influence of these characteristics on the outcome of investigations utilizing the gnotdbiotic pig as an experimental ani- mal should also be considered. Only after the results of these studies become known, can the gnotobiotic pig assme its greatest value in the field of research. It is beyond the scape of the present investigation to attempt to explore all the possible areas in which gnotobiotic pigs differ from conventionallybraised pigs. It seemed important, however, to explore some of the areas most likely to show differences between the 2 groups' of animals. In view of results reported in the literature on studies in germfree animals of other species, it seemed advisable to study total bOdy weights and the weights (both absolute and relative) of certain body organs. The importance of the lymphatic system in the body's de- fensive mechanism.suggested that this system should be a part of such a study. Interest has been expressed.in the significance of the compare- tively large amount of connective tissue seen in the pig's liver. Therefore, this organ seemed to merit investigation. The intestinal 82 ‘ . I :‘F. ”.1. 83 tract in germfree animals of other species has been sham to differ from the conventional. The findings relative to the intestinal tract of the gnotobiotic pig are the subject of a separate report. , J) 439‘ II. W 01“ LITERATURE A. Growth Rates of Germfree Animals (hastafsson (l9h6-l9h7), in discussing the coxnparison of gem- free animals with other animals, stated that little value can be at- tached to investigations where conclusions have been drawn from com- parisons between germfree animals and control animals reared by the mother. He suggested the use of 1; groups of Caesarean-derived animals for adeqmte comparisons. One group included the animals raised in a sterile environment on a sterilized diet. The second group consisted of animals raised in a non-sterile enviroment on a sterilized diet. The third group included those raised in a non-sterile environment on a non—sterilized diet. The fourth group consisted of animals taken by Caesarean section and raised by another female. In Gustafsson's work the 3 groups of rats fed the artificial diets were all about the same weight at the end of 28 days. The littermates raised by another female rat were somewhat heavier. Reyniers e_t g. (1916) fomd that their germfree rats and the animals maintained in a non-sterile enviroment but fed a sterilized diet grew more slowly than the normally suckled rats. Pleasants (1959) reported the successful weaning of hand-reared rats, mice, and rabbits. He found that, although growth and deve10pment were subnomal during the suckling period, few cases of permanent handi- cap resulted. Gordon (1959) noted that third- to eighth-generation germfree rats and mice showed a retardation of growth in comparison to normal animals also fed a sterilized diet. Glimstedt (1936) found that his gemfree guinea pigs grew more slowly than did the conventional animals. Later, however, the growth at an t, as “a- 4. 85 rate became balanced with the conventional animals. Miyakawa (1959b) reported that for the first 20 days the weight gains of his germfree guinea pigs were below those of conventional animals. After 30 days the weight gains became parallel to those of conventional animals on the same diet. The body weight of Miyakawa's germfree guinea pigs was always less than that of conventional animals of the same age. The growth of germfree chickens (Gordon, 1959) was found to compare favorably with the normal-stock controls fed a sterilized diet. Reyniers gt 5;. (1960) found that the body weight of comparable gamm- free and conventional chickens generally showed no essential difference up to the age of one year. B. 9.132 Ieights of Germfree Animals Gordon (1959) stated that, in terms of weight, the organs of his germfree animals fell into 2 distinct groups: one harboring, or in close association with, an abundant flora in normal life showed reduced weight in the germfree animal, and the other group of organs that is normally remote from bacteria showed practically no difference between the germfree and normal stock. As examples of the former group, Gordon listed various parts of the digestive tract and its associated lymph nodes and, in some instances, the liver. As examples of the latter group, Gordon listed the heart and brain. He also found that the adrenal glands of germfree rats were heavier than those of the normal- stock controls fed the sterilised diet. These conclusions were drawn from work with chickens, rats, and mice. Reyniers gt_gl. (1960) found a lower weight in the ileo-cecal-eolic junction (harboring the "cecal tonsils") in germfree chickens than in conventional birds. The heart, 86 brain, thymus, spleen, and endocrine glands displayed similar weights in the 2 groups. In rearing germfree guinea pigs, Glimstedt (1936) found that the liver, kidney, adrenal, heart, and lungs showed a moderate inhibi- tion of development. The development of the intestinal canal was in- hibited to a greater degree in the germfree animal. The thymus in the BO-day-old germfree animals weighed h? to 56 per cent of that found in the conventional animals. The corresponding figure for the 60-day-old animals was 39 to h6 per cent. The total lymphatic tissue amounted to 22 to 26 per cent in the 30-day-old animals and 25 to 3h per cent in the 60-dayaold animals of that seen in the control guinea pigs. Cor- responding values for the spleen wereh9 to 65 per cent and S7 to 66 per cent, respectively. In 2 germfree animals, Glimstedt found that the amount of splenic white pulp was increased, while in the remaining ani- mals it was reduced to 67 to 72 per cent of the mean value of the con. trol animals. From the above results Glimstedt (1936) concluded that the normal, nonpathogenic bacteria occurring in the guinea pig appeared to influence favorably the development of the animals. His investigation did not clarify the mechanism of this influence. Thorbecke and Benacerraff (1959) found that the combined rela- tive weight of liver and spleen was significantly lower in germfree than in control mice, chiefly because of a difference in relative liver weight. The liver weights per 20 gm. body weight in 9 control mice were 1180 t 80 mg., and in 15 germfree mice they were 900 i 130 mg. 87 C. Histolog 2f the Lymphatic m Since a study of the lymphatic system.forms a part of the present investigation, and since this system has been the subject of many scientific papers in the past, it seemed appropriate to review the highlights of the histology of the lymphatic system. Flemming (1885) studied the microscopic structure of lymph nodes and described light secondary nodules or germinal centers which serve as the principal foci of proliferation of lymphocytes. He emphasized that these are not con. stant parts of the lymphatic nodules but show fluctuations. Flemming also described large, peculiarlybstaining granular formations in cells of the germinal centers. These he called ”tingible Kerper". Maximow (1932) stated that these are phagocytosed inclusions in large macro- phages and that they are mostly darkly-staining particles of chromatin originating from degenerated lymphocytes. Hellman (1921, 1930) attached a different significance to the light centers found in lymphatic nodules. According to his theory, these centers are of no importance for the regeneration of lymphocytes, which occurs everywhere in the diffuse lymphoid tissue. They are, on the other hand, centers of reaction to bacterial and toxic stimuli acting upon the lymphoid tissue. If the stimulus is light, productive changes occur; if it is intense, the cells of the center display de- generative phenomena. Maximow (1927, 1932) has described in.detail the structure of lymph nodes and stated that the stroma of lymphoid tissue contains 2 types of cells. One of these is the reticular cell which is large and stellate or spindle shaped and has the ability to store vital dyes. The other is the cell of the reticular syncytium'which has not undergone .. .. ifflwhflvurwwmn ....r 88 differentiation and has no ability to store vital dyes. Maximow de- scribed the primary nodule or follicle of lymphocytes with its central area or secondary nodule which stains lighter than the periphery. This secondary nodule contains large pale nuclei with numerous mitoses. Haximow agreed with Flemming that these centers are the chief place of lymphocyte proliferation. He further stated that such centers are not found in embryos or newborn mammals and that they reach their height of development in the young animal. According to Maximow (1932) the lymphoid tissue in the germi- 4 nal centers under goes cyclic transformations and all the germinal cen— ters of a lymph node, perhaps of the whole body, show similar conditions at a given time. He described the ”active phase” of this cycle in which numerous mitotic divisions, mostly in medium-sized lymphocytes, occurs. The reticular syncytium may also show mitosis. During the "resting phase" no mitoses are present, and the arteriole in the center of the nodule is surrounded by a small pale area containing nuclei of the reticular syncytium and a few degenerated small lymphocytes. The rest of the nodule is made up of small lymphocytes and reticular nuclei. Maximow also recognized the ”beginning of a new active phase" and the ”transition from active into resting conditions". In the latter phase the medium-sized lymphocytes are transformed into small lymphocytes either by mitosis or without mitosis. Maximow (1927) also described the transformation of the un- differentiated reticular syncytium of the lymphoid tissue into lympho- cytes, myeloid cells, macrophages, and monocytes. Conway (1937) described the voluminous literature on lymphatic tissue as ".....a maze of contradictory experimental and descriptive C #5.... ...... ..-: . .. . . . I «M :finmi fiI 89 data, opinions, and theories". She described a "bare" germinal center as one without a surrounding area of densely—packed, small lymphocytes. Conway demonstrated that, in an induced bacterial infection in rabbits, the lymphatic nodule is primarily a focus of rapidly-proliferating lym- phocytes and only secondarily does it become a reaction center. She thus attempted to reconcile the 2 opposing views of Flemming and Hellman. She also stated that the reticular fibers of the lymphatic nodules are not arranged in a permanent pattern but are mechanically pushed apart by the growth pressure of the lymphocytes. Bloom (1938) reviewed the literature on lymphatic tissue and attempted to clarify the confusion of terminology appearing in the literature. Osogoe st 21. (1960) stated that secondary nodules are formed as a reaction to bacterial infections and that the term ”reaction center" is preferred to "genuinal center". They counted the mitotic figures in sections of rat mesenteric lymph nodes and found more mitoses in the pale-staining centers than in the remainder of the lymphatic tissue. Gyllensten (1950) found that the lymphatic system of newborn and young guinea pigs exhibits regional differences in regard to the de- gree of maturity. The maturity decreases in the following order: Peyer's patches and cervical lymph nodes, lymph nodes of the extremities, tracheal and mesenteric lymph nodes, and white splenic pulp. He stated that secondary nodules occur between the first and 23d day after birth in guinea pigs. One of the aspects of lymphoid tissue which has received con- siderable attention has been the function of this tissue. laximow and Bloom (1957) listed one of the functions of lymph nodes as the production 90 of lymphocytes. In some conditions, the lymph nodes also become the site of the formation of granular leukocytes. The lymph nodes also serve as filters in which various particles, arising locally or brought with the lymph from other parts of the body, are taken up and often de- stroyed. laximow and Bloom (1957) stated that, just like all the other organs containing many macrophages, the lymph nodes probably elaborate antibodies. They further suggested that lymphocytes possibly contain antibodies. Ham (1957) described the chief function of lymphatic tissue as the filtering of lymph before it is returned to the blood. The lymph nodes also add lymphocytes to the lymph that flows through them. Miller and Bale (l9Sh) studied the synthesis of the plasma proteins and found that all fractions are synthesised by the liver with the exception of gamma globulin. They concluded that if antibody pro- teins are regarded as gamma globulins, antibodies are formed by extra- hepatic tissue, most likely by radiosensitive cells of the hematopoietic system. Mclaster and Hudack (1935) demonstrated the formation of agglup tinins within the draining lymph nodes of mice following intradermal injections of killed cultures of microorganisms. Numerous attempts have been made to determine which specific cells within the lymph nodes are responsible for the elaboration of antibodies. Fagraeus (l9h8) listed 3 groups of cells which have been suggested as the site of antibody formation. These were (1) different cells belonging to the reticulo-endothelial system, (2) lymphocytes, and (3) plasma cells. He further stated that, in human pathology, there is never an increase in the level of serum globulin associated with an in- crease in the number of lymphocytes alone. Fagraeus concluded that 91 antibody formation occurs in cells belonging to the reticulo-endothelial system. On the occasion of intense antibody formation, a develoment of reticulo-endothelial cells into plasma cells occurs, and, in the course of this process, antibodies are being formed. Ortega and Mellors (1957) used fluorescent antibody techniques to study the cellular sites of the formation of gamma globulin in lym- phatic tissue. They found that gama globulin is formed in ”intrinsic" cells ofthe germinal centers of lymphatic nodules. They distinguished these cells from medium and large lymphocytes and the primitive reticu- lar cells that occur elsewhere and do not produce gamma globulin. They also found that gamma globulin is formed in the cytoplasm of mature and immature plasma cells. Coons (1955), White 23 51' (19558, 19551)), and Leduc e_t a}. (1955) have studied antibody production by the injection of various antigenic materials into laboratory animals. Their conclusion was that the formation of antibody is closely associated with the proliferation of plasma cells in the lymph nodes, spleen, liver, and in case of some antigens, at the site of injection. Carlson and Gyllensten (1958) studied the plasma cells in the growing lymphatic system of young guinea pigs. These cells were de- tected by their pyroninOphilia. They found no mature plasma cells in newborn animals, but these cells began to form in the medullary cords of the lymph nodes and in the red pulp of the spleen imediately after birth. The number of plasma cells increased during the first and second months of life. In white splenic pulp, lymph node cortex, Peyer's patches, and tlvmus, mature plasma cells were found only exceptionally, and immature cells were very rare. r...“ O ‘4. ......) 92 Speirs (1958) stated that eosinophils accumulate in lymph nodes draining an antigen-injected area or an area of infection and in the spleen following an intravenous injection of foreign material. He described 2 stages in antibody formation. In the first the antigen re- acts with certain cells (possibly eosinophils), producing enzymatic char~es or forming an enzymatic template. In the second step the speci- fic enzymes are utilized for synthesis of the antibody. This takes place in the lymphoid and reticulo-endothelial tissues, and the eosino- phil is phagocytized. D. Histolog of Germfree Animals Numerous histological studies have been made on germfree ani- mals, and most of these studies have involved either the lymphatic sys- tem or the intestinal tract. Glimstedt (1936) found that the cellular canposition of the lymphatic tissues of germfree guinea pigs was the same as in control animals but that quantitative differences existed in the numbers of cells. The number of lyinphocytes was reduced so that the cortex and medullary cords appeared narrow, the boundary between cortex and medulla was indistinct, and the intermediary sinus was wide. The number of large free cells in this sinus was also reduced. There was also a reduction in the number of lymphocytes in the white pulp of the spleen. Glimstedt stated that secondary nodules were not found in either the lymph nodes or spleen of germfree animals. Conway (1937) has disputed this claim on the basis of the various sizes of lymphocytes seen in some of Glimstedt's published photomicrographs. Miyakawa (1959b) and Miyakawa e_t 5.1: (1957) also studied the lymphatic tissue of germfree guinea pigs. They found an underdevelopment 93 of lymphatic tissue in these animals and were unable to demonstrate re- action centers or Flemming-type nodules. Peyer's patches were poorly developed. liyakawa 32 31. (1957) stated that there was a smaller num- ber of sinus reticulum cells in the lymph nodes of germfree animals and the phagocytic power of these cells was reduced when compared to those found in normal animals. Gustafsson (19h8) found that the number of free cells in the lymphatic organs of germfree rats was reduced and the reaction centers were missing. Thorbecke (1959) found no such outstanding differences between germfree and conventional rats. He described secondary nodules in the spleen of germfree rats and stated that they were occasionally found in the lymphoid tissue of the appendixes and in the mesenteric lymph nodes. These nodules were not as numerous in germfree animals, nor were plasma cells as numerous. Gordon (1959) and Gordon and Bruckner-Kardoss (1958-1959) have studied the scattered reticulo-endothelial elements in the mucosa and submucosa of the intestine of germfree chickens. They found a reduction in the number of globule leukocytes within the epithelium and in the lymphocytes, plasma cells, and possibly the macrophages of the lamina propria and submucosa. Gordon (1959) also found a lower percentage of connective tissue and a higher percentage of epithelium in the small in- testine of germfree chickens and rats as compared to conventionally- raised animals. Thorbecke (1959) found no plasma cells or secondary nodules in the intestinal tract of germfree chickens up to 6 weeks of age. No secondary nodules were seen in the spleen at this age, and the numbers Olrl. I‘ii'iM ‘nlw xvi-5.». . 9h of plasma cells in the spleens of germfree chickens were always less than in the spleens of control chickens. No differences were observed in the size or histological features of the bursa of Fabricius in germ- free and control chickens at the age of 6 weeks. Thorbecke gt gl. (1957) stated that secondary nodules could sometimes be found in the spleen and ileo-cecal-colic junction of germfree birds. I Reyniers gt El. (1960) described a method for determining the lymphocyte concentration per unit volume of tissue. By using this method they found no differences in the lymphocyte concentration of the spleen of germfree and conventional animals. The ileo-cecal-colic Junc- tion of the germfree birds contained a lower concentration of lympho- cytes than did the same tissue of the conventionally-raised birds. 8. Histology of the Eymphatic System and the Liver 22 the Big 1. Histology 23 the Eymphatic System Richter (1902) studied the lymph nodes of domestic animals and described the cell-rich cortical substance which contains germinal cenp ters and occupies the central part of the lymph nodes, while the cell- poor substance occupies the peripheral part of the node. He stated that the medullary substance is very poorly developed in swine lymph nodes. Richter also described the fine-meshed reticulum of the medulla with its individual elements being more strongly built than those of the cortex. Beam and Hills (1908) also found the cortex to be centrally located and to reach the capsule in only a few areas. They described the medulla as surrounding the cortex and sometimes extending in streaks between areas of the cortex. The genuinal centers lie close to lymph sinuses surrounding the septa. Baum and Hills found no germinal centers 95 in very young pigs. In 3 to hsweek-old pigs these centers were de- scribed as being more distinct and numerous but not clear-cut or typi- cal. They also described fatty degeneration of lymph nodes in older ' animals. Trautmann (1926) described in detail the structure of the lymph nodes of swine. He noted the frequent fusion of small lymph nodes to form a node with a lobed appearance. His description of the cortex and medulla agreed with that of Richter (1902) and Baum and Hills (1908). Trautmann (1926) described the circulation of lymph through the lymph nodes of swine. The lymph enters through the afferent lymphatic vessels whose branches extend into the trabeculae, and the lymph then empties into the wide sinus system surrounding the septum or trabeculae. From here the lymph may go directly into the marginal sinus where the trabeculae reach the capsule, but usually it goes through the medullary substance, receiving ”washings" from the germinal centers. The marginal sinuses empty into the efferent lymphatic vessels. Trautmann (1926) also described the coloring of swine lymph nodes caused by the collec- tion of blood components in the sinuses. Goldkuhl (1927) described the histology of swine lymph nodes and stated that the afferent lymphatic vessels enter through one or more pseudohili. The efferent vessels leave the node here and there on its convex surface without hilus formation or through one or more small hilus formations. Hellman (1930) recognised the unusual arrangement of the cor- tex and medulla of swine lymph nodes and reviewed some of the earlier investigations of the lymph nodes of swine. 96 Trautmann and Fiebiger (1957) stated that the usual descrip— tion of lymph nodes does not apply to the nodes of swine. In this ani- mal the lymphatic tissue that contains germinal centers and corresponds to the cortex of the node in other species occupies a more central position. The peripheral zone is filled with a tissue that is compa- rable to that of the medulla of other species, although it has a some- what different structure.They'noted that the same type of nodular lymp phatic tissue always lies closer to the afferent vessels, whether in swine or in other species. Bouwman (1959) described the histology of various lymph nodes from pigs ranging in age from late fetal life to 3 years. He also studied the lymph flow in these nodes. Trautmann and Fiebiger (1957) described the spleen of swine and ruminants as being intermediate between the "storage" spleen of the horse, dog, and cat, and the "defense" spleen of the rabbit and man. The reticulum of the red pulp is especially well-developed in the pig. These authors also stated that the destruction of red cells is particu— larly well-demonstrated in the swine spleen. Calhoun and Smith (1958) stated that the spleen of swine con- tains considerable white pulp and relatively little red pulp. Snook (1950) described the pig spleen as being of the non- sinusoidal type and having penicillar ellipsoids. He stated that the ellipsoid sheaths are quite large and measure 195 by 62f4. 2. Histolog of _th_e .1119: Illing (1905) studied the histology of the pig's liver. He found that the lobules are 5, 6, or several-sided and are completely separated by heavy bands of connective tissue which are not as distinct 97 in the young animals. Illing found the swine liver lobule to be larger than that of other domestic animals, measuring an average of 0.872 mm. in suckling pigs and 1.573 mm. in adult animals. Johnson (1916-1917, 1919) studied the development of the lobules of the pig's liver and found that the lobules are fused together until late fetal life. At this stage the pig's liver resembles closely the liver of most adult mammals. The start of segmentation becomes ap- parent just before birth and the formation of connective tissue septa is not completed until several months after birth. Johnson found that the liver cells themselves take part in the separation of the liver parenchyma. The cells along the boundaries of the lobules become granu- lar and stain more deeply than the cells elsewhere. Soon these cells become arranged in parallel rows or sheets extending from one branch of a portal vein to another. The rows of cells become split apart by a thickening of the reticulum between them. Collagen fibers gradually spread into this thickened reticulum from around the portal veins. Johnson found that the lobules increase in size from 0.h3 mm. at one day of age to 1.2 mm. in the adult. Johnson (1918a, 1918b) described a method of separating the lobules in blocks of hepatic tissue so that the shapes of the lobules could be more readily studied. He found that the lobules are irregular polyhedrons with h to 15 or more surfaces. The surface lobules are ir- regularly prismatic in shape, but the deeper lobules seldom approach any geometrical solids. Compound lobules are formed due to incomplete con, nective tissue septa. New lobules are formed by bifurcation of the cen- tral veins and splitting of the lobules. 98 White (1938-1939) also described the development of a lobular pattern in the liver of young pigs and stated that the first indication of a lobular pattern was found at 5 to 7 days of age due to a columnar arrangement of the liver cells between branches of the portal vein. The first outgrowth of collagen fibers usually began at 20 to 2b days of age, but there were striking variations in the development of con- nective tissue, even in pigs of the same litter. The clearly marked lobulation seen in some animals before the outgrowth of collagen fibers is due to thick bundles of reticulum surrounding the lobules. White listed the diameter of the lobule as 0.37 mm. at 5 days of age and 1.55 mm. at 297 days of age. Elias gt 5;. (l95h) have stated that the liver of the pig, characterized by sharply defined lobules separated from each other by connective tissue septa, exhibits a form of portal cirrhosis of unknown etiology. They noted the similarity between this condition and cirrho- sis of the liver in man. III . EXPERIMENTAL A. Procedures 1. Animals 233d The source of the various groups of animals has been sum- marized in Table I. The procedures used in rearing the gnotobiotic pigs in groups A, C, E, and F has been discussed in detail in Part One, as has the rearing of the pigs in groups B and D. The 11 pigs in group C were reared by the saw to the age of 3 weeks under conditions of management similar to those ordinarily seen on the farm. Five different litters were represented. Pigs G-1 and G—2 were fran one litter, 0-3 and G—h from a second, G-5 from a third, 6-6, G-7, G—8, and G-9 fran another, and G-lO and G-ll from a fifth litter. The latter 2 animals were littermates of the )4 pigs in group F. After the animals were derived by hysterectomy, these 2 animals along with 3 others were placed on a saw which had farer on the same day. The first 9 pigs in group G were given supplemental iron by a combination of methods. One method was to give each pig a tablet con- taining iron, copper, and cobalt (FeCuCo, Ft. Dodge Laboratories, Ft. Dodge, Iowa). Another method consisted of swabbing the sow's udder once a day with a solution containing FeSOh, CuSOh, and sugar in water. Iron was also provided by placing sod in the pen with the pigs. Pigs G—lO and G-ll were each injected with 2 ml. of iron daxtran solution (Armidexan, Amour Laboratories, Kankakee, Illinois). 2. m 3n_d m Weights Pigs in groups A, B, C, and D were killed at 17 days of age, and pigs in groups E, F, and G were killed at 21 days of age. when 99 - Ht- 100 necropsies were to be performed, the animals were weighed with a spring- type milk scale, using a loop of cotton cord around the leg above the hook Joint to suspend each animal from the hook of the scale. Blood samples were then drawn for hematological examinations, using either heparin or a combination of amonium and potassium escalates as the anticoagulant. The animals were then anesthetised with sodium ponto- barbital. After anesthesia was accomplished, the pigs were exam- guinated, and the necropsies were performed. The weights of the various organs were determined by weighing them on a laboratory-type balance or a small torsion balance. The lymph nodes were dissected out, using the description of swine lymph nodes by St. Clair (1958) as a guide. The nodes were stripped of as much extraneous fat and fascia as possible before they were weighed. The pericardial sac was opened, and the parietal layer was reflected back. The large vessels were then cut off Just above the heart. The atria and ventricles were then opened and washed before the heart was weighed. The trachea was renoved at its bifurcation, and the mediasti- num and its lymph nodes were removed before the lungs were weighed. The gall bladder was removed from the liver before this organ was weighed. The other organs were dissected out, freed of extraneous fat and fascia, and weighed in their entirety. After the total body weights and organ weights were deter- mined, the data were analyzed statistically, using an analysis of variance and the "F" test (Snedecor, 191:8). The data were then checked 101 for significant differences between the various group averages, using the multiple range test as described by Duncan (1955). In order to compensate for the relatively slow growth rate of the gnotobiotic animals, 2 methods of expressing organ weights were used. In the first method, the weights were expressed as grams. In the second method, they were expressed as relative weights (milligrams of tissue per 100 gm. of total body weight). 3. Histological Procedures Blocks of tissue from the organs to be studied in this in- vestigation were saved in Zenker's fixative (Armed Forces Institute of Pathology, 1957) except that acetic acidnwas not added before the fixa- tive was used. In some cases blocks were also saved in 10 per cent buffered formalin and Carnoy's fixative (Armed Forces Institute of Pathology, 1957). The Zenker-fixed tissues were left in the fixative for 12 to 2h hours and were then washed for 12 to 2h hours in running tap water. The tissues were then stored in 80 per cent ethyl alcohol until the time of processing. After the blocks were trimmed to size, they were dehydrated in a graded series of alcohols, cleared in.xylene, and embedded in paraffin. Triplicate sections were cut at 6 micra with a rotary micro- tome, placed on slides, and stained with Delafield's hematoxylin and eosin, Heidenhain's aniline blue, and Foot's modification of Bielschowsky‘s method for reticulum. Some extra sections were also cut and stained for iron by Gomori's method, and Giemsa's stain was applied to a few sections. Formalin-fixed and Carnoybfixed blocks of liver tissue were sectioned and stained for fat and glycogen, respectively, 102 using the Sudan IV technique for the former and the Best's carmine technique for the latter. All staining techniques were carried out ac- cording to the Manual of Histologic and Special Staining Technics airmed Forces Institute of Pathology, 1957) with the exception of the Sudan IV stain which was carried out according to the description by Mallory (1938) with the substitution of Harris's hematoxylin for alum hematoxylin. B. Results l. 223! and 92532 Weights The total body weights are shown in Table XIII. At the bottom of the table is shown the range of the group averages, in ascending order. The method of expressing significance between the averages is that described by Duncan (1955). Any 2 averages not underscored by the same line are significantly different, while any 2 averages underscored by the same line are not significantly different. The significance at the 5 per cent level is shown first, followed by the 1 per cent level. If the latter is not shown, it is an indication that there were no sig— nificant differences between the group averages at that level. The various organ weights, both absolute and relative, are shown in Tables XIV through XXIV, and the same method of indicating significant differences was used. 2. 95223 Observations It was noted that, at the time of necropsy, there was very little odor associated with the pigs raised in the isolator. The odor which was present resembled more closely that of the sterilized milk than the somewhat offensive odor sometimes associated with young pigs. 3.: muse 8.m 8.: mm: 8.: om .m I H0>0H ma amt: mud 8.m 8.: mm: 8.: om.n .33 mm m a a o m 4 m "as mousse; macaw mo 3:83.253 omJH mmd mwi oo.m 8.4 om.m 00.: amass. o.» d" :5 2 «.3 m m 0.2 o N.NH h 93 0 mad m as: m6 m.n : find o.m m3: m.n m.: m 0.2 ad 1: o.m 0.: N: m.: N 025 06 4.... o.m m.m w.~ 5m .n a Mug 3mm 3 3mm...» a drug .3 E 3253an HHH acofismmxm HH snofiuomxm H 955399 333 3:33 .38 ~38 .HHHN Ema 10h TABLE XIV. weights of mandibular lymph nodes (smo) 235 N0. Group A Group B Group C Group D Group E Group F Group G 1 10 11 Average 0.11a 0.12 0.20 0.19 0.17 0.22 0.168 Significance of group average at: E 5% level '- 00160 00168 0. 215 0. 292 0.672 1% level - 0.160 0.168 0.26 0.17 0.17 0.215 0.215 0.1h 0.1h 0.17 0.19 0.160 F 0.292 0.21 0.h0 0.27 O. 28 0.35 0.36 0.32 0.292 G 0.672 0.69 0.52 0.78 0.81 0.50 0.88 0.85 0.96 0.3h o.h1 0.60 0.71 0.58 0.71 0.76 0.79 0.66 0.82 0.70 0.59 0.h2 o.ho 0.672 ‘ In case of paired organs, first weight is right side, second weight is left side. _“ h—-— 105 TABLE XV. Relative weights of mandibular lymph nodes (mg. per 100 gm. body wt.) Pig No. Group A Group B Group C Group D Group E Grouo F Group G 1 1‘07 1105 700 SOS 1391 5.1 11.5 7.0 7.7 9.9 2 9.6 7.5 9.1 12.8 13.6 9.1 7.5 10.2 8.6 18.2 3 7.6 11.0 13.5 9.9 13.8 lh.8 h 12.2 13.3 10.8 15.0 5 7.1 8.6 6 10.3 12.2 7 10.5 12.8 8 18.0 1‘45 9 11.9 1h.8 10 18.3 15.5 11 13.2 12.6 Average 7.67 9.50 8.32 10.30 12.90 Significance of group averages at: 2 £3. 2 F 9 5% level - 7.67 8.32 9.50 10.30 12.90 TABLE XVI. Heights of external inguinal lymph nodes (smo) Pig No. Group A Group B Group C GroupgD Group E Group F Group G 1 0.32 0.28 0.38 0.29 0.29 0.37 2 0.20 0.22 0.33 0.19 0.18 0.32 3 0.16 0.13 h 5 6 7 8 9 10 11 Average 0.215 0.2h2 0.350 Significance of group averages at: 2 - E E 5% level - 0.215 0.2h2 0.350 0.h05 15 level - 0.215 0.2h2 0.350 0.h05 0.32 0.h0 0.37 00 OO O \J'l t'w HS em 5 0.1.05 9 0.793 0.793 0.82 0.86 1.06 1.23 0.86 0.89 0. 88 0. 88 0.55 0.115 0.91 1.01 0.62 0.69 0.80 0.76 0.75 0.82 0.61 0.65 0.70 0.65 0.793 107 TABLE XVII. Pig NO 0 1 13.6 12.3 10 11 Average 9.60 Significance of group averages at: 9. P. 9.60 5% level - 10.68 12.3 12.8 9.7 7.9 10.68 1h.30 19.0 18.5 17.7 17.2 18. 10 9. 15.h0 11.7 18.7 1h.30 E. 18.10 5:51 ~07: Relative weights of external inguinal lymph.nodes (mg. per 100 gm. body wt.) Group.A Group B Gropp C Gropp D Group E Gropp F Gropp G 15.6 16.3 5r: as {3: O O 00 GUI ‘0"? VIN 1?“! 3"“ ‘0‘) a: PJhJ a: raox N O 0 U1 ...: U! r O 108 TABLE XVIII. Heights of prefemoral lymph nodes (en-) gig N0. Group A Group B Group C Group D Group E Group F Group G 1 0.18 0.16 0.21 0.15 0.15 0.17 0.15 0.20 0.18 0.13 2 0.18 0.15 0.10 0.20 0.25 0.20 0.11 0.10 0.18 0.23 3 0.12 0.18 0.16 0.08 0.18 0.16 h 0.18 0.15 0.11 0.15 5 6 7 8 9 10 11 Average 0.156 0.157 0.152 0.178 0.172 Significance of group averages at: 2 A C F E 5% level - 0.152 0.156 0.157 0.172 0.178 1% level - 0.152 0.156 0.157 0.172 0.178 0.36 0.38 0.56 0.5h 0.35 0.31 0.66 0.68 0.25 0.216 o.h8 0.h7 0.30 0.3h 0.61 0.h5 0.36 0.28 0.3h 0.35 0.18 0.16 0.38h 0.38h 0.3§g l IIIIIIILrnIu 1.1.73,” khgi..rfihvfi Munoz-w 1...- . #7 ..‘ 109 TABLE III. Relative weights of prefemoral lymph nodes (mg. per 100 gm. body wt.) Pig N . Group A Gropp B Group C Grapp D Group B Group F Group G 1 1007 509 9.2 705 505 608 10.1 6.64 8.8 9.0 68.8 702 2 8.8 7.2 h.h 10.7 8.0 9.8 9.8 6.7 8.8 9.7 7.3 9.h 3 6.1 8.1 6.3 5.9 1401 801 603 5.2 h 10.h 5.1 10.3 8.1 5.1 10.6 5 5.2 5.0 6 8.3 8.1 7 5.8 6.1 8 7.5 8.3 9 6.5 5.1 10 8.9 9.2 11 5.7 5.0 Average 6.01 7007 6970 9022 6.05 7025 Significance of group averages at: A F 2 .0. 9 2 5% level - 6.01 6.05 6.70 7.07 7.25 9.22 110 TABLE XX. Weights of hearts (gnu) 27.0 31.1 32.0 35.2 26.6 28.9 29.9 31.9 22.8 21.7 28.51 Pig No. Group A Group B Group C Group D Group E Group F Group G 1 12.0 12.h 13.0 20.3 2 13.5 12.1 12.8 21.7 3 18.5 17.1 h 19.1 5 6 7 8 9 10 11 Average 13.33 12.25 12.90 19.55 Significance of group averages at: 2 2 2 2 2 5% level - 12.25 12.90 13.33 19.55 28.51 1% level - 12.25 12.90 13.33 19.55 28.51 111 TABLE XXI. Relative weights of hearts (mg. per 100 gm. body It.) Pig No. Group A Group B Group c Group D Group E Group F Group G 1 508.3 51.6.3 650.8 7h5.2 512.7 2 6h6.h 533.0 687.7 692.7 5h3.7 3 651.8 672.6 538.0 h 6h7.2 5h9.9 5 516.0 6 1497.3 7 539.8 8 .... 9 575.9 10 597.9 11 682.8 Average 602.17 539.65 669.25 689.h2 555.h0 Significance of group averages at: 2 E E E E 5% level 539.65 555.h0 602.17 669.25 689.h2 112 TABLE XXII. weights of lungs (sm.) Pig No. Grppp A Group B Group C Group D Gropp E Gropp F Group G 1 3h.5 28.7 20.9 53.8 2 33.5 31.1 21.3 h2.8 3 33.6 3h.7 h 38.7 5 6 7 8 9 10 11 Average 33.87 29.90 21.10 h2.so Significance of group averages at: E 2 E E 9 511ml- 21.10 29.90 33.87 1.2.50 71.88 1% level - 21.10 29.90 33.87 h2.50 71.88 7h.3 77.5 78.8 88.8 65.h 79.1 73.0 78.5 82.h 5h.8 38.1 71.88 113 TABLE XXIII. Relative weights of lungs (mg. per 100 gm. body wt.) Pig No. Group A Group B Group C Group D Group E Group F Group G \OmNO‘U‘C'WNH H O 11 Average Significance of group averages at: 5% level - 1h61.h 160h.1 1510.h 126h.3 1370.0 10h6.3 11hh.3 1975.0 1366.3 136h.9 1311.h 1h10.8 135h.8 132h.9 1387 .2 1371.9 1361.2 1318.0 1hho.9 1h87.7 11:37.0 1198.9 1525.30 1317.15 1095.30 150h.b0 1372.12 2 1317.15 9 1372.12 150k.ho 1525.30 TABLE XXIV. 11h Weights of livers (emo) Pig No. Group A Group B Group C Group D Group E Gropp F Gropp G 1 \OCD-dOsU'lt‘U 10 11 Average Significance of group averages at: h7.o h7.5 11.5 3h.2 h3.30 5% level - 1% level - 59.5 57.5 5h.h 71.0 66.1 55.1 5h.7 95.3 59.9 82.6 83.7 61.83 56.30 5h.55 83.15 :2 E 2. E. El h3.3o 5h.55 56.30 61.83 83.15 h3.30 5h.55 56.30 61.83 83.15 .9 193.7 216.3 15h.8 193.3 . 1h1.1 1h0.3 127.0 156.h 152.3 102.8 82.8 150.98 150.98 150.98 115 TABLE XXV. Relative weights of livers (mg. per 100 gm. body wt.) Pig No. Group A Group B Group C Group D Group E Group F; Group G 1 2798.0 2520.3 2533.0 2723.3 2606.5 3678.0 2 2325.0 3165.1 2827.3 2938.6 30h2.2 3781.2 3 2279.5 2692.6 32178.9 2602. 8 8 1982.8 2836.3 3019.7 5 2959.9 6 2141MB 7 2293.0 8 2870. 8 9 27147.9 10 2695.6 11 2605.h Average 2386.22 2792.67 2h80.15 2830.95 2933.h8 2878.96 Significance of group averages at: i E E E 9 E 5% level - 23h6.22 2h80.15 2792.67 2830.95 2878.96 2933.h8 “.0, ..ud- ..r .391: Pig No. l \ooowmmz’wm H O 11 Average Significance of group averages at: 2.1 2.1 2.8 2.0 2.25 5% level - 1% level - TABLE XXVI. Group A Group B Group C Group D weights of spleens gm. Group E Group F Group G 3.70 5.35 h.5 6.7 h.h 5.8 5.35 .9 8.52 2.7 2.6 3.8 2.5 2.8 3.6 2.2 2.17 2.70 3.70 E 2 E E 2.25 2.1.7 2.70 3.70 5.35 2.17 2.70 8.52 8.6 15.3 8.1 13.6 7.0 7.6 7.0 7.2 7.6 5.6 6.1 8.52 117 TABLE XXVII. Relative weights of spleens (mg. per 100 gm. body wt.) Pig No. Group A Group B Group C Group D Group E Group F Group G 1 125.0 11h.h 2 102.8 119.7 3 1h3.h 93.9 h 115.9 5 6 7 8 9 10 11 Average 121.78 111.00 Significance of group averages at: 2 2 11h.5 123.3 118.90 A 190.2 193.h 191.80 G 165.2 213.9 173.1 196.5 187.18 F 163.3 267.5 136.2 212.5 1h6.8 130.8 126.8 132.2 137.2 1h6.8 191.9 162.87 E 5% level - 111.00 118.90 121.78 162.87 187.18 191.80 118 TABLE XXVIII. Heights of kidneys (gm) Pig No. Group A Group B Group C Group D Group E Group F Group G 113065 1 7.5 9.9 9.1 7.5 9.5 7.5 9.2 8.8 7.6 9.8 2 8.5 8.h 8.6 7.0 10.7 8.5 9.h 7.3 7.3 10-5 3 8.0 5.7 9.6 800 600 90h h 6.2 10.3 6.3 11.2 5 6 7 8 9 10 11 Average 7.56 8.10 8.58 7.35 10.12 Significance of group averages at: E A E 2 5: level - 7.35 7.56 8.10 8.58 10.12 1% level - 7.35 7.56 8.10 8.58 10.12 1h.65 18.0 16.2 20.7 21.6 17.0 16.8 20.3 20.3 13.1 12.9 13.9 15.6 13.2 13.2 13.5 1h.5 13.9 13.3 8.6 9.5 7.7 85 O 1h.65 TABLE XXIX. Relative weights of kidneys (mg. per 100 9!. bOdy It.) 119 ‘Pig No. Group A .Group B Group C Group D Group E Group F Group G l 10 11 Average Significance of group averages at: hh6.5 M6605 h16.l h16.1 h09.3 h09.8 359.8 365.2 h08.66 5% level - 1% level - hl9.3 h00.9 375.h 3h8.8 3h1.8 389.7 387.7 380.5 359.8 307.6 h21.h 378.9 376.1 3h1.6 361.9 h50.1 3h3.6 392.2 335.2 377.6 256.2 377.6 285.8 269.7 369.7 282.5 3h9.o 317.1 379.5 317.1 278.8 270.6 239.2 268.h 238.3 238.3 2h7.8 266.2 251.0 2ho.1 225.5 2h9.1 2hz.3 267.5 367.73 377.78 381.05 357.65 277.75 E .0. 2 E 2. 357.65 367.73 377.78 381.05 808.66 357.65 367.73 377.78 381.05 h08.66 . . a .1 ....w 7 . 3.4%.... 8...... ... . .J.’ t \ 120 TABLE XXX. weights of pancreases (8111.) P1; 30. Group A Group B Group C Group D Group E l 3.3 3.6 3.2 2 3.0 3.9 3.2 3.1 O\ V". 2" w x) 10 11 Average 3.13 3.75 3.20 Significance of group averages at: 022: Group F Group G 5.2 6.5 6.2 5.0 8.23 5% level - 3.13 3.20 3.75 5.72 11.6 16.5 7.7 11.11 7.6 7.0 5.6 7.3 6.7 5.2 3.9 8.23 Pig No. l ‘Om-QONV‘C'W 10 11 Average Significance of group averages at: IABLE XIXI. 121 Relative weights of pancreases (mg. per 100 gm. bOdy It.) Group A Group B Group C Group D Group E Group F Group G 51 level - C 139.03 138.0 183.7 139.8 139.03 0 155.58 158.6 160.2 171.8 171.9 165.20 166.05 .2 .§ 165.20 166.05 190.9 207.5 283.9 169.8 202.92 E. -2°2°92 220.3 288.8 129.5 178.1 159.8 120.5 101.1 138.0 121.0 136.8 122.7 155.58 122 TABLE xxxxx. Heights of thyroids (gm) Pig No. Group A Group B Group C Group D Group E Group F Group G 1 0.80 0.30 0.19 0.30 2 0.80 0.26 0.28 0.29 3 0.30 0.85 8 0.27 5 6 7 8 9 10 11 Average 0.382 0.337 0.235 0.295 Significance of group averages at: n g E g A 5; level - 0.235 0.260 0.295 0.337 0.382 0.555 0.22 0.30 0.32 0.20 0.260 0.78 0.71 0.61 0.68 0.37 0.88 0.87 0.98 0.55 0.30 0.25 0.555 123 TABLE XXXIII. Relative weights of thyroids (mg. per 100 gm. body wt.) Pig No. Group A Group B Group C Group D Group E Group F Group G 1 23.8 12.7 ' 8.8 15.0 8.1 18.8 2 19.6 12.8 12.3 15.6 9.6 12.8 3 15.8 20.2 12.6 10.3 8 15.7 6.8 10.0 5 7.8 6 8.3 7 8.5 8 17.3 9 9.9 10 7.9 11 7.9 Average 18.62 15.10 10.35 15.30 9.28 10.86 Significance of group averages at: F 0 0 g E A 5% level - 9.28 10.35 10.86 15.10 15.30 18.62 1% level — 9.28 10.35 10.86 15.10 15.30 18.62 128 TABLE XIXIV. Weights(of §drenals gm. Pig N . Group A Group B Group_§ Group_p Group E Group F Group G 1 0.18 0.13 0.21 0.16 0.25 0.35 0.18 0.17 0.21 0.15 0.23 0.33 2 0.20 0.16 0.18 0.28 0.25 0.38 0.20 0.16 0.15 0.23 0.26 0.37 3 0.22 0.19 0.22 0.22 0.22 0.20 0.20 0.26 8 0.18‘1 0.22 0.32 0.18 0.23 0.88 5 0.36 0.33 6' 0.28 0.29 7 0.29 0.25 8 - 0.27 0.25 9 0.28 10 0.35 0.32 11 0.32 0.30 Average 0.185 0.168 0.178 0.195 0.232 0.311 Significance of group averages at: E E A E E E 5% level - 0.168 0.178 0.185 0.195 0.232 0.311 1% level - 0.168 0.178 0.185 0.195 0.232 0.311 a Missing weight. It was assumed that this adrenal had the same weight as the left one from the same animal. .5 125 TABLE XXIV. Relative weights of adrenals (mg. per 100 gm. body wt.) Pig N0. Group A Group B Group 0 Group 0 Group B Group F Group 0 1 10.7 5.5 9.2 8.0 9.2 6.6 1007 702 902 705 802‘» 603 2 9.8 7.7 6.2 12.9 8.0 6.6 9.8 7.7 6.6 12.8 8.3 6.5 3 11.3 8.5 8.6 3.7 11.3 9.0 7.9 8.8 8 8.1“ 7.8 5.0 8.1 7.8 6.9 5 7.6 6.9 6 8.8 5.0 7 5.2 8.5 8 5.0 8.6 9 5.1 5.1 10 9.2 8.8 11 10.1 9.8 Average 9.98 7.60 7.80 10.20 8.20 6.22 Significance of group averages at: .9 9. P. E. A E 5% leve1 - 6.22 7.60 7.80 8.20 9.98 10.20 ‘jlissing weight. It was assumed that this adrenal had the same 'weight as the left one from.the same animal. .. 0| 9.1. 126 Since the findings on the intestinal tract are to be reported elsewhere, they will not be extensively described here. It may be men- tioned, however, that the wall of the intestinal tract appeared thinner than normal, and the posterior portion of the small intestine contained a dark material which somewhat resembled a hemorrhagic exudate. In the remaining tissues, the most constant gross lesion con- sisted of slight to moderate congestion or hemorrhage in the peripheral parts of the lymph nodes, especially those of the iliac group. Other nodes were sometimes involved else. These lesions appeared to be unre- lated to the conditions under which the animals were raised. Pigs G—3 and G—h showed marked enlargement of one small area of the mesenteric lymph nodes, and 2 nodes of the iliac group of pig Geh were enlarged. Tissue was not saved from these enlarged areas. The peripheral lymph nodes of pigs G—lO and G—ll showed slight to marked peripheral staining with a brown-colored.pigment. Pig G-h exhibited interstitial emphysema in the diaphragmatic lobes of the lungs, and there was one small area of consolidation on the dorsal surface of the lung above the right cardiac lobe. Pigs 0.1 and 0-2 each had small cysts on the surface of the liver containing what appeared to be bile. The umbilicus of pig G-lO was found to contain a.moderate amount of purulent material. Gross examination of the germfree animal left in the isolator 3 days after death revealed that the liver was quite friable, the in- testines were somewhat soft, and the lungs showed areas of atelectasis and some emphysema. In the animal dead 5 days, it was noted that the 127 kidneys were quite soft, the intestines were soft, and the liver was rather friable. No objectionable odors were noted from either pig. Hematological data obtained from blood samples drawn shortly before each animal was killed are shown in Table XXXVI to indicate that these data are, for the most part, within the normal range for animals of this age group. 3. Histology pg 323 Lymph Nodes SEE Spleen The histological picture of the lymph nodes was characterized by extreme variability. This variability existed not only between pigs but also between different lymph nodes in the same animal, and it was sometimes noted that different regions of the same lymph node presented somewhat different pictures. One point of variability concerned the amount of fusion seen in the individual lymph nodes. Of the 8 groups examined the external inguinal nodes exhibited the most fusion, consisting of several small nodes separated in some cases by small hands of fatty tissue. In other instances the capsules were fused, and in still other instances the capsules were absent at the area of fusion and the medullary regions of the smaller nodes were in contact. The same type fusion was quite ap- parent in the mandibular and prefemoral nodes, although it seemed to occur to a somewhat lesser degree. The mesenteric nodes exhibited a small amount of such fusion. Another point in which the lymph nodes varied was in the rela- tive amounts and location of the cortex and medulla. In most nodes the tissue corresponding to the cortex of other mammals was located cen- trally, with the medulla surrounding it. It was occasionally found, 128 (“Chi-{MO OHMOmMJMI-{MMH oocmcm .osz OOOOOOOOOOOOOOOHHJNHNOMHNOOO HOOOOOHOOOOOOOHHHHOOOOHOONOO OHHHOHNOHMHMMHNONOHmammmmMOr-i Os \0 a u a a .mom .mem .oeo: .nQqu OONJHHNNNONONmfihNMHM—QH u..mouleoc nHHn Ohesmz «use Huoamoaosaaom N: am on 0: :m «N 00 mm am an am am 0m 0m ma m4 ma on :a an on mm 50 mm 0m m: 0H mm u.wou 00m.0 a0.m 0m~.0 0m.0 oomma ms”: 004.5 Ha.m 0mm 0 as m 00H.0 Hm.m 00~.HH 00.m 0mm.sa ~m.0 00m.~ ~0.m 00m.~a ma.m 000.0 0m.m 000.m 0H.» 0ma.0 0m40 0mm.m sm.m 0mm.m 04.0 000.0 H0.m 0mm.m m0.4 000.; 0H.0 0m0.ma 0m.m 0m~.4 Ha.m 00s.m m0.m 0m0.0 m0.m 000.m~ 40.~ 08.2 00$ 00~.m mm.m 000.: ma.m 000.~ m0.0 .330 you geae\nsoaaafia .0.m.s. .0.m.m .stxxvmamaa R.Ho> .aox mmmaaocouxnxqmomxowmmo CNNQQQF—QONHOOOIGDF—O «.ma H sees... .00.... HHHHHr—i HH 4444mmmooonnmmhmmmooocoooccoo 129 however, that the cortex was on the outside or perhaps along one edge. The relative quantities of the 2 types of tissue showed little uni- formity, one node being predominantly cortex, another predominantly medulla. The above mentioned variations made it extremely difficult to determine, quantitatively and qualitatively, whether or not differences existed between the various groups of animals. Light-centered nodules corresponding to the germinal centers of Flemming (1885) or the reaction centers of Hellman (1921, 1930) were seen in all groups of animals. In the fans-raised animals, the mandi- bular nodes exhibited a greater degree of activity, as evidenced by the number of light centers and the amount of mitosis within these centers, than did the nodes of the external inguinal, prefemoral, and mesenteric groups. In the germfree animals, however, the mesenteric lymph nodes appeared to show more activity than did the other nodes. In the re- maining groups of animals either the mandibular or the mesenteric nodes of the h groups examined, seemed to show the greater activity. There was a definite tendency for the lymph nodes of the germfree animals to show more solid primary nodules consisting of dense collections of small lymphocytes, as in Figures 21 and 22. However, this type nodule was also found in the pigs raised on the artificial diet in open cages and occasionally in the farm-raised animals. The lymphatic nodules found in the nodes of farm-raised ani- mals exhibited numerous light-centered nodules as shown in Figure 23. However, such nodules were not absent from the gnotobiotic animals, as is evidenced by Figure 2h from a germfree animal and Figure 25 from an animal of group C (raised in the presence of Bacillus 52.). The $I' « u < '.§E§§€;I;Lh ... <59 .M' ,- .m-(‘I' Figure 21. Solid primary nodules in prefemoral lymph node of germfree pig E-l Hematoxylin and eosin. x 105 llllll 1“ 1" Figure 22. Solid primary nodule in mandibular lymph node of germfree pig E-2 Hematoxylin and eosin. x 215 Figure 23. Light-centered nodules in prefemoral lymph node of farm-raised pig G-l Hematoxylin and eosin. x 105 - . A ' ‘7.“\ . If I.» - . .‘4 g“. «g 1. ‘ A.“ . . ‘5}: “4;? J . ‘ ‘ «‘1 . if”. 2 , * “‘ . ‘ Y "" . 0‘ 7L- 1 u_ ‘7" 445%.. x - $wa 7‘49“». .23"- .- , £4 “'3 ,m. A "L“ 1"..‘. A? . . p 3.1;" ,2, ,M .44: ,, If ' “Que; . »_ - Figure 21;. Light-centered nodules in mesenteric lymph node of germfree pig E-l Hematoxylin and eosin. x 105 I I at“ . a .o . v ./0 .... ‘, v ,V . x . . .. r w . ‘$ .yvx‘flfi'... . . $4313.”... 4 o#\r5 ..NI .0 .. a u 1, ha . . \ d.‘ ..- l . Light-centered nodule in Figure 25. mandibular lymph node of pig C-3 x 360 Hematoxylin and eosin. One Note dark cells in center of nodule. or 2 of these represent mitotic figures, the remainder are pyknotic nuclei. 135 Opposite end of the lymph node pictures in Figure 2h consisted primarily of solid nodules as in Figures 21 and 22. Another type of nodule seen rather frequently consisted of a collection of reticular cells, macrophages, and some large lymphocytes. These cells were often surrounded by a capsule-like reticulum as seen in Figure 26. These nodules were sometimes ciroled by small lympho- cytes, but in mamr instances they were not. Mitotic activity was ordi- narily negligible in these centers. - Figure 25 represents the more typical germinal or reaction center. These centers also contained numerous reticular cells, macro- phages, and large lymphocytes. The mitotic activity in these centers was quite variable. In most instances at least a few mitotic figures could be seen, and sometime several mitoses were present. When such centers were observed under low magnification, it appeared that con- siderable numbers of prophase mitotic figures were present. Examination under the oil-immersion objective, however, revealed that only part of these structures were mitotic figures. The rest were. dark, apparently degenerating, nuclei. From the size and location of these nuclei, it appeared that some of them were from larger lymphocytes and some from the reticular cells or macrOphages. Figure 27 shows at least 2 mitotic figures, as well as several degenerating nuclei. Also included in the figure are several small, round to irregularly-shaped objects which stain quite darkly and appear to correspond to the "tingible Kbrper" described by Fleming (1885). These are apparently composed of chroma- tin material from degenerated cells which have been engulfed by macro- phlges o Figure 26. Light-centered nodule in mandibular lymph node of germfree pig E-l Hematoxylin and eosin. x 360 Note connective tissue fibers sur- rounding nodule, scarcity of mature lymphocytes, and lack of mitotic activity in nodule. 137 '5.‘ . ‘ a g. f. C ‘ t '. ~31. ' " “"a" ‘0 '3 ‘ J" t" at ' "" 3‘" 9'” M "35’. b _. ' «C‘s. ‘/ i‘ ;’ '. ‘ ‘ (1).? ' ' ‘E ‘ r I v " , A 09 a Figure 27. Cells in light-centered nodule from mesenteric lymph node of farm-raised pig G-lO Hematoxylin and eosin. x 960 A. Mitotic figure B. Degenerating nuclei C. "Tingible Kbrper" 138 Most of these germinal or reaction centers were surrounded by at least a small zone of mature lymphocytes. However, this zone was not usually as large or densely staining as that shown in Figure 25. This is probably due to the fact that some of the cells in this figure 'were hematOpoietic cells (to be discussed later). Sometimes this zone of.mature lymphocytes was absent, or present on one side and absent on the other. ‘lhen the latter was the case, the border containing no small lymphocytes was usually adjacent to a trabecular sinus. Although it was difficult to quantitate, it appeared that these typical germinal centers were larger and more abundant in the farmpraised pigs. No recognizable differences were found between the lymph nodes of animals in groups A and.C (each raised in the presence of one species of bacteria) and their littermates (groups B and.D, re- spectively) fed the sterilized diet in Open cages. Numerous cells of the granulocytic series were seen in the lymph nodes of these pigs. The most common cells were immature eosino- phils, most of which appeared to be metamyelocytes or band cells. These cells were scattered in the medulla and cortex but were very sel- dom seen in the lymph nodules themselves. They ranged from a very few to many in a section, and no correlation could be found.between the . numbers of these cells and the conditions under which the animals were reared. The granules in these eosinophilic cells were in most sections very distinct. Also noted were some neutrophils. These granulocytes appeared to‘be.more mature than the eosinophils and were primarily band forms with some segmented nuclei present. The granules were not dis- tinct. In.most sections these cells occurred in smaller numbers than did the eosinophils, and they had a tendency to be more in groups than 139 did the eosinophils. Here again, there was no correlation between the method of rearing the animals and the numbers of neutrophils present. Figure 28 illustrates these 2 types of cells in the cortex of a lymph node. In most sections small, darkly-staining cells, resembling mature lymphocytes under low magnification, were found. Examination of these cells under the oil-immersion objective, however, revealed that they possessed nuclei which stained so intensely that no detail could be made out. Some of them appeared to contain no cytoplasm, whereas others had a small rim of eosinophilic cytoplasm. Figures 29 and 30 show these cells, which could not be distinguished from the cells of the small hematopoietic centers in the livers of these animals. The cortices of a majority of the nodes contained at least a few of these dark cells. Sometimes they were accumulated in groups in the sinuses of the medulla, and collections of these cells were commonly found in the trabecular'sinuses as in Figures 29 and 30. In several of the lymph nodes these cells were so abundant that they could easily be seen under the scanning objective as dense black masses. In areas where these dark cells were quite densely packed, it was occasionally noted that spindle-shaped cells resembling degenerated fibroblasts accompanied these smaller cells. An amorphous, brown-staining material was seen in the periph- eral parts of all the lymph nodes of pigs 0-10 and G-ll, although the amount in the mesenteric nodes was much less than in the other 3 groups. Some of this material was free within the reticular sinuses, and part of it was intracellular. Gomori's iron stain showed this to be an iron-containing pigment (Figure 31). Figure 28. Eosinophils and neutrophils in medulla of mandibular lymph node of farm-raised pig G-2 Giemsa. x 1600 Figure 29. Hematopoietic cells in medulla of superficial inguinal lymph node of farm-raised pig G-l Hematoxylin and eosin. x 175 A. Trabecula with cells in surrounding sinus. Note dense collections of these cells in lower portion of figure. Figure 30. Hematopoietic cells in trabecular sinus of superficial inguinal lymph node of farm-raised pig Gul Hematoxylin and eosin. x 960 Note dark staining of these cells in comparison to surrounding cells. 'n a“. ..:.v..;.:~.» . , . \ ~ - ., .. ..,'___..n.‘ - , r. V .. . A;,§;;"‘§:"r~1§;Jg~§ ,Id’ 3% . ,4}... . “:st Q ~ i We». 73%“ ~. :- 1*. ‘_ 25:1“ ‘='-? 1'- Figure 30 1h3 “2.3: it its 3’ Figure 31. Iron-containing pigment in medulla of lymph node of farm-raised pig G—ll Gomori's iron stain. x 960 The Heidenhain aniline blue stain and the Foot's modification of Bielschowsky's stain failed to reveal any differences in the connec- tive tissue framework of the lymph nodes, but the latter stain empha- sized the reticular framework of the different parts of the node. As Figure 32 shows, there were very few fibers in the center of the lym- phatic nodules, but these fibers formed a slight capsule around the ex- terior of the nodule. The fibers in the remainder of the cortex were relatively coarse and widely spaced, while those in the medulla were fine and more closely spaced. The histolOgical picture of the spleen was much more uniform than that found in the lymph nodes. As can be seen from Figure 33, the red pulp occupied a larger part of the volume of the spleen than did the white pulp. Hitotic activity was noted in the lymphocytes of the splenic corpuscles. Sometimes the mitotic figures were scattered in the relatively mature lymphocytes. At other times light-centered areas were found in one portion of the splenic corpuscle. These light centers were usually smaller than those seen in the lymph nodes, but their cellular make-up was quite similar. No definite differences in the structure of the white pulp were noted between the various groups of animals. The ellipsoids were well-marked in the red pulp of the spleen. Some eosinophils and neutrOphils were noted in the red pulp, but their number was quite small in comparison to that found in the lymph nodes. A few small collections of cells similar to cells of the hematopoietic centers of the liver were seen in the red pulp along with an occasional me gakaryo cyte . v."- t . A O . e.‘ 9 43:. .3! i .. t . -» A. .{I .J. -.,s.-- . A :3 . - . , \N‘fi- \e " '. . ’94? fl" - l Figure 32. Reticular framework of prefemoral lymph node of farm-raised pig G-h Foot's modification of Bielschowsky's stain for reticulum. x 215 A. Germinal center B. Reticular fibers surrounding A C. Cortex D. Medulla E . Trabecula 1146 v - i ‘ ‘ -- ‘ . 't‘)‘: 1’ 59K. "1. .‘ 1 . the we. 34"“ >5 .‘n ‘\ . Pitt 5‘." . gtfil‘iv w >- . {l‘ l .5 s 1..) a was :45- . .. ("3. ... AP. -£a.‘-:’k ' $5 =“-‘*’«,~¢-s$‘£§ii’f ~ W .r-, 2511. v - ' ‘ 'Ifivv’jgr-I . ./ .3... at; ' '. .'. $355? ‘9 . ’4 r r. " 5“.” f4 4’; .- f..”fl’r§m?:’3 ‘ 5" "9 a . ‘ f; fl’ls‘ {36‘ 4F: {33- ~ “3' “‘ _. ' .' u l I . , w w‘; .i . s- o .1', Figure 33. Spleen of germfree animal B-l Hematoxylin and eosin. x 105 A. Splenic corpuscle with light-centered area a. Redpulp C . Ellipsoid 1h? The amount of ironpcontaining pigment, presumably hemosiderin, found in the spleens was quite variable. Pigs of group A, group C, and group D, all raised artificially, exhibited quite significant amounts of this pigment in the cytoplasm of cells of the reticulo-endothelial system. The pigment granules were rather small when compared to those seen in the lymph nodes of pigs G-lO and G-ll. Pigs of groups A and E, also raised artificially, had very little of this pigment in the spleen, and the same was true for the farmpraised pigs of group 0. Figure 3h demonstrates this material. . The aniline blue and reticular stains revealed no significant differences between the various groups. The white pulp was sometimes surrounded by an accumulation of reticular fibers, but more often these fibers were arranged concentrically within the splenic corpuscle itself. The red pulp contained fibers similar to those seen in the cortex of the lymph nodes, but they were finer. The ellipsoids contained reticular fibers so small as to not be noticeable except under very high magnifi- cation. h. Histology of the Liver The outstanding histological difference between the livers of the various groups of pigs involved the connective tissue septa between the lobules. In the farmpraised pigs these septa were clear and dis- tinct as in Figure 35 and were composed of from one or 2 to several rows of fibrdblasts. There often appearedto be slight separations between the rows of fibroblasts and also between the fibroblasts and the hepatic cells themselves. This tended to emphasize the septa. 1148 Figure 31;. Iron-containing pigment in spleen of ‘ gnotobiotic pig A-l Gomori's iron stain. x 960 lb? The interlobular septa were less distinct in the artificially- raised pigs, and in many cases the only indications of the borders of the lobules were a somewhat more foamy appearance of the cytoplasm of the peripherolobular hepatic cells and a tendency for the cells to line up in rows in these areas. Figure 36 illustrates this arrangement. The connective tissue stains emphasized this difference in the degree of development of the septa. Figures 37 and 38, both stained for reticulum, demonstrate the band of connective tissue in the farm- raised animal and the lining up of the reticular fibers between the hepatic cells at the edge of the lobule in the germfree animal. The underdevelopment of the septa was less distinct in the group F animals (those raised in the presence of Achromdbacteriaceae and an unidentified anaerobe). Although there was some deve10pment of the septa in these pigs, it was not as distinct as in the farm-raised animals. Best's carmine stain revealed the presence of glycogen in all the sections stained. The glycogen appeared to be in heavier concen- trations in the cells of the peripherolobular areas. The fat stains failed to demonstrate lipids in any of the sections stained. However, the hematorylin and eosin stain of the liver of pig B-3 revealed nu- merous vacuoles diffusely spread throughout the ldbules. These were quite suggestive of fatty changes, but formalin-fixed tissue was not available for lipid staining. Most of the liver sections examined contained several collec— tions of small, intenselybstaining cells (Figure 39). These were found in the sinusoids and wera presumed to be remaining evidence of the hema- topoietic function of the fetal liver. Figure 35. Liver of farm-raised pig G-9 Hematoxylin and eosin. x 105 Note clearly-defined interlobular septa. Figure 36. Liver of germfree pig E-2 Hematoxylin and eosin. x 105 A. Lining up of hepatic cells in peripherolobular area B. Hematopoietic centers “Ff-5113‘} ; , ' , . ,fi’ 53“)” .13; 8‘ ‘. x- . a, .' a I n a. a (a, .2" “1 N I; " ‘. SQ . ‘ “194' ‘L—‘ . ~\ . , the? 2'3“? fiimfit .: i» ..._ warsh- * ‘ #{lgfi‘ .93; a 43‘ ~ ‘ fist-gt! t .... 3 1.1;; . ‘19. :2 . '1‘- , “I - ~!\" .7 . ‘ '1 s' {1. .. *4.” as . a ' t a j {45" I ' ~ 4 \ fifiwgfgg ._ IV:— is *r V n" .0“ has.” 152 Figure 37. Liver of farmpraised pig G—8 Foot's modification of Bielschowsky's stain for reticulum. x 105 Note well—developed interlobular septa. Figure 38. Liver of germfree pig E-2 Foot's modification of Bielschowsky's stain for reticulum. x 105 Note lining up of reticular fibers in peripherolobular areas. .. ............ .. ...... . .., ,. .... . ,. . ANNA...“- . , ,, r , 4. ... An.” .X. 1.x; \ ...awv. . .3. " :‘h-h e .- ( ~ 3. fl a. w . x v 5 Figure 37 t \ at.) . .. \ ... ii 7.3.x... .. .a .. _ .8 . . . .a . Am 3 I Am. . . . . r . paw..cv .. ..aflezuwnwsi J.. a mm» . a. gene... ; .V 2. F. .ntuya. when}, . Figure 38 ‘5 e ‘ I!" " I. ’1 -5 a: . .9 e; » ’.-Lqu II! a “ “it... , a we , *‘m '6', ‘ ' 0'33"“. : ‘ 1,, ...,e if? I. >>> APPENDIX Necropsy Numbers of Animals PigN. HH l-‘O‘OmflGmrmeC’WNI-‘NHNHWNHUNHF’WNH 179 Necropsy N . D6261 m 49E; 1 ‘ A. 0:3 Jan/576 67 JUL 3 '55. ‘ “H; g 3:1 '“7T‘C77 ARIES H 1I ‘H J ‘ MICHIC‘SAN' STATE UNIVERS‘ITY‘L‘IVBR“ ‘IWI$i‘1llilil4HIE5IIWHIlHNlilHlllllHllllH " ‘3‘12’9'3‘0317'8 '15'23