LESEONS 0F DCPERIMENTAL memo ‘ coumczuosss m NEONATAL. Gastoato‘nc PIGS: A mums. , ‘ ELECTRON -MECRGSCOP!C>STUDY _ - ThesEs for the Degree of M. S. MlCHlGAN STATE-BMVERSITY' LOUIS E. NEWMAN 1975 IhESIS UBRARV amoens 'i kSPRING'UFT, MICHIGIJ' ABSTRACT LESIONS OF EXPERIMENTAL ENTERIC COLIBACILLOSIS IN NEONATAL GNOTOBIOTIC PIGS: A SCANNING ELECTRON MICROSCOPIC STUDY BY Louis E. Newman Scanning electron and light microscopy were utilized to View sections from the stomach, duodenum, anterior jejunum, posterior jejunum, ileum, cecum, and spiral colon from 18 gnotobiotic pigs. Five of the pigs were reared as controls and 13 were infected at 6 days of age by the oral administration of 1.6 x 106 colony forming units of Escherichia coli 0138:K81:NM. The pigs were euthanatized at selected intervals after EXPO“ sure by the injection of sodium pentobarbital intravenously. The pigs were immediately exsanguinated and the abdominal viscera was exposed and removed. Two adjacent full thickness sections were removed from each of the 7 locations. The sections were fixed in either 10% formalin-sodium acetate solution or Karnovsky's fixative. The formalin-fixed tissues were embedded in paraffin and stained with hematoxylin and eosin for light micro— scopic examination. Whole thickness sections of the tissues for scanning electron microsc0pic examination were attached to aluminum discs and dehy— drated in a series of ethanol solutions. The ethanol was replaced Louis E. Newman with amyl acetate. A critical point drying apparatus was used to dry all tissue sections. The sections were individually coated with 400 A of gold and viewed with the scanning electron microscope. The onset of diarrhea, flushed skin and erect hair coats and the gross lesions were consistent with reports by other investigators. The surface of the gastric mucosa of the pyloric portion of the stomach when viewed by scanning electron microscopy presented an irregular pattern of ridges and furrows. The appearance of the rugae, gastric pits and mucosal extensions between the gastric pits correlated well with the appearance of the gastric mucosa by light microscopy. The infiltration of leukocytes into the mucosa of the stomach seen with the light microscope has not been previously reported. Scanning electron and light microscopic observations of the small intestine were consistent with expectations and the literature with three exceptions. One pig at 16 hours after exposure had lost a number of epithelial cells resulting in distinct holes in the mucosa of the villi in the anterior jejunum. One pig 24 hours after exposure had lost epithelial cells from the tips of villi in the anterior jejunum which resulted in exposure of the lamina propria. Many sections of the ileum from both control and infected pigs contained a number of collapsed cells in the area around the extrusion zone at the tips of the villi. These cells were more numerous in the infected pigs, and more cells appeared to have been sloughed from the area of the extrusion zone resulting in exposure of the lamina propria. Louis E. Newman No changes of the cecum or spiral colon were apparent with either scanning electron or light microscopy. However, the appearance of the large intestine under scanning electron microscopy was par— ticularly interesting. The convoluted folds of the spiral colon in some sections and the ridge upon fold appearance of others were considered to be normal variations. The alterations of the mucosa of the intestinal tract of gnotobiotic pigs infected with E. coli, as visualized by scanning electron microscopy, were not considered consistent enough to be of diagnostic significance. LESIONS OF EXPERIMENTAL ENTERIC COLIBACILLOSIS IN NEONATAL GNOTOBIOTIC PIGS: A SCANNING ELECTRON MICROSCOPIC STUDY BY Louis E. Newman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1975 ACKNOWLEDGEMENTS The author wishes to express his gratitude to his major pro- fessor, Dr. Allan L. Trapp, for his guidance and assistance in this investigation. His patience and advice are greatly appreciated. My sincere thanks go to the other members of my academic committee: Doctors Robert F. Langham and Glenn L. Waxler of the Department of Pathology and Dr. Gordon R. Carter of the Department of Microbiology and Public Health, for their support; and Dr. C. C. Morrill, former chairman of the Department of Pathology, for providing the facilities and supplies for this research. Dr. Glenn L. Waxler and Dr. David P. Olson, a colleague in the Department of Large Animal Surgery and Medicine and a fellow graduate student, also deserve special thanks for their advice and help with the gnotobiotic pigs and scanning electron microscopy. I also wish to thank Mrs. Helen L. Davidson, medical tech- nologist in the Department of Pathology, and Mr. William S. McAfee, supervisor of the Scanning Electron Microscope Laboratory, for the technical assistance which was so necessary to the completion of this investigation. I am also indebted to a number of other persons for their technical assistance: Mr. James Southern, animal caretaker at the Veterinary Research Farm; Mrs. Mae Sunderlin and Mrs. Frances ii Whipple, histopathologic technicians; and all members of the Department of Pathology. I particularly appreciate the patience and support I received from my wife, Jane, and our seven children during the course of these studies. iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . . . 3 Introduction. . . . . . . . . . . . . . . . . . . . . 3 Escherichia coli. . . . . . . . . . . . . . . . . . . 4 Clinical Signs. . . . . . . . . . . . . . . . . . . . 5 Gross Lesions . . . . . . . . . . . . . . . . . . . . 6 The Mucosa of the Intestine . . . . . . . . . . . . . 7 Histologic Lesions. . . . . . . . . . . . . . . . . . 9 Scanning Electron Microscopy. . . . . . . . . . . . . ll Scanning Electron Microscopic Description of the Intestinal Mucosa. . . . . . . . . . . . . . . 13 Summary . . . . . . . . . . . . . . . . . . . . . . . 13 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . 15 Experimental Animals. . . . . . . . . . . . . . . . . 15 Determination of Sterility. . . . . . . . . . . . . . l7 Infective Agent . . . . . . . . . . . . . . . . . . . 18 Necropsy Procedures . . . . . . . . . . . . . . . . . 20 Light Microscopy. . . . . . . . . . . . . . . . . . . 21 Scanning Electron Microscopy. . . . . . . . . . . . . 21 ESULTS O O O O O O O O O O O O O O O O O O O O O O O O O O O 25 Clinical Signs and Gross Lesions. . . . . . . . . . . 25 Scanning Electron and Light Microscopy. . . . . . . . 27 Stomach. . . . . . . . . . . . . . . . . . . . 27 Duodenum . . . . . . . . . . . . . . . . . . . 42 Anterior Jejunum . . . . . . . . . . . . . . . 42 Posterior Jejunum. . . . . . . . . . . . . . . 55 Ileum. . . . . . . . . . . . . . . . . . . . . 58 Cecum. . . . . . . . . . . . . . . . . . . . . 68 Colon. . . . . . . . . . . . . . . . . . . . . 68 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . 84 Clinical Signs and Gross Lesions. . . . . . . . . . . 84 Scanning Electron and Light Microscopy. . . . . . . . 85 iv SUMMARY. . . BIBLIOGRAPHY VITA . . . . Table LIST OF TABLES Page Number, sex, weight and grouping of pigs in experiment. . . . . . . . . . . . . . . . . . . . . . 16 Karnovsky's fixative. . . . . . . . . . . . . . . . . 22 Location of nuclei and size and distribution of vacuoles in the epithelial cells of the mucosa. . . . 86 vi LIST OF FIGURES Figure Page 1 109A (116367A) Photomicrograph of the pyloric portion of the stomach of a control gnotobiotic pig. Note the rugae (R), gastric pits (G) and mucosal extensions (M) between the gastric pits . . . 28 2 109A (103) Scanning electron micrograph of the stomach of a control gnotobiotic pig. These are the mucosal extensions (M) and gastric pits (G) of a single ruga. . . . . . . . . . . . . . . . . . . 29 3 109A (116367A) Photomicrograph of the stomach of a control gnotobiotic pig showing a portion of one ruga. The appearance seen here results from roughly parallel crevices (gastric pits) being sectioned at right angles to their long axes. . . . . 30 4 109A (104) Scanning electron micrograph of the pyloric portion of the stomach of a control gnotobiotic pig showing the ridge appearance of the mucosal extensions . . . . . . . . . . . . . . 31 5 109A (116367A) Photomicrograph of the pyloric portion of the stomach of a control gnotobiotic pig showing the tips of the mucosal extensions between the gastric pits. . . . . . . . . . . . . . . 32 6 109A (105) Scanning electron micrograph of the pyloric portion of the stomach from a control gnotobiotic pig. Compare Figure 6 with Figure 9. . . 33 7 110A (116368A) Photomicrograph of the pyloric portion of the stomach of a control gnotobiotic pig showing the normal gastric epithelium and gastric pits on a single ruga. Note the blunt mucosal extensions. Compare with Figure 3 and note the normal variation between pigs. . . . . . . . 34 8 110A (88) Scanning electron micrograph of the pyloric portion of the stomach of a control gnotobiotic pig. The material in the gastric pits is gelatin from the mounting procedure . . . . . 35 vii Figure 10 11 12 13 14 15 16 17 Page 110A (89) Scanning electron micrograph of the pyloric portion of the stomach of a control gnoto- biotic pig. There is gelatin in the gastric pits from the mounting procedure. Note the prominent cell outlines. These cells lack the hemispherical bulging appearance of cells seen in the epithelium from the stomachs of other piglets. Compare with Figure 6. . . . . . . . . . . . . . . . . . . . . . . . 36 202A (93) (36 hours after exposure) Scanning electron micrograph of the stomach of an infected pig showing a greater number of cells undergoing degeneration and extrusion than was seen in the control pigs (arrows) . . . . . . . . . . . . . . . . . 37 202A (94) (36 hours after exposure) Higher mag- nification of a portion of Figure 10. . . . . . . . . . 38 209A (106) (48 hours after exposure) Scanning electron micrograph of the stomach of an infected pig showing the smaller number of degenerating, bulging, hemispherical cells 48 hours after exposure than was seen in the pigs 36 hours after exposure. . . . . . . . . . . . . . . . . . . . . . . . 39 209A (109) (48 hours after exposure) Scanning electron micrograph of the stomach of the same infected pig as seen in Figure 12, but at higher magnification. . . . . . . . . . . . . . . . . . 40 202A (117666A) Photomicrograph of the pyloric portion of the stomach of a gnotobiotic pig 36 hours after exposure. Note the lymphocytes (L) and neutrophils (N) in the lamina propria . . . . . . . 41 209A (117673A) Photomicrograph of the pyloric portion of the stomach of a gnotobiotic pig 48 hours after exposure. There is a large collec- tion of neutrophils just below the epithelial surface of the mucosa (N) . . . . . . . . . . . . . . . 43 109B (117) (control) Scanning electron micro- graph of the duodenum from a control gnotobiotic pig. The extrusion zones (E), goblet cell orifices (G) and transverse furrows (T) are visible on the villi. . . . . . . . . . . . . . . . . . 44 109B (118) (control) Scanning electron micro- graph of the same villus as in Figure 16, but at higher magnification . . . . . . . . . . . . . . . . 45 viii Figure 18 19 20 21 22 23 24 25 Page 2098 (152) (48 hours after exposure) Scanning electron micrograph of the duodenum of an infected pig. Note the shortened villi and increased space between villi when compared with controls . . . . . . . 46 209B (153) (48 hours after exposure) Scanning electron micrograph of the duodenum from an infected pig. Note the extrusion zone (E) at the tip of the villus and the discharging goblet cell (G). . . . . 47 1098 (1163678) Photomicrograph of the duodenum of a control gnotobiotic pig. Note the transverse furrows (T) and the basal location of the nuclei in the epithelial cells (N) . . . . . . . . . . . . . . 48 2098 (1176738) Photomicrograph of the duodenum of a pig 48 hours after exposure. Note the slightly more vacuolated basal portion of the epithelial cells, the central location of the nuclei within the epithelial cells, the vacuoles beneath the basement membrane of the epithelium, and the large number of neutrophils (N) present in the lamina propria of the villus . . . . . . . . . . . . . . . . . 49 110C (3) (control) Scanning electron micrograph of the anterior jejunum of a control gnotobiotic pig 0 O O O O O O O O O O O O O O O O O C O O O O I O o 50 110C (4) (control) Scanning electron micrograph of the anterior jejunum of a control gnotobiotic pig. Note the long, fingerlike villus, the transverse furrows (T), the extrusion zone (B), the goblet cell openings (G) and the bulging hemispherical appearance of the individual cells (C) . . . . . . . . . . . . . . . . . . . . . . . . . . 51 109C (116367C) Photomicrograph of the anterior jejunum from a control gnotobiotic pig. Note the large number of vacuolated epithelial cells on the villi (V) and the change of position of the nucleus from basal or central to apical (N) when the cell becomes vacuolated. . . . . . . . . . . . 53 104C (116362C) Photomicrograph of the anterior jejunum of a pig 24 hours after exposure. Note the large vacuoles, pyknotic nuclei and cell damage with the loss of epithelial cells exposing the lamina propria at the villal tips . . . . . . . . . 54 ix Figure Page 26 101C (8) (16 hours after exposure) Scanning electron micrograph of the anterior jejunum of an infected pig. The loss of cells resulting in holes (H) in the epithelial surface may be an artifact. . . . . . . . . . . . . . . . . . . . . . . . 56 27 104C (10) (24 hours after exposure) Scanning electron micrograph of the anterior jejunum of the same infected pig as in Figure 25. Note the exposed lamina propria which is the result of the epithelial cells having sloughed off the tip of the villus. . . . . . . . . . . . . . . . . . . . . . . 57 28 109D (l4) Scanning electron micrograph of the posterior jejunum from a control gnotobiotic pig. There is some charging (distortion) in this photo- graph. The epithelial cells Of the villi are swollen, bulging and hemispherical in shape . . . . . . 59 29 109D (1163670) Photomicrograph of a single villus from the posterior jejunum of a control gnotobiotic pig. The apical nuclei (N), vacuola- tion and distention of the epithelial cells are clearly visible. The extrusion zone (E) at the tip of the villus is also visible . . . . . . . . . . . 60 30 109E (1163678) Photomicrograph of the ileum of a control gnotobiotic pig. The numbers of cells with vacuoles and the size of the vacuoles in the cells are both greater than they were in the epi- thelium of the villi of the anterior portion of the small intestine. Most of the nuclei (N) are basal in position within the cell . . . . . . . . . . . 61 31 109E (78) Scanning electron micrograph of the ileum of a control gnotobiotic pig. The epithelial cells of the villi are swollen and hemispherical in shape. A few of the cells near the extrusion zone at the tips of the villi appear collapsed (C). . . 62 32 101E (116359E) Photomicrograph of the tip of one villus from the ileum of a pig 16 hours after exposure. Markedly swollen cells (S), collapsed cells (C) and the increased width of the lamina propria (L) are visible here. . . . . . . . . . . . . . 63 33 101E (28) (16 hours after exposure) Scanning electron micrograph of the ileum exhibiting the crowding of villi, the swelling of the individual epithelial cells, and a number of collapsed epi- thelial cells . . . . . . . . . . . . . . . . . . . . . 64 Figure 34 35 36 37 38 39 40 41 Page 101E (27) (16 hours after exposure) Scanning electron micrograph of the ileum; greater magni- fication of the tip of the villus in Figure 33. . . . . 65 104E (ll6362E) Photomicrograph of a villus from the ileum of a pig 24 hours after exposure. Note the swelling of the individual cells (C), the large number of neutrophils (N), and the increased width of the lamina propria (L) . . . . . . . . . . . . 66 104E (39) (24 hours after exposure) Scanning electron micrograph of one villus from the ileum. This photograph is of a villus from the same pig as in Figure 35. The appearance is very similar to that of scanning electron microscopy of the ileum of control pigs. Compare with Figure 37. There is a loss of microvilli from the individual epithelial cells (C) closest to the extrusion zone (E) . . . . . . . . . . . . . . . . . . . . . . . . . . 67 201E (42) (36 hours after exposure) Scanning electron micrograph of the ileum. There are more collapsed cells than were seen in the controls; however, the presence of collapsed cells following infection was not a consistent finding. . . . . . . . . 69 201B (43) (36 hours after exposure) Scanning electron micrograph of a villus from the ileum. . . . . 70 201E (132) (36 hours after exposure) Scanning electron micrograph of the tip of a villus from the ileum. Note collapsed cells (C) and loss of microvilli (M). . . . . . . . . . . . . . . . . . . . . 71 201E (117665E) Photomicrograph of the tip of a villus from the ileum of a pig 36 hours after exposure. Note the vacuolation and swelling of the individual cells (S), the collapsed cells (C) near the tip of the villus, the exposed lamina propria (L) in the area of the extrusion zone, and the hypercellularity and concentration of nuclei (N) in the lamina propria of this area . . . . . 72 202B (44) (36 hours after exposure) Scanning electron micrograph of the tip of a villus from the ileum. There has been a greater loss of cells from the area surrounding the extrusion zone than seen in sections from control pigs. This has resulted in exposure of the lamina propria (L) at the tip of the villus. Compare with Figure 40. . . . . 73 xi Figure Page 42 201E (133) (36 hours after exposure) Scanning electron micrograph of the tip of a villus from the ileum of a pig infected with E. coli. None of the control pigs had this number of cells under- going necrobiosis at the tips of the villi. Note the loss of microvilli (M) on some cells. . . . . . . . 74 43 lllF (116369F) Photomicrograph of the mucosa of the cecum from a control gnotobiotic pig. . . . . . . . 75 44 llOF (62) Scanning electron micrograph of the cecum of a control gnotobiotic pig. The ridges of the mucosa are readily visualized. . . . . . . . . . 76 45 110F (63) Scanning electron micrograph of the cecum of a control gnotobiotic pig. The orifices of discharging goblet cells (G) are visible at this magnification . . . . . . . . . . . . . . . . . . . . . 77 46 1106 (1163686) Photomicrograph of the colon of a control gnotobiotic pig. The nuclei are basal in position in the epithelial cells, there are many goblet cells (G), and the mucosa is ridged in appearance between the crypts . . . . . . . . . . . . . 78 47 110G (69) Scanning electron micrograph of the colon of a control gnotobiotic pig. Note the ridge- shaped appearance of the mucosa . . . . . . . . . . . . 79 48 1106 (70) Scanning electron micrograph of the colon of a control gnotobiotic pig. The individual cell outlines (C) and the orifices of discharging goblet cells (G) are readily discernible. . . . . . . . 80 49 108G (116366G) Photomicrograph of the colon of a pig 8 hours after exposure. No changes were apparent as the result of the infection. Note the appearance of ridges upon folds of the mucosa in the colon of this pig . . . . . . . . . . . . . . . . . 81 50 1016 (71) (16 hours after exposure) Scanning electron micrograph of the colon of a pig. The appearance of ridges upon folds is a normal varia- tion of the ridge-shaped mucosa of the colon. . . . . . 82 xii INTRODUCTION Diarrheal disease is one of the most common causes of mor— bidity and mortality of man and higher animals throughout the world. Enteric colibacillosis caused by infection with Escherichia coli is a leading cause of diarrheal disease in man and animals. The ubiquitous nature of E. coli has contributed to diagnostic and research problems in determining the role of this organism in health and disease. The ability to differentiate the serotypes of E. coli, to differentiate the enteropathogenic from the nonenteropathogenic strains, to rear germfree or gnotobiotic animals, and to examine specimens by scanning electron and transmission electron microscopy has contributed to the understanding of this disease. This study utilized the gnotobiotic pig as a model to study the effect of a specific serotype of E. coli (0138:K81:NM) on the intestinal tract. The clinical signs exhibited and the changes seen with light microscopy and scanning electron microscopy were recorded. The objectives of this investigation were: 1. To depict the normal light and scanning electron microscopic appearance of the surface of the stomach, small intestine and large .intestine of the gnotobiotic pig. 2 2. To determine whether any changes of diagnostic significance occurred in or on the surface of the intestinal tract as a result of infection with E. coli. 3. To further elucidate the changes which can be seen with the light and scanning electron microscope in the intestinal tract of the gnotobiotic pig infected with E. coli. LITERATURE REVIEW Introduction Colibacillosis is one of the most serious causes of economic losses in newborn animals (Fey, 1972). In spite of the economic importance of colibacillosis, the actual yearly losses are not known. A survey of Michigan dairy herds revealed a calf mortality rate of 17.7% between birth and 60 days of age (Oxender et a1., 1973). A diagnosis of colibacillosis or infectious calf diarrhea was reported in 50% of the calves submitted to the Michigan State University Veterinary Diagnostic Laboratory for postmortem examina- tion. It has been reported that 15 to 25% of piglets born alive died before 8 weeks of age (Barnum et al., 1967). A very high percentage of these losses were attributed to colibacillosis. Illinois records indicate that 75 to 80% of the cases of baby pig diarrhea are diagnosed as colibacillosis (Leman, 1970). The greatest mortality of animals born alive occurs in the neo- natal period (Barnum et al., 1967). The disease syndromes desig- nated as enteric colibacillosis are among the most common infectious diseases of this age group. The term colibacillosis has been used to designate a group of diseases caused by infection with bacteria classified as Escherichia coli. The definition of this term has been broadened to include syndromes occurring in all animals. 4 The term colibacillosis is most often used to describe E. coli infection of the digestive system (Barnum et al., 1967). Colibacillosis of swine includes several disease syndromes: diarrheal disease of newborn pigs; syndromes resulting from septi- cemia and bacteremia in newborn pigs; edema disease; and coliform enteritis of weaned pigs. Enteric colibacillosis caused by entero- pathogenic E. coli is primarily manifested as diarrhea in neonatal to postweaning pigs (Dunne, 1970). Escherichia coli was first incriminated as a cause of diarrhea in young pigs in 1899. The most common definition of the patho- genesis of neonatal enteric colibacillosis involves a susceptible pig infected with an enteropathogenic strain of E. coli which has the capacity to proliferate in the proximal small intestine and to produce and release enterotoxins in adequate amounts to cause an alteration in the normal fluid and electrolyte transport functions of the intestine (Christie, 1969; Gyles, 1972). Escherichia coli Escherichia coli was first described in 1885 by, and later named after, Escherich. The early generic name for this organism, which still appears in some literature, was Bacterium coli. EScherichia coli was first associated with disease (calf scours) in 1891 by Jensen in Denmark. Jensen was also able to distinguish between pathogenic and nonpathogenic strains of E. coli. Gay (1965) divided the different syndromes associated with E. coli infection in the newborn calf into 3 groups based upon bacterio- logic, clinical and pathogenetic findings: 1) the coli-septicemic 5 form; 2) the enteric-toxemic form; and 3) the enteric form. Indi- viduals who would like a detailed description of E. coli including precise information about the cultural, biochemical and serological characteristics of this organism are referred to Identification of Enterobacteriaceae (Edwards and Ewing, 1972) and to the monograph pub- lished by Sojka (1965). Limited discussions of the toxic substances produced by E. coli (endotoxins, enterotoxins, colicines, hemolysins and edema disease principle) and the typing of E. coli (serotyping, phage typing, colicine typing and hemolysin typing) are presented by Fey (1972) and Barnum et a1. (1967). A description of drug resistance and the transfer of drug resistance may also be found (Fey, 1972). Most E. coli isolated can be grouped by serologic methods which identify the 0 group (cell wall antigens), K group (capsular antigens) and H group (flagellar antigens) (Kohler, 1972). Clinical Signs Colibacillosis of the newborn pig is most frequently seen as a diarrheal disease occurring primarily during the first week of life. The disease may occur only sporadically on a given farm or it may be enzootic. Morbidity ranges from zero to one hundred percent. Not all litters farrowed during an enzootic are affected, and all pigs in one litter may not be affected. The percentage of affected pigs that recover depends upon the severity of the diarrhea and the success of therapy employed. Recovery or death may occur up to several days after the onset of diarrhea, and mortality may range from zero to one hundred percent (Leman, 1970; Barnum, 1971). 6 The first sign usually noted is a whitish or yellowish fluid diarrhea. As the clinical signs progress the piglets become dehydrated and emaciated and develop a roughened hair coat. The posterior portions of the animal often become pasted with feces. The tail may become necrotic and slough. Younger pigs, especially during the first 3 days of life, are more likely to become moribund and die than older pigs (Kohler, 1966; Stevens, 1972). Gross Lesions The gross lesions of enteric colibacillosis have been recorded and reviewed by Barnum et a1. (1967). Dehydration, pasting of the rear quarters with feces and distention of the intestine with fluid are characteristic of enteric colibacillosis in piglets. The stomach often appears normal and is filled with coagulated milk. The small intestine is distended with fluid, mucus, gas and particles of coagulated milk; the large intestine is also distended with gas and fluid. In some cases fluid contents and distention of the spiral colon may be the only intestinal abnormalities noted. No congestion or other evidence of inflammation is usually noted if the animals are examined immediately after death. The clinical signs of the disease produced by oral exposure of gnotobiotic neonatal pigs to E. coli 0138:K81:NM closely resemble the clinical syndrome as it is described in the literature (Christie, 1969). The time elapsed between death and postmortem examination may influence the gross lesions at necropsy (Cross and Kohler, 1969). Kohler and Bohl (1966) reported no lesions in the small and large intestines of gnotobiotic pigs examined immediately 7 after death while lesions suggestive of enteritis in the jejunum and ileum and congestion in many of the other abdominal organs were seen in pigs dead for 4 to 12 hours prior to necropsy. The Mucosa of the Intestine The small intestine is the main organ for absorption of dietary nutrients. The villus and the epithelial cells on the surface of the villus constitute the functional absorptive unit (Kenworthy, 1967). The mucosa of the tubular crypt is continuous with that of the villus. The integrity of the villus is essential to the normal function of the intestinal tract. Intestinal epi- thelial cells in general have a very fast turnover rate (1 to 6 days). The whole epithelial lining is renewed every few days as the result of rapid cell replacement (Leblond, 1958; Moon, 1971). The crypt epithelium has the specialized function of cell division, so that as cells are lost from the extrusion zone of the villus they are replaced by continual movement of epithelial cells from the crypt. The epithelial cells arise from the repeated division of relatively undifferentiated cells in the crypts of Lieberkfihn and, once division has taken place, differentiation is complete relative to eventual function (Kenworthy, 1967). The crypts are almost solely for epithelial cell production while the villi provide a large surface area almost wholly for absorption. The cells lost from the tips of the villi disintegrate rapidly so that they are rarely recognizable in the lumen of the small intestine (Moon, 1971). 8 The lamina propria of the villus is covered with simple columnar epithelium bearing a discernible brush border of microvilli. In the pig the brush border of the epithelial cells in the duodenum and jejunum appears to be more pronounced than it is on epithelial cells of the ileum. Goblet cells are interspersed among the epithelial cells on the villus. There are increasing numbers of goblet cells in the more distal portions of the small intestine. The epithelium of the villus rests on a basement membrane. The lamina propria of the villus consists of connective tissue and capillaries, lymphatic vessels, a central chyle vessel termed a lacteal, contractile fibers termed Breucke fibers, and variable numbers of reticuloendothelial cells and lymphocytes (Ham, 1965). The villi are longer and the crypts are shorter in gnotobiotic pigs than in naturally raised pigs (Christie, 1967; Moon, 1970). Large intracytoplasmic vacuoles are present to a considerable extent in the intestinal mucosa of germfree and monocontaminated neonatal pigs (Moon, 1973). This vacuolation is more marked in infected pigs and is more common in sections taken from more caudal sites in the small intestine (Christie, 1967). These vacuoles have been described as the result of either normal intestinal absorption or of necrobiosis in the germfree pig. The more marked vacuoliza- tion seen in pigs monocontaminated with E. coli has been suggested to bethe result of absorption vacuolization, necrobiosis or hydropic degeneration associated with the toxic activities of the enteric organisms (Christie, 1967). Christie (1969) was relatively unsuccessful in attempts to determine by histochemical means the 9 nature of the contents of these vacuoles. He concluded that the fluid contents of the vacuoles were of a serous nature. Histologic Lesions Early workers concluded that diarrhea occurs in E. coli infected pigs without morphologic evidence of enteritis. However, Christie (1967) described histologic changes found in the gastro- intestinal tract. The lesions varied from acute hemorrhagic and necrotic enteritis to a histologic picture consistent with that observed in normal gnotobiotic pigs. The most frequent change was hydropic degeneration in the epithelium of the villi. Subepithelial edema of the villi and neutrophilic infiltration of the lamina propria were also observed. Kohler (1967) reported a mild neutro- philic infiltration of the lamina propria in the duodenum of pigs infected with either enteropathogenic or nonenteropathogenic E. coli. He suggested this reaction was the normal host defense response to the presence of bacteria. Drees (1969) reported considerable histologic variation in the epithelial cells in the different regions of the small intestine. The epithelial cells in the duodenum were columnar and the cyto- plasm rarely contained visible vacuoles. The nuclei were centrally located. The jejunal epithelial cells were similar morphologically, but a few contained large apical cytoplasmic vacuoles which appeared to have pushed the nucleus to the base of the cell. The ileum con- tained a larger number of these vacuolated cells and the number increased as the pigs aged. The ileal epithelial cells were almost 10 all vacuolated in many pigs by 48 hours after birth. Histologic changes were first observed in pigs which had been infected for 10 hours or longer. The most consistent change was edema of the lamina propria and dilatation of the central lacteal near the tips of the villi. Occasional neutrophilic infiltration of the lamina propria of the villi was observed. These changes were most prominent in the tips of the villi in the posterior jejunum and ileum. Moon (1972, 1973) has also reported the vacuolated villous epithelium of the small intestine of young pigs. Drees (1969) concluded that E. coli does not have to invade the intestinal epithelium to cause diarrhea. He reported that the organism rarely invaded the epithelium of the duodenum and invaded the epithelium with increasing frequency at more distal levels of the intestine. Invasion of the epithelium occurred only in the upper third of the villi and was much more frequent in pigs infected at an early age. Christie (1967) reported that the clinical signs of profuse watery diarrhea, dehydration and erect hair coat char— acteristic of colibacillosis in the neonatal pig were not apparent until the organism became well established in the gastrointestinal tract. From histologic studies made with E. coli 0138:K81:NM it is clear that there are inflammatory changes associated with coli— bacillosis although these changes may be inadequate to be readily apparent in tissues taken from field cases of the disease (Christie, 1967; Christie, 1969). There are no lesions pathognomonic of the infection which may be seen at the light microscope level. 11 Scanning Electron Microscopy The scanning electron microscope (SEM) is designed primarily for visualization of the surface structure of an object (Pease, 1971). The scanning electron microscope has been used in the past primarily for the investigation of hard tissues. Recently, interest has centered on the value of this technique in the study of soft tissues (Toner and Carr, 1969). The scanning electron microscope allows the three-dimensional character of histologic tissue struc- tures to be visualized; this represents the electron optical advance of the dissecting microscope (Demling et al., 1968). Increased depth of focus, better resolution and greater magnification have been made possible by scanning electron microscopy (Asquith et al., 1970). The principles of the technique of scanning electron microscopy were known in the 19305, but the first commercial scanning electron microscope did not become available until 1965 (Kavin et al., 1970). The scanning electron microscope differs in principle from the con— ventional transmission electron microscope. In the conventional transmission electron microscope the electron beam passes through the specimen and it is the scattering of these electrons as they pass through the tissue that produces the image. Although the scanning electron microscope can be used to examine sectioned and freeze-fractured specimens to reveal internal details of tissue organization, it is perhaps best suited to the study of natural surfaces. The mechanical principle of the scanning electron micro- scope involves an electron beam emitted from a hot tungsten filament 12 in a conventional electron gun. The beam is reduced in size and focused by a series of electromagnetic lenses into a very fine probe. As the beam scans the surface of the specimen, secondary electrons are emitted and accelerated into a collector. The secondary electrons are then accelerated into a cathode-ray tube. By switching over to a second cathode-ray tube the image can be recorded as points of lines on photographic film. Since the depth of focus of the SEM is many times that of the optical microscope, accurate focusing can be obtained upon all parts of the specimen at the same time. The three-dimensional effect of the image is caused by the bright-dark contrast. The brightness of the image depends upon the angle at which the primary beam strikes the specimen. If the beam grazes the objective, the number of secondary electrons and thus the brightness is high. If the primary beam is Perpendicularly incident or if the distance is large, the displayed POints are darker (Demling et al., 1969). By turning and tilting the specimen it can be examined from all sides. Magnifications of 20x to 100,000x are possible. The preparation of intestinal specimens for scanning electron microscopy has been described by Toner and Carr (1969) , Demling et a1 . (1969), Kavin et a1. (1970) , Asquith et al. (1970) , Waxler (1972) and Olson et al. (1973) . Animal tissues present a number of Problems not encountered in the investigation of hard tissues. All Of the methods of specimen preparation include a controlled fixa- tion procedure, complete drying and coating with a thin layer of vaporized metal . l3 Scanning Electron Microscopic Description of the Intestinal Mucosa The ultrastructural surface of the small intestine of the young pig has been described by Waxler (1972), Olson (1972), Olson et a1. (1973) and Spotts (1974). They described long, fingerlike villi of varying length and configurations projecting from the basilar crypt area. The surface of each villus was interrupted by irregular trans— verse furrows of varying depth and the openings of numerous goblet cells. The cleft forming the extrusion zone at the tip of each villus was also described. The surface of the villi in the lower jejunum and ileum was not flat and smooth but was interrupted by many hemispherical projections. These projections were caused by the outward protrusion of the luminal surface of the absorptive cells (Mouwen, 1971). Summary Enteric colibacillosis continues to be a major cause of illness and death among neonatal animals. Research has elucidated means of determining enteropathogenic and nonenteropathogenic strains of E. coli and the means to serotype and identify these strains. It is believed that an enteropathogenic strain of E. coli must colonize the intestinal tract in large numbers to produce illness. However, the precise effect of the organism or toxin upon the digestive tract and the exact nature of the cellular changes which take place are still to be determined. The diagnosis of enteric colibacillosis is most often based upon clinical signs and the recovery of E. coli in almost pure culture from the anterior small intestine. Gross and histopathologic l4 lesions are not of diagnostic significance. Researchers have indi- cated the usefulness of scanning electron microscopy in the study of the intestinal tract. Gnotobiotic techniques and the scanning electron microscope may be useful in studying changes in the intestinal tract as the result of enteric colibacillosis. MATERIALS AND METHODS Experimental Animals Twenty-one gnotobiotic pigs were delivered from 2 crossbred sows by hysterotomy into sterile plastic isolators by the technique described by Waxler et al. (1966). Surgery was performed on the 112th day of gestation. Epidural anesthesia was accomplished with 25 ml of 2.5% procaine hydrochloridea injected into the epidural space at the lumbosacral articulation (Getty, 1963). Two hundred fifty milligrams of promazine hydrochlorideb was administered to each sow as presurgical tranquilization. Surgery was performed in the left flank area. The initial incision through the bottom layer of plastic of the surgical isolator and skin of the sow was made with an electric cautery unit. As the pigs were removed from the uterus, self-locking clampsc were placed on the umbilical stump to control hemorrhage. Eighteen of the 21 pigs delivered survived and were used in this study. Table 1 lists information concerning the experimental and control animals, including number, sex, weight, and group. a . . . . Epidural, Haver Lockhart Company, Kansas City, Missouri. bSparine, Wyeth Laboratories, Philadelphia, Pennsylvania. C"Double-Grip" Disposable Cord Clamp, Hollister, Inc., Chicago, Illinois. 15 16 Table 1. Number, sex, weight and grouping of pigs in experiment Pig Initial wt. Time after exposure no. Sex (lbs.) to euthanasia (hrs.) Status 101 F 3.5 16 exposed 102 F 3.0 24 exposed 103 M 3.0 16 exposed 104 M 4.0 24 exposed 105 F 4.0 4 exposed 106 F 4.0 8 exposed 107 M 4.0 4 exposed 108 M 3.0 8 exposed 109 F 2.0 8 control 110 M 3.0 24 control 111 M 1.5 16 control 201 F 2.6 36 exposed 202 F 3.0 36 exposed 203 F 2.6 —- died 204 M 1.6 24 control 205 F 2.6 48 control 206 M 2.4 -- died 207 F 2.9 16 exposed 208 M 2.4 24 exposed 209 M 3.5 48 exposed 210 F 3.1 -- died 17 The sterile plastic isolator containing the newborn pigs was moved to the rearing room as soon as possible after surgery. The pigs were aseptically transferred into plastic rearing isolators. Each pig was placed into an individual cage within the plastic isolator such that no isolator contained more than 4 pigs. Each isolator also contained the equipment for feeding, inoculation and sample collection. The temperature of the rearing units was main- tained at 37 C for the first 3 days, then gradually reduced to 29 C. All pigs were fed a sterile semisynthetic dieta and observed 3 times each day. The pigs were taught to drink from pans and fed 3 ounces of the liquid diet at each feeding the first day, then fed 4 ounces 3 times daily beginning the second day. Determination of Sterility Composite specimens of fecal and waste material were collected from the excrement trays beneath each rearing cage in each isolator with sterile swabs. Sterile swabs were also used to collect a rectal specimen from each pig before it was inoculated with E. coli and again prior to its removal from the isolator. Material was streaked on tryptose blood agarb plates and inoculated into thio- glycollate medium.C The media were incubated aerobically and anaerobically at room temperature, 37 C and 56 C. Material was aSPF-Lac, Borden Company, New York, N.Y. bTryptose Blood Agar Base, Difco Laboratories, Detroit, Michigan; Defibrinated Sheep Red Cells, Colorado Serum Company, Denver, Colorado. CBactofluid Thioglycollate Medium, Difco Laboratories, Detroit, Michigan. 18 also inoculated into PPLO brotha and incubated aerobically at 37 C for 5 days. A blind passage was made from this broth onto a PPLO plate and into another PPLO broth and incubated for an additional 5 days. This procedure was repeated twice. All media were observed for 3 weeks. The organisms isolated from the fecal samples (rectal swabs) from the individual pigs were further examined by the follow- ing biochemical tests: triple sugar iron (TSI) agar, indol test, methyl red test, Voges-Proskauer test, Simmons citrate agar, and motility test. Infective Agent The organism used in all experiments was E. coli 0138:K81:NM which had been isolated from experimental pigs at Michigan State University and lyophilized in glass vials. The contents of each vial were inoculated into 7 m1 of liquid brain-heart infusion medium (BHI broth)a and 0.1 ml of this suspension was transferred to a second tube of 7 m1 of BHI broth. MacConkey's agar and blood agar plates were streaked at the same time. All culture media were incubated at 37 C for 16 hours. The BHI broth cultures were washed twice with sterile 0.85% NaCl solution. The cultures were centrifuged for 20 minutes using a relative centrifugal force (g-force) of 1440 and the BHI broth was removed aseptically with a pasteur pipette. The sediment was resuspended in 10 ml of sterile 0.85% NaCl solution and mixed with a Vortex mixer. This procedure was repeated a second time. a . . . . . Difco Laboratories, DetrOit, Michigan. 19 Following the second washing, the bacteria-saline suspension was compared with McFarland nephalometer tubes. The suspension matched tube number 6 in this series (approximately 1.8 billion organisms per milliliter). In order to obtain a 1:600 dilution of the suspension, 0.1 ml of the suspension was added to 9.9 m1 of sterile 0.85% NaCl solution, then 1.0 ml of this dilution (1:100) was added to 5.0 m1 of sterile 0.85% NaCl solution, resulting in a 1:600 dilution or an estimated 3 x 106 organisms per milliliter of solution. MacConkey agar and blood agar plates were streaked from the 1:100 dilution vial. Gram-stained slides were also prepared from this dilution. Five milliliter quanta of the 1:600 dilution were heat sealed in ampules. Five milliliter quanta of sterile 0.85% NaCl solution were heat sealed in sterile ampules to be used as inoculum for the con- trol pigs. One ampule of E. coli inoculum was saved to verify the dilution by a plate count. The pour plate method was used in this experiment. The infective dose used in this experiment based on the plate count was 1.6 x 106 colony forming units. The swabs for culture were removed from the isolators and the ampules for inoculation were sprayed with peracetic acid and trans- ferred into the isolators. One milliliter of the inoculum was administered orally to the pigs 3 hours following the removal of the cultures from the incubator. A disposable plastic syringe was used to deposit the inoculum on the poasterior aspect of the tongue. 20 Necropsprrocedures The pigs were euthanatized at the selected intervals by the injection of 1.5 ml of sodium pentobarbitala intravenously into the anterior vena cava. The intervals between the time of exposure and euthanasia of the pigs are given in Table l. The pigs were immedi- ately exsanguinated and then placed in dorsal recumbency and the sternum and ventral abdominal wall incised and reflected caudally exposing the abdominal viscera. The intestinal tract was removed by severing it at the cardia of the stomach and between the rectum and spiral colon and then incising along the mesenteric attachment. The intestine was placed on a table with the small intestine placed in a Z pattern so that the 3 sections were approximately of equal length. Care was taken not to stretch or crush the intestine. Two adjacent full thickness sections approximately 2 cm long were removed at each of 7 locations for scanning electron and light microscopy. The sections were removed from: 1) the pyloric por- tion of the stomach; 2) the duodenum 5 cm posterior to the pylorus; 3) one-third of the distance from the pylorus to the ileocecal valve (anterior jejunum); 4) two-thirds of the distance from the pylorus to the ileocecal valve (posterior jejunum); 5) ileum 5 cm anterior to the ileocecal junction; 6) cecum; and 7) spiral colon. The time interval between euthanasia and complete removal of all sections did notexceed 20 minutes. One of the paired sections from each area of the intestinal tract was placed in 10% formalin-sodium acetate solution for aHaver Lockhart Laboratories, Kansas City, Missouri. 21 histopathologic examination. The other section was fixed for scanning electron microscopy. The intestine was cut along the mesenteric border to expose the mucosal surface. Each section was fastened to a styrofoam block, mucosal surface uppermost, with 6 to 8 stainless steel pins. The mucosal surface was gently washed with 0.85% NaCl solution at room temperature. The styrofoam blocks were then floated with the tissue down in Karnovsky's fixative until dehydration procedures were started (Table 2) (Karnovsky, 1965). Light Microscopy The fixed tissues were embedded in paraffin and stained with hematoxylin and eosin (H&E) according to established procedures (Luna, 1968). Scanning Electron Microscopy Whole thickness sections approximately 1.0 cm in diameter were cut from the fixed intestine and attached to roughened aluminum discs with a small drop of 10% gelatina (Nowell et al., 1970). The mounted tissues were transferred into Karnovsky's fixative for storage. Sections were dehydrated by placing them for 1 hour each in a series of 25, 40, 60, 80, 90, 95, 100 and 100% ethanol solutions, followed by a final 100% ethanol solution for 12 hours. The ethanol was replaced with amyl acetate by placing the tissues in solutions containing 2 parts ethanol to 1 part amyl acetate, 1 part ethanol to 1 part amyl acetate, 1 part ethanol to 2 parts amyl acetate, and 2 solutions of 100% amyl acetate. The tissues aDifco Laboratories, Detroit, Michigan. 22 Table 2. Karnovsky's fixative The phosphate buffer (pH 7.2): 0.2M NAZI-{PO4 78 ml 0.2M KHZPO4 28 m1 Modified Karnovsky's fixative: Paraformaldehyde 40 gm Distilled water 400 m1 Phosphate buffer (pH 7.2) 300 ml The paraformaldehyde, distilled water and phosphate buffer are heated to 70 C in an Erlenmeyer flask on a heated magnetic stirring unit. Stirring is continued until the solution is almost clear (it will have a milky appearance; it does not com- pletely dissolve). It is then cooled to room temperature, allowed to stand overnight and filtered. Add: 25% glutaraldehyde 200 m1 Phosphate buffer 100 ml Dilute 1:4.5: Stock fixative 1000 m1 Distilled water 1000 ml Phosphate buffer 3500 ml Adjust to pH 7.2 (:_0.1) with l N HCl. Store at 4 C. Use at room temperature; filter before use. 23 were held for 1 hour in each solution with agitation on a mechanical shakera at low speed. The final 100% amyl acetate solution was used as tissue storage prior to drying. A critical point drying apparatus was used to dry all tissue sections (Nemanic, 1972). The procedure of Anderson (1951) was followed except that it was necessary to wrap each specimen in lens paper to prevent foreign material contained in the CO from being 2 deposited upon the tissues. Liquid carbon dioxide, at not less than 850 psi, was released into the drying chamber for 15 minutes to exchange and bathe the tissues. Displaced amyl acetate and CO2 were then allowed to escape for 15 minutes. This procedure was continued at 15-minute intervals for 3 hours, or approximately twice the time required for the amyl acetate odor to disappear. Dried tissue specimens were stored prior to scanning electron microscopy under vacuum in a desiccator containing anhydrous calcium sulfate. The aluminum discs on which the tissues were mounted were then glued to aluminum stub specimen holdersb with a conducting adhesive.c O I O O I O 0 d C The sections were indiVidually coated in a vacuum mini-coater with 400 A of gold. The sections were viewed with the scanning electron a . . . Eberback Corporation, Ann Arbor, Michigan. bErnest F. Fullam, Schenectaday, New York. CTelevision Tube Koat, G. C. Electronics Division, Hydrometals, Inc., Rockford, Illinois. dFill-Vac, Englewood, New Jersey. 24 microscopea using beam-accelerating voltages of 6, 10 or 21 kv at magnifications ranging from lOOX to 5000X. Scanning electron micro- graphs were made on black and white film.b aModel AMR-900, American Metals Research Corporation, Burlington, Massachusetts. bPolaroid 4X5 Land Film, Type 55 P/N Polaroid Corp., Cambridge, Massachusetts. RESULTS Clinical Signs and Gross Lesions All pigs remained active, behaved normally, and ate well throughout the experiment. The bowel movements of the control pigs remained semisolid and normal. On postmortem examination, the stomachs contained normal curds. The small intestine generally contained relatively little fluid or ingesta and almost no gas. The contents of the large intestine were semisolid. There was no fluid or gas accumulation and no distention of the intestinal tract. Four hours after exposure no pigs were visibly ill and there was no visible diarrhea when the pigs were removed from the isolator. On postmortem examination there was a slight distention of the small intestine and early indications of slightly more fluid feces in the large intestine. Eight hours after exposure the pigs had a small amount of pasty material around their hindquarters. 0n postmortem examination there were minimal changes; they were similar to the controls except for slightly more dilatation and fluid in the cecum and colon. Twelve hours after exposure the pigs had an obvious scouring condition, flushed skin and erect hair coats. On postmortem exami- nation the stomach contained a normal curd and the small intestine 25 26 appeared normal, but the contents were watery. The colon and cecum were dilated with gas and fluid. Sixteen hours after exposure the pigs had diarrhea. On postmortem examination the stomach contents were normal and the small intestine contained a large quantity of fluid, but no gas. There was some gas in the spiral colon. Twenty-four hours after exposure the pigs had diarrhea with pasty material adhered to their posterior quarters. Hair coats were erect and rough. On postmortem examination there was a normal curd in the stomach and the small intestine looked normal and contained little fluid or ingesta. There was distention of the cecum and colon with gas and fluid. Thirty-six hours after exposure the pigs had diarrhea. On post- mortem examination the stomach contents appeared normal; however, the stomach was distended with some gas. The anterior small intestine appeared normal without distention, but the posterior one-half of the small intestine was dilated with fluid, although it contained relatively little gas. Forty-eight hours after exposure the remaining pig was scouring; however, the diarrhea was less profuse than the diarrhea seen in pigs 36 hours after exposure. There was dilatation of the posterior one-half of the small intestine with fluid and the large intestine and cecum contained fluid and gas; however, there was less fluid and gas than in the pigs 36 hours after exposure. 27 Scanning Electron and Light Microscopy Stomach The appearance of the pyloric portion of the stomach when viewed by scanning electron microscopy was both interesting and unexpected. The surface of the gastric mucosa presented an irregular pattern of ridges and furrows of varying depths. In retrospect, the appearance of the rugae, gastric pits and mucosal extensions between the gastric pits correlated very well with the appearance of the gastric mucosa by light microscopy (Figures 1 through 6). Considerable normal variation existed among control pigs. In some pigs the mucosal extensions between the gastric pits were ridge-shaped and lacked the hemispherical bulging cells on the top of the ridges (Figures 7 through 9). No diagnostic changes were visible on the mucosa of the stomach upon scanning electron microscopy. It did appear that more indi- vidual cell damage occurred at intervals longer than 16 hours after exposure. Cell damage was most severe 36 hours after exposure (Figures 10 and 11). However, this was only slightly more severe than the normal degeneration and extrusion of cells seen in some of the control pigs. By 48 hours after exposure few degenerating or bulging hemispherical cells were visible (Figures 12 and 13). Histopathological changes were noted in the tissues with light microscopy. Changes were first noted 36 hours after exposure and consisted of a few isolated neutrophils and lymphocytes in the lamina propria of the mucosa (Figure 14). By 48 hours after exposure there were several accumulations of a few leukocytes in the lamina propria. 28 Figure 1. 109A (116367A) Photomicrograph of the pyloric portion of the stomach of a control gnotobiotic pig. Note the rugae (R), gastric pits (G) and mucosal extensions (M) between the gastric pits. HsE stain; x 50. 29 Figure 2. 109A (103) Scanning electron micro- graph of the stomach of a control gnotobiotic pig. These are the mucosal extensions (M) and gastric pits (G) of a single ruga. x 200. Fr" 30 Figure 3. 109A (116367A) Photomicrograph of the stomach of a control gnotobiotic pig showing a portion of one ruga. The appearance seen here results from roughly parallel crevices (gastric pits) being sectioned at right angles to their long axes. H&E stain; x 125. 31 Figure 4. 109A (104) Scanning electron micro- graph of the pyloric portion of the stomach of a control gnotobiotic pig showing the ridge appearance of the mucosal extensions. x 500. 32 Figure 5. 109A (116367A) Photomicrograph of the pyloric portion of the stomach of a control gnoto- biotic pig showing the tips of the mucosal extensions between the gastric pits. H&E stain; x 500. 33 Figure 6. 109A (105) Scanning electron micro- graph of the pyloric portion of the stomach from a control gnotobiotic pig. Compare Figure 6 with Figure 9. x 1000. 34 Figure 7. 110A (116368A) Photomicrograph of the pyloric portion of the stomach of a control gnotobiotic pig showing the normal gastric epithelium and gastric pits on a single ruga. Note the blunt mucosal extensions. Compare with Figure 3 and note the normal variation between pigs. H&E stain; x 125. _h-I_u A! l—JT‘FJ 3S Figure 8. 110A (88) Scanning electron micro- graph of the pyloric portion of the stomach of a control gnotobiotic pig. The material in the gastric pits is gelatin from the mounting procedure. x 200. 36 Figure 9. 110A (89) Scanning electron micro- graph of the pyloric portion of the stomach of a control gnotobiotic pig. There is gelatin in the gastric pits from the mounting procedure. Note the prominent cell outlines. These cells lack the hemispherical bulging appearance of cells seen in the epithelium from the stomachs of other piglets. Compare with Figure 6. x 1000. .—-_,- ._-_ .. A 37 Figure 10. 202A (93) (36 hours after exposure) Scanning electron micrograph of the stomach of an infected pig showing a greater number of cells under- going degeneration and extrusion than was seen in the control pigs (arrows). x 200. 38 l D I tmuln \ . \ )~‘,§.u'.l’0‘ (36 hours after exposure) 202A (94) Higher magnification of a portion of Figure 10 Figure 11. x 500. 39 Figure 12. 209A (106) (48 hours after exposure) Scanning electron micrograph of the stomach of an infected pig showing the smaller number of degenerat- ing, bulging, hemispherical cells 48 hours after exposure than was seen in the pigs 36 hours after exposure. x 200. 4O Figure 13. 209A (109) (48 hours after exposure) Scanning electron micrograph of the stomach of the same infected pig as seen in Figure 12, but at higher magnification. x 2000. 41 Figure 14. 202A (117666A) Photomicrograph of the pyloric portion of the stomach of a gnotobiotic pig 36 hours after exposure. Note the lymphocytes (L) and neutrophils (N) in the lamina propria. H&E stain; x 500. 42 There were a few accumulations of neutrophils just below the surface of the gastric epithelium (Figure 15). The epithelial surface was still intact above these accumulations of neutrophils and no erosions were seen. These areas were not possible to identify by scanning electron microscopy. Duodenum There were no consistent changes visible in the duodenum upon scanning electron microscopy. In some pigs there did appear to be a shortening of villi and an increased space between villi when the sections were observed by scanning electron microscopy (Figures 16 through 19). Most of the nuclei in the epithelial cells of the duodenum were basal or central in position. and when large vacuoles occurred in these cells, they occurred at a position apical to the nucleus (Figure 20). No histopathologic changes were noted until 36 hours after exposure. lu:that time a few neutrophils were seen at the bases of villi and in the lamina propria of an occasional villus. There were no changes in the epithelial cells. Forty-eight hours after exposure there were numerous neutrophils in the lamina propria of villi (Figure 21). Anterior Jejunum The villi of the anterior jejunum were long fingerlike pro- jections. The cell outlines were more distinct than in the duodenum, and the individual cells had a bulging hemispherical appearance (Figures 22 and 23). 43 Figure 15. 209A (117673A) Photomicrograph of the pyloric portion of the stomach of a gnotobiotic pig 48 hours after exposure. There is a large collec- tion of neutrophils just below the epithelial surface of the mucosa (N). H&E stain; x 500. 44 Figure 16. 109B (117) (control) Scanning electron micrograph of the duodenum from a control gnotobiotic pig. The extrusion zones (E), goblet cell orifices (G) and transverse furrows (T) are visible on the villi. x 500. 45 Figure 17. 109B (118) (control) Scanning electron micrograph of the same villus as in Figure 16, but at higher magnification. x 1000. 46 ; a . . ‘ J J ,I if! Q! :2 3 I . Figure 18. 209B (152) (48 hours after exposure) Scanning electron micrograph of the duodenum of an infected pig. Note the shortened villi and increased space between villi when compared with controls. x 500. .. .. a...“ -mmomurgrmgx. - . 47 Figure 19. 209B (153) (48 hours after exposure) Scanning electron micrograph of the duodenum from an infected pig. Note the extrusion zone (E) at the tip of the villus and the discharging goblet cell (6). x 1000. 48 ‘ '2 !,b- “q .. ' w'“' "O' ‘1 ‘Nfiy,,\ Figure 20. 1098 (1163678) Photomicrograph of the duodenum of a control gnotobiotic pig. Note the transverse furrows (T) and the basal location of the nuclei in the epithelial cells (N). H&E stain; x 125. 49 Figure 21. 2098 (1176738) Photomicrograph of the duodenum of a pig 48 hours after exposure. Note the slightly more vacuolated basal portion of the epithelial cells, the central location of the nuclei within the epithelial cells, the vacuoles beneath the basement membrane of the epithelium, and the large number of neutrophils (N) present in the lamina propria of the villus. H&E stain; x 500. 50 Figure 22. 110C (3) (control) Scanning elec- tron micrograph of the anterior jejunum of a control gnotobiotic pig. x 200. 51 Figure 23. 110C (4) (control) Scanning elec- tron micrograph of the anterior jejunum of a control gnotobiotic pig. Note the long, fingerlike villus, the transverse furrows (T), the extrusion zone (E), the goblet cell openings (6) and the bulging hemispheri- cal appearance of the individual cells (C). x 500. 52 The light microscopic examination of sections from the anterior jejunum were of interest primarily because of the change in location of the nuclei in the epithelial cells. As the cells progressed towards the tip of the villi, they became more vacuolated. When vacuoles appeared in these cells, the location of the nucleus changed from basal or central in position within the cell to apical in position. This change occurred in most cells, but not all cells. The nuclei in the epithelial cells on the distal portion of the villi were almost all apical in position in both the anterior and posterior jejunum. This was not true in the duodenum and was true to a much lesser extent in the ileum. This was characteristic of all of the gnotobiotic pigs in this study in all portions of the jejunum (Figure 24). The vacuolation occurred on the upper one-half of the villi in the anterior jejunum in the control gnotobiotic pigs. In the infected pigs there was greater vacuolation and it occurred in the cells on the upper three-quarters of the villi. With increasing numbers of hours after exposure the vacuoles became larger, pyknotic nuclei appeared and cell damage was more apparent (Figure 25). By 16 hours after exposure there were a few neutrophils in the lamina propria of the villi and there appeared to be some disruption of cells and necrobiosis of cells at the tips of the villi. Twenty—four hours after exposure there were a few neutrophils at the bases of villi and in the lamina propria of the villi. The increased vacuolation, destruction, death and sloughing of cells seen with light microscopy and scanning electron microscopy appeared to be most severe 24 53 Figure 24. 109C (116367C) Photomicrograph of the anterior jejunum from a control gnotobiotic pig. Note the large number of vacuolated epithelial cells on the villi (V) and the change of position of the nucleus from basal or central to apical (N) when the cell becomes vacuolated. H&E stain; x 125. 54 Figure 25. 104C (116362C) Photomicrograph of the anterior jejunum of a pig 24 hours after exposure. Note the large vacuoles, pyknotic nuclei and cell damage with the loss of epithelial cells exposing the lamina propria at the villal tips. H&E stain; x 125. 55 hours after exposure. The changes were still apparent 36 and 48 hours after exposure, but appeared to be less severe. There were 2 changes noted on scanning electron microscopy that might be classified as artifacts. These changes were not seen in all pigs, but rather each change was seen in only 1 pig. In 1 pig 16 hours after exposure there were a number of cells on the villi which had apparently degenerated to the point that the entire cell had sloughed into the lumen of the intestine. However, the other cells around these cells had remained intact resulting in distinct holes in the epithelium of the villi (Figure 26). Several areas of the anterior jejunum from 1 pig 24 hours after exposure contained villi which had lost the epithelial cells from the tips of the villi. The lamina propria was exposed and pro- truded above the remaining epithelial cells (Figures 25 and 27). Posterior Jejunum Scanning electron microscopy of sections of the posterior jejunum from control pigs revealed long fingerlike villi with bulg- ing hemispherical epithelial cells (Figure 28). Light microscopy revealed very swollen vacuolated absorptive epithelial cells with apical nuclei (Figure 29). The vacuoles were larger than those in the epithelial cells of the anterior jejunum and the epithelial cells of the entire villus were vacuolated. Very few changes were apparent in the posterior jejunum follow- ing infection. Loss of a few epithelial cells from the tips of villi 36 and 48 hours after exposure could be seen on scanning electron microscopy. Very few changes were visible by light microsc0py. A 56 ' . ,i r 4(0/ ’ss '1 Figure 26. 101C (8) (16 hours after exposure) Scanning electron micrograph of the anterior jejunum of an infected pig. The loss of cells resulting in holes (H) in the epithelial surface may be an artifact. x 500. 57 Figure 27. 104C (10) (24 hours after exposure) Scanning electron micrograph of the anterior jejunum of the same infected pig as in Figure 25. Note the exposed lamina propria which is the result of the epithelial cells having sloughed off the tip of the villus. x 1000. 58 few neutrophils were present in the villi 48 hours after exposure, but they were sporadic in any section. There were no focal concen- trations of neutrophils. listen; The villi in the ileum from control pigs did not appear as long as those in the anterior small intestine. The villi were broader with more leaf-shaped forms. The cells were more bulging and hemispherical in shape. Most of the nuclei were basal in position within the cell. Vacuoles occupied the epithelial cells in the 4/5 of the villus closest to the tip (Figure 30). Some of the cells close to the tips of the villi appeared collapsed (Figure 31). There appeared to be more lymphoid tissue in the lamina propria of the ileum. Sixteen hours after exposure the villi appeared to be more crowded, there was an increased width of the lamina propria and there was increased swelling of the individual epithelial cells. Greater numbers of the epithelial cells were collapsed in sections from some pigs, but this was not a consistent finding (Figures 32, 33 and 34). Although the epithelial surface of the ileum of pigs 24 hours after exposure showed very little change with scanning electron microscopy, a number of changes were visible by light microscopy. There appeared to be a widened lamina propria and many neutrophils in the villi and at the bases of the villi (Figures 35 and 36). Thirty-six hours after exposure more neutrophils were present in this area of the intestine. There appeared to be more than the 59 Figure 28. 109D (l4) Scanning electron micro- graph of the posterior jejunum from a control gnoto- biotic pig. There is some charging (distortion) in this photograph. The epithelial cells of the villi are swollen, bulging and hemispherical in shape. x 200. 6O Figure 29. 109D (116367D) Photomicrograph of a single villus from the posterior jejunum of a control gnotobiotic pig. The apical nuclei (N), vacuolation and distention of the epithelial cells are clearly visible. The extrusion zone (E) at the tip of the villus is also visible. H&E stain; x 500. 61 Figure 30. 109E (116367E) Photomicrograph of the ileum of a control gnotobiotic pig. The numbers of cells with vacuoles and the size of the vacuoles in the cells are both greater than they were in the. epithelium of the villi of the anterior portion of the small intestine. Most of the nuclei (N) are basal in position within the cell. H&E stain; x 125. 62 Figure 31. 109E (78) Scanning electron micro- graph of the ileum of a control gnotobiotic pig. The epithelial cells of the villi are swollen and hemispherical in shape. A few of the cells near the extrusion zone at the tips of the villi appear col- lapsed (C). x 500. 63 Figure 32. 101E (116359E) Photomicrograph of the tip of one villus from the ileum of a pig 16 hours after exposure. Markedly swollen cells (8), collapsed cells (C) and the increased width of the lamina propria (L) are visible here. H&E stain; x 500. 64 Figure 33. 101E (28) (16 hours after exposure) Scanning electron micrograph of the ileum exhibiting the crowding of villi, the swelling of the indi- vidual epithelial cells, and a number of collapsed epithelial cells. x 200. 65 Figure 34. 101E (27) (16 hours after exposure) Scanning electron micrograph of the ileum; greater magnification of the tip of the villus in Figure 33. x 1000. 66 Figure 35. 104E (1163628) Photomicrograph of a villus from the ileum of a pig 24 hours after exposure. Note the swelling of the individual cells (C), the large number of neutrophils (N), and the increased width of the lamina propria (L). HsE stain; x 500. 67 Figure 36. 104B (39) (24 hours after exposure) Scanning electron micrograph of one villus from the ileum. This photograph is of a villus from the same pig as in Figure 35. The appearance is very similar to that of scanning electron microscopy of the ileum of control pigs. Compare with Figure 37. There is a loss of microvilli from the individual epithelial cells (C) closest to the extrusion zone (E). x 1000. 68 usual number of epithelial cells sloughing from the tips of the villi leaving the lamina propria exposed in the area of the extrusion zone. There also appeared to be a concentration of nuclei at the tips of the villi and a hypercellularity of the lamina propria at this point (Figures 37 through 42). gene Scanning electron microscopy added a third dimension to the appearance of the ridges in the mucosa of the cecum. The individual epithelial cells contained few vacuoles and the nuclei were basal or central in position within the cell (Figures 43 through 45). Many goblet cells were present in the crypts. No differences between control and infected pigs were observed with either light microscopy or scanning electron microscopy. 29.1.2 The mucosa of the colon was thrown up into many folds and ridges. The pattern of these ridges differed somewhat between pigs when viewed by light and scanning electron microscopy (Figures 46 through 50). The nuclei of the individual epithelial cells were basal in position and there were many goblet cells between the epithelial cells (Figure 46). The appearance of the mucosa was one of many branching ridges (Figure 47). The orifices of discharging goblet cells were readily apparent on scanning electron microscopy (Figure 48). The mucosa of the colon of some pigs appeared as many small ridges upon larger ridges or folds (Figures 49 and 50). No changes between the control gnotobiotic pigs and pigs 4 hours to 48 69 Figure 37. 201E (42) (36 hours after exposure) Scanning electron micrograph of the ileum. There are more collapsed cells than were seen in the con- trols; however, the presence of collapsed cells following infection was not a consistent finding. x 200. 7O Figure 38. 201B (43) (36 hours after exposure) Scanning electron micrograph of a villus from the ileum. x 500. 71 Figure 39. 201B (132) (36 hours after exposure) Scanning electron micrograph of the tip of a villus from the ileum. Note collapsed cells (C) and loss of microvilli (M). x 1000. 72 Figure 40. 201E (117665E) Photomicrograph of the tip of a villus from the ileum of a pig 36 hours after exposure. Note the vacuolation and swelling of the individual cells (S), the collapsed cells (C) near the tip of the villus, the exposed lamina propria (L) in the area of the extrusion zone, and the hypercellularity and concentration of nuclei (N) in the lamina propria of this area. H&E stain; x 500. 73 Figure 41. 202E (44) (36 hours after exposure) Scanning electron micrograph of the tip of a villus from the ileum. There has been a greater loss of cells from the area surrounding the extrusion zone than seen in sections from control pigs. This has resulted in exposure of the lamina propria (L) at the tip of the villus. Compare with Figure 40. x 2000. 74 Figure 42. 201E (133) (36 hours after exposure) Scanning electron micrograph of the tip of a villus from the ileum of a pig infected with E. coli. None of the control pigs had this number of cells under- going necrobiosis at the tips of the villi. Note the loss of microvilli (M) on some cells. x 2000. 75 lllF (116369F) Photomicrograph of Figure 43. the mucosa of the cecum from a control gnotobiotic H&E stain; pig. x 125. 76 Figure 44. 110E (62) Scanning electron micro- graph of the cecum of a control gnotobiotic pig. The ridges of the mucosa are readily visualized. x 200. 77 Figure 45. llOF (63) Scanning electron micro- graph of the cecum of a control gnotobiotic pig. The orifices of discharging goblet cells (G) are visible at this magnification. x 2000. 78 Figure 46. 110G (116368G) Photomicrograph of the colon of a control gnotobiotic pig. The nuclei are basal in position in the epithelial cells, there are many goblet cells (G), and the mucosa is ridged in appearance between the crypts. H&E stain; x 125. 79 Figure 47. 1106 (69) Scanning electron micro— graph of the colon of a control gnotobiotic pig. Note the ridge-shaped appearance of the mucosa. x 100. 80 Figure 48. 1106 (70) Scanning electron micro- graph of the colon of a control gnotobiotic pig. The individual cell outlines (C) and the orifices of discharging goblet cells (G) are readily discernible. x 1000. 81 Figure 49. 1086 (1163666) Photomicrograph of the colon of a pig 8 hours after exposure. No changes were apparent as the result of the infec- tion. Note the appearance of ridges upon folds of the mucosa in the colon of this pig. H&E stain; x 50. 82 Figure 50. 1016 (71) (16 hours after exposure) Scanning electron micrograph of the colon of a pig. The appearance of ridges upon folds is a normal variation of the ridge-shaped mucosa of the colon. x 100. 83 hours after exposure were observed with either light microscopy or scanning electron microscopy (Figures 46 through 50). DISCUSSION Clinical Signs and Gross Lesions The clinical signs and gross lesions observed in this investi- gation were similar to those described in other studies in which gnotobiotic pigs were infected with E. coli 0138:K81:NM (Christie, 1967; Christie, 1969; Drees, 1969). All infected pigs, except those euthanatized up to 8 hours after exposure, developed diarrhea, but remained active and maintained a good appetite throughout the experi- ment. The effects were probably less severe than those reported by other workers because of the older age (6 days) at which the pigs were infected. The first evidence of pasty material on the rear quarters was noted 8 hours after exposure but the pigs were not visibly scouring until 12 hours after exposure. They also had developed flushed skin and erect hair coats by this time. The earliest gross lesions were observed 4 hours after exposure and consisted of slight intestinal dilatation and fluidity of the feces. Watery contents of the small intestine and large intestinal dilatation with gas and fluid were not noted until 12 hours after exposure. The clinical signs and gross lesions lead one to speculate that by 36 hours after exposure the clinical signs had peaked and by 48 hours the pigs had started to recover. 84 85 ScanningfiElectron and Light Microscopy Scanning electron microscopy of the stomach, small intestine and large intestine of the gnotobiotic pig produced some unexpected results. The appearance correlated well with light microscopic examination, but the three-dimensional appearance of the luminal surface was not as smooth a pattern as had been anticipated. The scanning electron microscopy of the stomach, cecum and colon of the control gnotobiotic pig has not been previously reported, and the stereoscopic appearance of these mucosal surfaces was interesting. In the stomach time mucosal extensions formed an interwoven branch- ing series of narrow ridges. The gastric pits between the mucosal extensions appeared variable in size and depth. A branching, ridged mucosa with a cobblestone appearance was visualized in the ceca of the gnotobiotic pigs. The mucosa of the colon appeared to be thrown up into branching folds and, in some cases, branching ridges upon the folds. The changes visible upon scanning electron microscopy were not consistent in all infected pigs and were not of diagnostic signifi- cance. The cell loss and the baring of the lamina propria were surprising when compared with other studies, but it is thought that these changes are not artifacts. Similar changes have been seen in a scanning electron microscopic study of the intestinal tract of virus-infected gnotobiotic calves (Mebus, 1974). The light microscopy confirmed the appearance of the mucosa seen by scanning electron microscopy; however, it was not possible to envision the three— dimensional appearance of the mucosa from light microscopy alone. 86 The location of nuclei and the size and distribution of vacuoles in the epithelial cells of the mucosa of the different segments of the intestinal tracts were of interest (Table 3). This may be related to the absorptive function of the cells. The dome- shaped projections on the villi of the posterior jejunum and ileum were the bulging hemispherical apices of the epithelial cells which were the result of vacuolation of the cytoplasm of these cells. Table 3. Location of nuclei and size and distribution of vacuoles in the epithelial cells of the mucosa Area of villus covered Area POsition of Vacuole by epithelial cells sectioned nucleus in cell size containing vacuoles Stomach basal none --- Duodenum basal none --- Anterior apical moderate tip one-half jejunum Posterior apical large all of villus jejunum Ileum basal large tip four-fifths Cecum basal none --- Spiral colon basal none --- The subepithelial changes were consistent with the findings of other investigators in all areas except the stomach (Christie, 1969). The presence of leukocytes in the mucosa of the stomach, which has not been reported by other investigators, 36 and 48 hours after 87 exposure may have been an indication of a more proximal coloniza- tion of E. coli or the presence of enterotoxins late in the course of the infection. The numerous leukocytes in the lamina propria of duodenal villi may have been a reaction to bacteria or to entero- toxin in the lumen of the duodenum. Scanning electron and light microscopy of the intestine of the gnotobiotic pig did not illustrate any pathognomonic lesions of enteric colibacillosis and did not provide much promise as a diag- nostic tool. This study also confirms the usefulness of villous atrophy as an aid in the differential diagnosis of transmissible gastroenteritis and enteric colibacillosis. SUMMARY Scanning electron and light microscopy were utilized to view sections from the stomach, duodenum, anterior jejunum, posterior jejunum, ileum, cecum and spiral colon from 18 gnotobiotic pigs. Five of the pigs were reared as controls and 13 were infected at 6 days of age by the oral administration of 1.6 x 106 colony forming units of Escherichia coli 0138:K81:NM. The onset of diarrhea, flushed skin and erect hair coats and the gross lesions were consistent with the reports by other investi- gators. The infiltration of leukocytes into the mucosa of the stomach seen with the light microscope has not been previously reported. The irregular pattern of the mucosal surface of the stomach formed by the gastric pits and mucosal extensions between the pits on the individual rugae revealed with scanning electron microscopy was different than anticipated following light micro- scopic studies. Scanning electron and light microscopy of the small intestine were consistent with expectations and the literature with 3 exceptions. One pig at 16 hours after exposure had lost a number of epithelial cells resulting in distinct holes in the mucosa of the villi in the anterior jejunum. One pig 24 hours after expo- sure had lost epithelial cells from the tips of villi in the anterior jejunum which resulted in the lamina propria being exposed. 88 89 Future work will be required to rule out the possibility that these may have been artifacts. Many sections of the ileum from both control and infected pigs contained a number of collapsed cells in the area around the extrusion zone at the tips of the villi. These cells were more numerous in the infected pigs and more cells appeared to have been sloughed from the area of the extrusion zone resulting in exposure of the lamina propria. No changes were apparent with either scanning electron or light microscopy of the cecum or spiral colon. However, the architecture of the large intestine was particularly interesting. The folds of the cecum and discharging goblet cells were clearly visualized. The convoluted folds of the spiral colon in some sections and the ridge upon fold appearance of others were considered to be normal variations. The alterations of the mucosa of the intestinal tract of gnotobiotic pigs infected with E. coli as visualized by scanning electron microscopy were not considered consistent enough to be of diagnostic significance. BIBLIOGRAPHY BIBLIOGRAPHY Anderson, T. F.: Techniques for the Preservation of Three- Dimensional Structure in Preparing Specimens for the Electron Microscope. Trans. N.Y. Acad. Sci., Ser. II, 13, (1951): 130-134. Asquith, P., Johnson, A. 6., and Trevor Cooke, W.: Scanning Electron Microscopy of Normal and Celiac Jejunal Mucosa. Am. J. Dig. Dis., 15, (June, 1970): 511-521. Barnum, D.A.: The Control of Neonatal Colibacillosis of Swine. Ann. N.Y. Acad. Sci., 176, (1971): 385-400. Barnum, D. A., Glantz, P. J., and Moon, H. W.: Colibacillosis. Ciba Veterinary Monograph Series. Ciba Foundation, Summit, N.J., 1967. Britt, A. 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Olson, D. P., Waxler, G. L., and Roberts, A. W.: Small Intestinal Lesions of Transmissible Gastroenteritis in Gnotobiotic Pigs: A Scanning Electron Microscopic Study. Am. J. Vet. Res., 34, (October, 1973): 1239-1245. 93 Oxender, W. D., Newman, L. E., and Morrow, D. A.: Factors Influenc- ing Dairy Calf Mortality in Michigan. J.A.V.M.A., 162, (March, 1973): 458-460. Pease, R. T. W.: Fundamentals of Scanning Electron Microscopy. Proc. 4th Ann. SEM Symposium IIT Res. Institute. Scanning Electron Microscopy/1971. Chicago, Ill., (1971): 11-16. Sojka, W. J.: Escherichia coli in Domestic Animals and Poultry. Commonwealth Bureau Anim. Hlth., Rev. Series No. 7, (1965). Spotts, Donald E.: Regeneration of Small Intestinal Villi in Gnotobiotic Pigs Infected with Transmissible Gastroenteritis Virus: A Scanning Electron Microscopic Study. M.S. Thesis, Michigan State University, East Lansing, Mich., 1974. Stevens, J. B., Gyles, C. L., and Barnum, D. A.: Production of Diarrhea in Pigs in Response to Escherichia coli Entero- toxin. Am. J. Vet. Res., 33, (1972): 2511-2526. Toner, P. 6., and Carr, K. E.: The Use of Scanning Electron Microscopy in the Study of the Intestinal Villi. J. Path., 97, (April, 1969): 611-617. Waxler, G. L., Schmidt, D. A., and Whitehair, C. K.: Technique for Rearing Gnotobiotic Pigs. Am. J. Vet. Res., 27, (1966): Waxler, 6. L.: Lesions of Transmissible Gastroenteritis in the Pig as Determined by Scanning Electron Microscopy. Am. J. Vet. Res., 33, (July, 1972): 1323-1328. VITA The author, Louis E. Newman, was born in Schenectady, New York, on November 14, 1930. He received his primary and secondary education in Schenectady, New York, and Marblehead, Massachusetts, and graduated from Marblehead High School in 1948. The author graduated cum laude with a Bachelor's Degree in Animal Husbandry from the University of New Hampshire in 1952. He received the Doctor of Veterinary Medicine Degree from Cornell University in 1956. Following graduation from veterinary school, the author practiced veterinary medicine in a two-man large animal practice in WOrland, wyoming. In 1957 he moved to Glasgow, Montana, where he founded and operated Glasgow Veterinary Clinic, Glasgow Veteri- nary Supply and Flying N Ranch. In 1969 the author joined the staff of Michigan State University as Project Leader of Veterinary Medicine Extension in the Department of Large Animal Surgery and Medicine. The author is married to the former Jane Ann Yarhouse and has seven children between six and eighteen years of age. 94 MICHIGAN STATE UNIVERSITY LIBRARIES O 1 3 3 45 8 38 l IIII 3 1293