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Pathology degree in (QAQ-fififl‘nwx Major professor Date lolllIQG ‘ \_ MS U is an Affirmaii’w Action/Equal Opportunity Institution 0— 12771 ‘V" V 'W ‘v“ v 'v ' ~— v "V“ H Hmr— v W‘V‘ v v— 'c-r'v-OV'V‘VVVV I O V V U V PLACE Ii RETURN BOX to move this checkout from your record. TO AVOID FINES rotum on or baton doto duo. DATE DUE DATE DUE DATE DUE MSU ioAn Affirmative Action/Equal Opportunity instituion Wm: THE EFFECT OF RETINOL PALMITATE AND/OR PUTRESCINE ON SMALL INTESTINAL REGENERATION IN PIGLETS BY Soegiarto A DISSERTATION Submitted to Michigan state University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1996 ABSTRACT THE EFFECT OF RETINYL PALMITATE AND/OR PUTRESCINE ON SMALL INTESTINAL REGENERATION IN PIGLETS BY Soegiarto This research examined the effect of daily oral administration of retinyl palmitate and putrescine alone or in combination on the clinical signs, lesions and intestinal mucosal epithelial regeneration in rotavirus-infected piglets. For the first. 48 hours of life, 128 newborn piglets consumed colostrum. Starting on day 3, the piglets were divided into 4 groups of 32 piglets. Group RP was given retinyl palmitate, group P was given putrescine, group RPP was given retinyl palmitate and putrescine and group MO was given milk only. On day 5 half of the piglets in each group were infected with porcine rotavirus. The piglets were then euthanatized on day 6, 7, 8 and 13 (1, 2, 3 and 8 days postinoculation [DPI]). Treatment did not prevent or shorten the course of the disease, and did not‘affect the morphology of the intestinal lesions. Treatment did not have any significant effect on villous height, crypt depth, complexity index or goblet cell number in the small intestine. Infection increased the small intestinal mucosal complexity indices (P<0.05). Infection reduced the relative volume of the small intestinal mucosa (P<0.0l). Treatment increased the mitotic index in the small intestinal crypt epithelium (P<0.01). Mucosal protein and RNA concentrations were greater in piglets 8 DPI than in piglets 1 and 2 DPI, regardless of treatment group, The mucosal DNA concentration was decreased by infection status (P<0.01), but it was not affected by treatment or time. Liver vitamin A concentration was influenced by treatment and time but not by infection status. Piglets in groups given RP and RPP had higher liver vitamin A concentrations than piglets given P and MO (P<0.01). Piglets in groups given RP and RPP that were 8 DPI had higher concentrations of liver vitamin A than piglets 1 DPI (P<0.01). Putrescine tended to decrease the concentration of liver vitamin A in infected animals. Infected piglets given RPP had significantly lower vitamin A concentrations than infected piglets given RP (P<0.01). Dedicated to my parents Ibuk Soewarti and Bapak Tamid iv ACKNOWLEDGMENTS I would like to extend a special thanks to Dr. Sheila Grimes, my major professor, for her encouragement, guidance and dedication to assisting me during the course of this study. I would also like to thank the other members of my guidance committee, Dr. Jon Patterson, Dr. Robert Holland, Dr. Thomas Herdt, Dr. Gretchen Hill and Dr. Gary Watson for their suggestions, support and contributions to this research. Special thanks are extended to Dr. Robert Holland for his help in performing the surgical procedure in my pigs. I would like to thank Dr. David Benfield for the rotavirus inocula used in this study. I would also like to thank Dr. Stuart Sleight for his support in this study. I am indebted to Josselyn Carrasco, Amy Porter, Kathy Campbell, Leann Lumbert, Ann House, Julie B. Moore, Kathleen O'Hare, David A. Brigham, Ralph Common, Sharon Thon, John Allen and Alan Snedegar for their technical assistance. I would also like to thank Supriyandono's family, Dr. Eric Kufuor-Mensah, and my family and friends, whose moral support, encouragement and understanding were invaluable in the completion of this degree. I greatly appreciate the financial support I received from the Indonesian government during my graduate studies at Michigan State University. vi TABLE OF CONTENTS Page LIST OF TABLES ........................................... ix LIST OF FIGURES................ .......... ........ ........ Xi LIST OF ABBREVIATIONS .................................. xiii INTRODUCTION.. ............................................ 1 LITERATURE REVIEW............. ............................ 4 SMALL INTESTINAL MUCOSAL KINETICS ..... .................. 4 Organization of cell proliferation in the small intestine .............................................. 4 Factors affecting cell proliferation in the small intestine .................... . ......................... 6 Cell proliferation assessment in the small intestine.. 12 VITAMIN A ............................... ’ ............... 13 Metabolism ........ . ...... ........ .......... . .......... 15 Vitamin A homeostasis ................................. 20 Effect of vitamin A status in the intestine ........... 22 Molecular biology of vitamin A ........................ 22 POLYAMINES ............................................. 24 Metabolism ............................................ 26 Enzymes for polyamine biosynthesis .................... 29 Role of polyamines in cell proliferation..... ......... 33 ASSOCIATION OF VITAMIN A AND POLYAMINE BIOSYNTHESIS ....34 ROTAVIRUS .. .............. . ........... . ................ 35 Classification and serotyping ......................... 36 Genome and viral proteins..... .......... ....... ....... 38 Viral entry and growth. ..... ......................... 39 Factors affecting infection ........................... 41 Clinical signs ........................................ 43 Lesions ............................................... 44 Diagnosis ............................................. 46 CHAPTER 1: EFFECT OF DAILY DIETARY ADMINISTRATION OF RETINYL PALMITATE AND/OR PUTRESCINE ON THE CLINICAL SIGNS, LESIONS AND ROTAVIRAL ANTIGENIC EXPRESSION IN ROTAVIRUS-INFECTED PIGLETS Abstract ............................................... 48 Introduction ........................................... 49 Materials and methods .................................. 51 Results ................................................ 59 Discussion ............................................. 77 vii CHAPTER 2: EFFECT OF DAILY DIETARY ADMINISTRATION OF RETINYL PALMITATE AND/OR PUTRESCINE ON THE SMALL INTESTINAL MUCOSAL MORPHOMETRY OF ROTAVIRUS-INFECTED PIGLETS Abstract ............ ........ ... ........................ 83 Introduction ......................................... ..84 Materials and methods ..................................86 Results ................................................91 Discussion ............................................ 104 EFFECT OF DAILY DIETARY ADMINISTRATION OF RETINYL PALMITATE AND/OR PUTRESCINE ON INTESTINAL MUCOSAL NUCLEIC ACID AND PROTEIN, AND ON HEPATIC VITAMIN A CONCENTRATIONS OF ROTAVIRUS INFECTED PIGLETS CHAPTER 3 ......... ................. ...... . ....... .......111 Abstract 0 O O O O O O O O O O O O O ..... O O ...... O 0 0 O 0 O O O O O O O ..... O O 1 1 1 Introduction .......................................... 112 Materials and methods ............ ...... ............... 113 Results ........................ ..................... .. 120 Discussion ................ . ................ . .......... 131 CONCLUSIONS ............................................. 138 APPENDIX ................................................ 140 Materials and methods ................................ . 140 Results ...... ......................................... 143 Discussion ................... .. ....................... 145 REFERENCES....... ........... ............................147 viii LIST OF TABLES Nutritional composition of milk used in the experiments ........... .... ....... ........... Number of diarrheic piglets 1 - 8 days postinoculation with the SDSU strain of porcine rotavirus...................... ..... Average of combined fecal and urinary weights (grams) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ......... ............ ...... Average of daily body weights (grams) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ........ Average of daily body weight gains (grams) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus... Enterocyte vacuolation of control piglets vs. piglets infected with the SDSU strain of porcine rotavirus...................... ..... Vacuolar degeneration of enterocytes in piglets infected with the SDSU strain of porcine rotavirus.............. ..... . ....... Immature enterocytes in piglets infected with the SDSU strain of porcine rotavirus... Number of piglets with rotavirus-antigen- positive enterocytes, based on an immunohistochemistry............ ............ Expression of rotavirus-antigen in enterocytes of piglets infected with the SDSU strain of porcine rotavirus ............ Complexity indices in the small intestines of control piglets vs. piglets infected with the SDSU strains of porcine rotavirus ....... ix Page 54 6O 62 63 64 68 7O 73 75 76 92 Table Page 2.2 Villous length (um) in the small intestines of control piglets vs. piglets infected with the SDSU strain of porcine rotavirus........ 95 2.3 Crypt depth (um) in the small intestines of control piglets vs. piglets infected with the SDSU strain of porcine rotavirus........ 97 2.4 Mitotic indices per crypt in the small intestines of control piglets vs. piglets infected with the SDSU strain of porcine rotavirus......................... ..... ..... 102 2.5 Goblet cells per crypt in the small intestines of control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ................................... 105 3.1 Small intestinal mucosal protein concentrations (mg/g mucosal dry matter) from control piglets vs. piglets infected with the SDSU strain of porcine rotavirus... 121 3.2 Small intestinal mucosal DNA concentrations (mg/g mucosal dry matter) from control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ................. 123 3.3 Small intestinal mucosal RNA concentrations (mg/g mucosal dry matter) from control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ................. 125 3.4 Protein:DNA, RNA:DNA and protein:RNA ratios from control piglets vs. piglets infected with the SDSU strain of porcine rotavirus... 128 3.5 Hepatic vitamin A concentrations (pg/gram dry weight) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ................................... 129 Figure LIST OF FIGURES Major metabolic transformations of vitamin A Mode of action of retinoid receptors ........ Metabolic pathway of polyamines ............. Structure of polyamines and related compounds ................................... Experimental design ......................... Photomicrograph of a section of ileum from a rotavirus-infected piglet in the retinyl palmitate and putrescine group, 2 DPI. Normal enterocyte vacuolation ............... Photomicrograph of a section of jejunum from a rotavirus-infected piglet in the putrescine group, 2 DPI. Notice dilated villous lymphatic and enterocyte vacuolar degeneration .......... . ..................... Photomicrograph of a section of jejunum from a rotavirus-infected piglet in the milk-only group, 8 DPI. Notice the atrophic villi covered by squamous to cuboidal enterocytes. Photomicrograph of a section of duodenum showing the presence of rotaviral-antigen... Photomicrograph of a section of ileum with a Weible graticule superimposed on the image to illustrate the use of a Weible graticule. Pooled small intestinal mucosal complexity indices (SIMCI) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ........... ....... ......... Pooled small intestinal mucosal villous length (SIMVL) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ........................... xi Page 16 25 27 28 52 67 69 72 74 89 93 96 Figure Page 2.4 Pooled crypt depth for control piglets vs. . piglets infected with the SDSU strain of porcine rotavirus........... ..... .. ......... 98 2.5 Photomicrograph of a section of jejunum with a high number of mitotic figures ............ 100 2.6 Photomicrograph of a section of jejunum with a low number of mitotic figures ............. 101 2.7 Pooled mitotic indices of control and infected piglets (mean i standard error).... 103 3.1 Outline of nucleic acid and protein analyses (Munro and Fleck, 1966, with some modification) ............................... 116 3.2 Pooled small intestinal mucosal protein concentrations (SIMPC) for control piglets vs. piglets infecyed with the SDSU strain of porcine rotavirus ...... . ......... .. ......... 122 3.3 Pooled small intestinal mucosal DNA concentrations (SIMDC) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus ........................... 124 3.4 Pooled small intestinal mucosal RNA concentrations (SIMRC) for control piglets vs. piglets infecyed with the SDSU strain of porcine rotavirus ........................... 126 3.5 Pooled hepatic total vitamin A concentration of control and infected piglets (mean 1 standard error)................ ............. 130 xii CDCD CF CRABP CRBP DFMO DMBA DNA DPI DU ELISA FA FABP GD HRP HTC IBM 19 IU LI LJ MI mRNA MO NB NSP ODC OSU PBS PRN RBP RNA RP RPP RXR SAM-DC SDSU TGF TTR UI UJ VP LIST OF ABBREVIATIONS Colostrum-deprived caesarean-derived Complement fixation Cellular retinoic acid-binding protein Cellular retinol-binding protein DL-a—Difluoromethylornithine dimethylbenzanthracene Deoxyribonucleic acid Day postinoculation Duodenum Enzyme linked immunoassay Fluorescent antibody Fatty acid-binding protein Gel-diffusion Horseradish peroxidase Hepatoma cell culture Immunoelectron microscopy immunoglobulin International unit Lower ileum Lower jejunum Mid intestine Messenger ribonucleic acid Milk only Naturally born Nonstructural polypeptides Ornithine decarboxylase Ohio State University Putrescine Phosphate-buffered saline Plaque reduction neutralization Retinoic acid receptor Retinol-binding protein Ribonucleic acid Retinol palmitate Retinol palmitate and putrescine combined Retinoid X receptor. S-adenosylmethionine decarboxylase South Dakota State University Transforming growth factor Transthyretin Upper ileum Upper jejunum Viral polypeptides xiii INTRODUCTION Small intestinal mucosal regeneration is essential for animals and human beings, either for continual physiological renewal or for recovery from intestinal injury. Intestinal injury with mucosal damage can be caused by various protozoal, bacterial and viral pathogens (Holland, 1990). Major efforts to reduce intestinal damage have focused on immunization to prevent enteric diseases or the use of antibiotics and chemotherapeutic agents to treat the diseases. Vaccination against some intestinal diseases has proven to be beneficial, but has a limited protective effect in immunosuppressed individuals. Mishandling of vaccines may also contribute to vaccine failure or inadequate protection. In addition, the current concern with chemical residues has restricted antibiotic use in food animals. Therefore, the use of natural chemicals, such as vitamin A and/or putrescine, to prevent or to minimize intestinal mucosal damage would be extremely beneficial. The scientific literature has suggested, but not conclusively proven, that vitamin A and putrescine. may enhance intestinal epithelial regeneration. We hypothesized that the oral administration of vitamin A and/or putrescine in the diet would minimize the effect of rotavirus on the enhancing ‘mucosal regeneratitun Rotavirus, like several other viral and protozoal enteric diseases, causes small intestinal damage which results in villous atrophy. Various forms of vitamin A are known to have an effect on vision, reproduction and the maintenance and growth of epithelial cells. Most studies involved the use of vitamin A deficient animals. Vitamin A deficiency reduced cellular DNA, RNA and protein synthesis in the intestinal mucosa. Readministration of vitamin A in the animals restored cellular DNA, RNA and protein synthesis (Johnson et a1, 1969, Zachman, 1967). Recently, vitamin A was reported to protect the small intestine of rats from morphological, biochemical and physical damage caused’ by methotrexate administration (Tsurui et a1, 1990). Methotrexate, as an antitumor drug, inhibits mitosis of epithelial cells in small intestinal crypts, disrupts the steady state system of the epithelium and progressively reduces the size of crypts and villi (Jeynes and Altmann, 1978). Polyamines, spermidine and spermine, and their diamine precursor, putrescine, are found in virtually all cells of eukaryotic organisms. Their cellular concentrations are highly regulated. They play an important role in cellular growth and differentiation, but their cellular functions at the molecular level are not known (Heby, 1981). In the present study, vitamin A and/or putrescine were given to young piglets in an attempt to prevent or decrease the effects of rotavirus infection. The objectives of this study were: 1) 2) 3) To determine if the clinical signs, lesions and rotavirus antigen expression in the small intestinal mucosa produced by rotavirus infection in piglets could be altered by the daily dietary administration of vitamin A and/or putrescine; To determine the effect of the daily dietary administration of vitamin A and/or putrescine on the small intestinal mucosal morphometry of rotavirus- infected piglets; To determine the effect of the daily dietary administration of vitamin A and/or putrescine on hepatic vitamin A, and small intestinal DNA, RNA and protein concentrations of rotavirus-infected piglets. n) 'rv (I) [O LITERATURE REVIEW SMALL INTESTINAL MUCOSAL KINETICS Mature intestinal mucosal cells on the tips of villi are continuously sloughed and replaced by new epithelial cells which migrate from the crypts by a complex mechanism. Apart from normal physiological processes, the sloughing or destruction of mature or immature intestinal epithelial cells may also be caused by such factors as weaning (Hampson, 1986 and Kenworthy, 1976), stress (Wang and Johnson, 1991), ionizing radiation (Thomassen, 1972; Kent and Moon, 1973) and starvation (Clarke, 1970b). Enterocyte destruction may also be caused by various infectious agents, including intestinal parasites such as coccidia (Fernando and McCraw, 1973), pathogenic bacteria such as Serpulina hyodysenteriae and Campylobacter species (Neef et a1, 1994; Eaton et a1, 1989), bacterial toxins such as ClOstridium perfringens type C exotoxin (Bergeland, 1972) and Escherichia coli heat-stable enterotoxin b (Rose et al, 1987) and viral agents such as coronavirus (Thake, 1968) and rotavirus (Theil et a1, 1978). Organization of cell proliferation in the small intestine Epithelial renewal systems in the small intestine are usually divided into proliferation, maturation and functional compartments. In the small intestine, the functional compartment is found in the villus, whereas, both proliferation and maturation compartments are located in the crypt. In the maturation compartment, cells lose their proliferative capacity and acquire characteristics of mature functional cells (Leblond et a1, 1967, Lesher, 1967). A steady flow of cells migrates from the proliferation compartment into the maturation compartment and then to the functional compartment in the villus. Subsequently cells will exfoliate from the tip of the villus. A second functional compartment or terminally differentiated cell compartment, referred to as the Paneth cell zone, is present in the base of the small intestinal Crypt (Cheng and Bjerknes, 1980). Mitosis in the lower portion of the proliferation compartment yields two new proliferating cells. Mitosis in the middle portion of the proliferation compartment, corresponding to the distance from the base, yields cells with a declining probability for proliferation. Mitosis in the top portion of the proliferation compartment yields terminally differentiated cells (Cheng and Bjerknes, 1980). The small intestinal crypt contains Paneth cells, goblet cells, enteroendocrine cells, intra-epithelial lymphocytes and columnar epithelial cells. At least 4 of these cell types - Paneth, goblet, enteroendocrine and columnar epithelial cells - originate from a multipotential stem cell situated at or near the crypt base (Cheng, 1974a,b; Cheng and Leblond, 1974a,b,c). Paneth cells are absent in cats, dogs and pigs (Magee and Daley, 1986). Most of the columnar epithelial, goblet and enteroendocrine cells differentiating from the stem cells migrate in an upward direction to populate the villi. All of the Paneth cells, some columnar epithelial and a few goblet and enteroendocrine cells migrate downward into the stem cell zone at the base of the crypt (Cheng and Bjerknes, 1980). Factors affecting cell proliferation in the small intestine Multiple factors, such as species, animal age, nutrition, hormones, intestinal flora and the degree of epithelial damage, influence intestinal mucosal regeneration. These factors affect. each. other, but the underlying mechanism leading to an increase or a decrease in the number of cells proliferating in the crypts is still unknown. CSpecies and age of animal The effect of species and age on small intestinal epithelial cell proliferation has been studied by several investigators. In albino rats, the rate of epithelial cell production in the small intestine is approximately 36 cells per crypt per hour (Clarke, 1970a). The replacement time, starting with the synthesis of DNA in the crypt until the cell reaches the tip of the villus, varies depending upon the age of the animal, the species and the region of the intestine. Moon and Skartvedt (1975) showed that the replacement time of small intestinal mucosal cells of l-daye old chickens was faster than the replacement time in 3-week- old and 6-month-old chickens. In 1-day-old chickens, epithelial cell replacement was nearly complete iJiES days, whereas during the same period of time, epithelial replacement in 3-week-old and 6-month-old chickens was only 75 and 50% complete, respectively. Clarke (1967) noted that with age duodenal villi in chickens varied in size but not in number. In adults, villi were larger in size, but the total number of villi was approximately the same as during the late embryonic stage. Duodenal crypts, on the other hand, increased greatly in number and depth with age. In contrast to chickens, small intestinal epithelial migration in older rats was faster than in young rats. Within 48 hours, the enterocytes of 210 day-old-rats had migrated to the top of the villus, whereas the 7 day-old rats' enterocytes had reached only about 25% of the length of the villus (Koldovsky et a1, 1966). Similar to rats, small intestinal mucosal cell replacement in young pigs is slower than in older animals. In day-old pigs, the epithelium is replaced in 7-10 days, while in 3-week-old pigs it takes only about 2-4 days (Moon, 1971). The site of enterocyte proliferation is different during the various stages of development of the animal and in various portions of the intestinal tract. DNA synthesis and cell proliferation occur in epithelial cells along the entire length of the villus of fetal rats. However, in 1- day-old suckling rats replication is confined to the extreme base of the villus (Hermos et al, 1971). Kenworthy (1976) observed that the mitotic indices of 2-week-old weaned and unweaned pigs were higher in the jejunum and ileum than in the duodenum. In addition, the overall mitotic indices in the small intestine of weaned pigs were higher than in unweaned pigs. Using tritiated labeled thymidine, Imondi and Bird (1966) reported that the mucosa of 2-day-old Chick's jejunum 'was replaced approximately in 48 hours, whereas the replacement time for the epithelial cells in the ileum and duodenum was longer than 48 hours. Studies from various other animals have also revealed that many intestinal mucosal functions are affected by age. Chen et a1 (1990) showed that there was a greater rate of transepithelial transport of D-glucose and 3-O-methyl-D- glucose in young growing mice compared to older animals. These changes were accompanied by decreases in the number and height of villi with age, which resulted in a decrease in the mucosal surface area. The ability of intestinal mucosal epithelial cells to ingest macromolecular complexes is also associated with age (William and Beck, 1969). Studies in rats, rabbits and guinea-pigs, utilizing trypan blue as a marker for pinocytic activity, indicated. that epithelial maturation was complete about 3-4 weeks after birth. Perozzi et a1 (1993) described the temporal expression of intestinal functions during fetal and postnatal development in the pig. Results of studies using Northern hybridization indicated the presence of the messenger’ ribonucleic acids (mRNAs) for cellular' retinol binding protein II (CRBP II), for the digestive enzyme aminopeptidase N, and for the microvillar proteins, villin and ezrin, in the small intestine of both weaned and 40-day fetal pigs. The mRNAs for two types of fatty acid-binding proteins (FABPs), I-FABP and L-FABP, which are involved in the metabolism of long chain fatty acids, were detected only in weaned animals, whereas the mRNA for CRBP I was detected only in the fetal intestine. Nutrition Starvation in rats leads to marked loss of small intestinal weight, and RNA and protein content (Steiner et al, 1968). The presence of food in the intestine may exert either direct or indirect effects on the growth of the small intestinal mucosa. The direct effect of the presence of food in the intestinal lumen on the_mucosal growth may be a result of epithelial desquamation, or food may provide local nutrition and direct stimulation by particular dietary constituents may act as growth factors. The indirect effect of the presence of food in the intestinal lumen may be a result of nerve stimulation and the release. of gastrointestinal peptides (Henning et al, 1994). Dietary nucleotides, adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, uridine monophosphate 10 and inosine monophosphate, appear to be modulators of intestinal development following chronic diarrhea (Nufiez et al, 1990). Administration of vitamin D to vitamin D deficient chicks for more than 110 hours partially restored the size of villi and improved epithelial cell migration (Spielvogel et a1, 1972). Intestinal flora The intestinal flora affect intestinal epithelial transit time. Bacterial colonization in the small intestine causes 'physiologic inflammation', a condition that leads to higher rates of intestinal epithelial proliferation and migration (Abrams et al, 1963). Inn addition, the transit time of cells moving from the crypts to the villous tips is faster in conventional mice than in germ-free animals (Lesher et al, 1964). Feedback control mechanism Epithelial cell renewal is also believed to be regulated by a feedback control mechanism of the functional villous cell. Irradiation is known to kill actively dividing cells. Following cell depletion in the crypt after irradiation, initial development of crypt epithelium was similar in germ-free and conventional rats. However, subsequent proliferation and maturation of the cells were slower in germ-free rats. This probably was associated with the cellular density of the maturing non-dividing cells, determined by the level of esterase activity which is higher 11 in germ—free rats (Galjaard et al, 1972). This phenomenon was also observed by Sherman and Quastler (1960), who concluded that the continuous migration of intestinal epithelium is not dependent upon a push from dividing crypt cells but is a response to the loss of villous epithelial cells. Villous epithelial loss caused by intestinal parasites enhances epithelial regeneration. Intestinal epithelial turnover time in chickens infected with Eimeria acervulina was faster than the epithelial turnover time of normal chickens (Fernando and McCraw, 1973). Epithelial cell number in the crypts and villi of rats that have had their intestines partially resected is higher than normal. The increase in epithelial Cell number begins as early as 2-4 days postoperation, reaching its maximum in 12 days, and. is proportionate to the amount of tissue removed (Hanson et al, 1977a,b). Chalones, soluble substances that act as endogenous inhibitors of mitotic activity, have long been implicated as effectors of' a negative feedback mechanism on intestinal crypts (Brugal, 1976; Houck, 1973). Recent in vitro and in vivo studies indicate that transforming growth factor beta (TGF B) acts as a chalone. Exogenous addition of TGF B has been shown to inhibit the proliferation of a non-transformed rat jejunal cell line (Lamprecht et al, 1989) and of a primary culture of rat small intestinal epithelium (Booth et a1, 1995). Studies in mice shortly after whole body 12 irradiation have shown that TGF B-l was an effective inhibitor of the small intestinal crypt proliferation of unirradiated and early regenerating animals (Potten et al, 1995). Using Northern blot analysis and immunohistochemistry in murine small intestines, Barnard et al (1993) showed that TGF B-l, TGF B-2 and TGF 5-3 were expressed in the small intestinal epithelium, most prominently in the enterocytes located on the villous tip. No TGF B expression was detected in crypts. Cell proliferation assessment in the_emell inteetine Intestinal mucosal regeneration has been studied extensively using morphological, kinetic or biochemical assessments. Most morphological studies were either based on epithelial cell counting (Hampson, 1986), linear measurements of mucosal thickness, villous height and crypt depth (Hart and Kidder, 1978) or mucosal surface complexity assessments (Bertram et al, 1991; Hart and Kidder, 1978; Rose et a1, 1987; Dunnill and Whitehead, 1972). Mucosal surface complexity was assessed by determining the surface and volume ratio based on the method developed by Chalkley (Chalkley et al, 1949) with the use of a Weibel graticule (Weibel, 1963). Kinetic assessments were done with autoradiography, using tritiated thymidine to measure the rate of epithelial cell migration and replacement (Abrams et al, 1963). Biochemical analyses have been used to assess a () "fl Lil 13 intestinal DNA, RNA, mucosal protein and disaccharidase activities (Nufiez et al, 1990). In addition, a D-xylose absorption test has been used to assess intestinal malabsorption of carbohydrates (Bolton et al, 1976). VITAMIN A Vitamin A was first discovered by McCollum and Davis in 1913 in animal fat and fish oil as a factor that had growth promoting activity. The substance was called vitamin A by Drummond in 1920. In 1930, Moore showed that B-carotene extracted from plants had provitamin A activity. Vitamin A or its precursors may be present in various forms, snob as B-carotene, retinaldehyde, retinol, retinyl acetate and retinoic acid. The major sources of vitamin A or its precursors in the diet are carotenoid pigments, such as B-carotene from plants, and preformed vitamin A, mostly in the form of retinyl esters from animals. The various forms of vitamin A are involved in vision, reproduction and the maintenance and growth of epithelial cells. Only the role and the mechanism of retinaldehyde on the visual cycle have been clearly explained (Morton, 1944; Wald, 1953), whereas the action of the other forms of vitamin A are still under investigation. ' Interconversion between vitamin A forms is known to exist but the functional activity of certain forms of l4 vitamin A cannot be replaced by other forms. For example, retinol and retinaldehyde can be utilized by all target tissues, whereas administration of retinoic acid to vitamin A deficient rats successfully maintained the growth of the rats but failed to maintain visual and reproductive functions (Dowling and Wald, 1960). Either a deficiency of vitamin A or excessive dietary vitamin A can cause various clinical signs and lesions. Vitamin A deficiency causes night blindness which then proceeds to xerophthalmia (Fridericia and Holm, 1925). Also, vitamin A deficiency increases the incidence of diarrhea and respiratory disease (Sommer et al, 1984; Bloem et al, 1990), as well as the mortality rate among preschool children in developing countries (Sommer et al, 1983). Furthermore, vitamin A supplementation in children decreases the incidence of keratOmalacia (Vijayaraghavan et al, 1984), diarrhea, respiratory diseases (Bloem et a1, 1990) and childhood mortality (Sommer et al, 1986). Vitamin A also causes abnormalities if administered in excess. Clinical cases and experimental studies of vitamin A intoxication in human beings and animals have been documented and reviewed by many authors (Clarke, 1971; Bendich and Langseth, 1989). Excess vitamin A causes teratogenic effects (Rosa et al, 1986), bone deformities (Irving, 1948, Tang et al, 1985), intracranial hypertension (Bhettay and Bakst, 1988) and hepatic fibrosis (Bioulac-Sage et a1, 1988). Most of the previously listed conditions were e ”r. 15 caused by the consumption of animal liver rich in vitamin A or by the excessive ingestion of a vitamin A preparation. Most conditions were reversible with. 21 cessation of consumption. Knowing the deleterious effects of either vitamin A deficiency or toxicity, many authors have reviewed the recommended daily intake for human beings (Bendich and Langseth, 1989; Sklan, 1987) and for animals (Baumann, 1953). Herdt and Stowe (1991) reported the range of serum vitamin A concentrations in dairy cattle. They also indicated that various factors, such as physiologic state, diet and environmental conditions should be considered prior to vitamin A supplementation in dairy cows; Similar studies have not been done with pigs. Studies on the metabolism and mode of action of vitamin A have led to the discovery of various vitamin A carrier proteins, including retinol binding protein (RBP) (Kanai et al, 1968), transthyretin (TTR), CRBP (Bashor et al, 1973; Hollander et al,-1978b), CRBP II (Ong et a1, 1987), CRBP III (Nishiwaki et al, 1990), cytoplasmic retinoic acid binding protein (CRABP) (Chytil and Ong, 1984) and the nuclear receptor for retinoic acid (Giguere et al, 1987; Petkovich et al, 1937). Metabolism The metabolism of vitamin A (Figure 1) has been extensively reviewed by many authors (Blomhoff et al, 1990; U. CurRRhDLmn.UPLC 16 GLUCORON I C AC I D , U / TAURINE' ETC. ACTIVE : INACTIVE I i MGP : C11 1 I : RE RP : RPM i C 1 4 i E I i RG 5 C16 i / i I CONJUGATION ' WITH ROL-BP ROL RAG i c19 t l I I I I I I Figure 1. Major metabolic transformations of vitamin A. Metabolites with high biological activity are on the left saide of dotted line. ROL, retinol; RAL, retinaldehyde; RA, retinoic acid; RE, retinyl esters; RP, retinyl phosphate; RG, Retinyl B-glucoronide; RAG, retinoyl B-glucoronide; I{PM, retinyl phosphomannose; ROL-BP, cellular retinol- lbinding protein; RAL-BP, cellular retinaldehyde-binding IIrotein; RA-BP, cellular retinoic acid-binding protein; MGP, Huannosylated glycoprotein; 4-HRA, 4-hydroxyretinoic acid; 4- ORA, 4-oxoretinoic acid; 5,6t-ERA, 5,6-epoxyretinoic acid; (319, C16, etc., oxidized metabolites of retinoic 'acid Chantaining 19, 16, etc., carbon atoms (Olson, 1986). 17 Frolik, 1984; Goodman, 1969; Olson, 1969; Sklan, 1987; Herdt and Stowe, 1991). The absorption of B-carotene by the intestine requires the contribution of fat and bile salts to form mixed-micellar solutions (El-Gorab and Underwood, 1973). In all species, B-carotene appears to reach the intestinal mucosa in the same manner, that is through a passive diffusion phenomenon which is directly proportional to the concentration of B-carotene in the intestinal lumen (Hollander and Ruble, 1978). There are two pathways for B—carotene absorption from the intestine before it reaches the circulation. The B- carotene is either directly transported unchanged to the lymph and blood or it may be cleaved prior to absorption. Direct transport of B-carotene from the small intestinal mucosa to circulation occurs in human beings in which significant amounts of carotene are present in the lymph and blood (Goodman et al, 1966) . However, in rats virtually no intact B-carotene is absorbed beyond the intestinal mucosa (Huang and Goodman, 1965). Within the intestinal mucosa, B-carotene is cleaved into 2 molecules of retinaldehyde by a soluble enzyme, B- Carotene 15-15'-oxygenase. Immediately after it is formed, retinaldehyde is reversibly converted to retinol by a retinaldehyde reductase enzyme. Also, some retinaldehyde is 18 irreversibly oxidized to retinoic acid. Evidence suggests that the intestinal mucosa also serves as a target organ for retinoic acid. As an intermediate active metabolite in the intestinal mucosa, retinoic acid is converted to its isomer and glucoronyl derivatives (Cullum and Zile 1985), as well as to 5,6-epoxyretinoic acid (Napoli et al, 1978) . Dietary preformed vitamin A, primarily in the form of retinyl esters, is hydrolyzed to retinol by digestive esterase enzymes originating from the pancreas (Hollander and Muralidhara, 1977) and brush border intestinal membranes (Rigtrup and Ong, 1992). Both sets of enzymes are stimulated by the presence of bile salts. In pharmacological concentrations, absorption occurs through passive diffusion. However, under physiological concentrations, absorption occurs through a carrier- mediated-process using an intraluminal retinol carrier (Hollander and Muralidhara, 1977) . Inside intestinal epithelial cells, retinol formed from B-carotene or dietary retinyl esters is carried by CRBP to the endoplasmic reticulum (Hollander and Ruble, 1978). Within the endoplasmic reticulum, retinol is esterified mainly to form retinyl-palmitate (Ong, 1985), irrespective Of other types of esters ingested (Mahadevan et a1, 1963) . Almost all retinyl esters, derived from B-carotene or dietary retinyl esters, are incorporated with acylglyceride- rich chylomicrons prior to entering the lacteals and 19 reaching the circulation through the thoracic duct (Goodman, _et a1 1966). After extrahepatic hydrolysis of many of the chylomicron acylglycerides, the chylomicron remnants containing retinyl ester are then taken up by hepatocytes (Goodman et al, 1965) via receptor-mediated endocytosis (Windler et al, 1980). The retinyl esters are subsequently transferred. to 'perisinusoidal stellate cells for storage (Blomhoff et al, 1982; 1985). Prior to the transfer, retinyl esters are hydrolyzed to retinol in the hepatocytes and then transferred to the stellate cells (Norum et al, 1986). Transfer from the hepatocytes to stellate cells is influenced by the vitamin A status of the animal (Blomhoff et a1, 1982). From the liver, retinol is mobilized and delivered to target organs by a specific carrier, RBP (Kanai et al, 1968). One molecule of RBP carries one molecule of retinol. The RBP-retinol is found in the circulation as a ternary complex 'with one ‘molecule of 'thyroxin-binding prealbumin (transthyretin, TTR). The RBPéretinol-TTR complex is a stable complex and it is not susceptible to glomerular filtration (Wolf, 1984). Besides retinol, RBP can also bind retinaldehyde, retinoic acid and retinyl acetate. Cytoplasmic binding proteins for retinol and retinoic acids are present in a wide variety of tissues (Ong et al, 1982). Cytoplasmic retinol binding protein is highly specific, binding only to retinol but not to retinaldehyde, retinoic acid nor retinyl esters. However, CRABP can bind to 13-cis- 20 retinoic acid, as well as to all-trans-retinoic acid (Wolf, 1984). Vitemin A homeostasis The concentration of vitamin A in plasma is highly regulated. Plasma retinol concentration is fairly constant, independent of diet and liver stores. With very high consumption of vitamin A, plasma concentrations of vitamin A increase simultaneously with the appearance of retinyl esters in the circulation. Decreases of serum vitamin A concentration are observed when liver stores are completely exhausted (Sklan, 1987). The presence of retinyl esters in the circulation in human beings and rats indicates hypervitaminosis A (Sklan, 1987). However, even under normal physiological conditions, vitamin A in the circulation of dogs is in .the form of retinyl esters (stearate and palmitate) bound to lipoprotein (Schweigert et al, 1990). Under normal - physiological conditions, retinol_ circulates in the blood as 1:1:1 trimolecular complex with RBP and TTR (Goodman, 1984). Four processes inside hepatocytes regulate the release of stored retinol to the circulation. The processes are 1) hydrolysis of stored retinol to free retinol, 2) synthesis of RBP, 3) attachment of retinol to RBP and 4) release of retinol-RBP-TTR complex. A review by Wolf (1984) postulates that the normal homeostatic set point for vitamin A may be regulated by an 21 extrahepatic signal, since the plasma retinol concentration is independent of the hepatic concentration of vitamin A. Retinol metabolites such as retinoic acid may also serve as a regulatory signal for' homeostasis. Administration of retinoic acid lowered the plasma homeostatic set point, presumably due to a reduction of the tissue utilization and mobilization of retinol. Gerlach and Zile (1990) suggested that the kidney may have a direct or indirect role on vitamin A homeostasis. In a study using nephrectomized rats or rats with signs mimicking acute renal failure, they found that an increase in circulatory retinol concentrations was not associated with increased enzyme activity necessary for the deposition and/or release of vitamin A. They suggested that the kidney may serve as a producer of a specific regulatory substance. A decrease in the substance may act as a stimulus for the release of liver retinol or possibly the kidney fails to remove a peripheral signal that increases the homeostatic set point. Transthyretin plays an important role in retinol transport and homeostasis. Disruption of the transthyretin (ttr) gene in mice results in a depression of plasma retinol and RBP to 3% and 6% of normal concentrations, respectively (Episkopou, 1993). 22 Effect of vitemin A status in the inteetine Using tritiated thymidine in rats, Zile et a1 (1981) indicated that at a very early stage of vitamin A deficiency, the DNA labeling index in jejunal crypt cells was not altered. However, a similar study has revealed that in more advanced stages of vitamin A deficiency, the jejunal crypt cell cycle is slowed by 14% due to a lengthening of DNA synthesis rate (Zile et al, 1977). Vitamin A deficiency decreased the number of goblet cells in the duodenal mucosal epithelium and reduced the synthesis of protein by membrane bound polyribosomes in the rat intestinal mucosa (De Luca et al, 1969). Readministration. of vitamin A to vitamin A deficient rats increased nuclear RNA Synthesis in the intestinal epithelial mucosa (Johnson et al, 1969; Zachman, 1967) and increased the number of goblet cells (De Luca et al, 1970). However, Rojanapo et a1 (1980) showed that reduced numbers of goblet cells in the duodenal mucosal cells of rats was only apparent 4 days after the withdrawal of retinoic acid from the diet. Subsequently, goblet cell numbers returned to normal. Furthermore, they suggested the presence of two different populations of goblet cells in the intestine, one relatively insensitive and one sensitive to vitamin A deficiency. Molecular biology of vitemin A De Luca (1977) reviewed the molecular role of retinol and retinoic acid. He suggested that retinol is involved in 23 the direct transfer of mannosyl residues to membrane glycoproteins through phosphorylation and glycosylation pathways. Retinoic‘ acid might also have a similar involvement in mannosyl transfer. However, it is not known whether the receptor molecule(s) is a constituent of the cell membrane or a secretory product. Increased mannose incorporation is also associated with an increase in adhesive properties of spontaneously transformed mouse fibroblasts (De Luca et al, 1979). Substantial evidence has been reported for the nuclear activity of retinoids. In vivo and in vitro studies indicate that retinol increases RNA synthesis and uridine incorporation into RNA in the mucosa of the small intestine and colon of vitamin. A deficient rats (Zachman, 1967). Similarly, potassium retinoate increases uridine incorporation into the intestinal mucosa and liver "rapidly labeled" nuclear RNA (Johnson et a1, 1969). Furthermore, the finding' of a nuclear receptor for retinoic acid in the human chrbmosome, hRAR (Petkovich et' al, 1987) or hRR (Giguere et a1, 1987), has partially elucidated the molecular mechanism of retinoids in cellular proliferation and differentiation. The retinoid receptors are structurally similar' to steroid and thyroid hormone receptors which may act as either a transcriptional mediator or as a transcriptional regulator where their interaction with retinoic acid induces a cascade of regulatory events controlling morphogenesis and homeostasis or acts as a 24 negative regulator of oncogenesis. A review by Desbois (1993) classified these receptors as the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) which are apparently universal among vertebrates. The proposed physiological ligand for RARs is all-trans-retinoic acid and the ligand for RXRs is the stereoisomer 9-cis—retinoic acid. All-trans-RA binds only to RAR, whereas 9-cis-RA binds both RARs and RXRs. However, both RARs and RXRs can be activated by either all-trans-RA or 9-cis-RA. :n: is often observed that the induction of cell differentiation is associated with a decrease in cell proliferation after the addition of retinoids, and it has been suggested that retinoids may disturb both processes independently. ' Additional data indicated that both RARs and RXRs positively control genes involved in promoting cell differentiation through their binding to retinoid-response elements. However, RARs also indirectly repress expression. of .AP-l-regulated. genes by inactivating AP-l transcription factor (Figure 2). Genes involved in the control of cell division are included in the genes regulated by AP-l transcription factor. POLYAMINES The polyamines, spermidine and spermine and diamine putrescine, are found :hj all prokaryotiC’ and eukaryotic cells. They are polycationic, aliphatic, nitrogenous compounds with a low molecular weight. Polyamines play an important role in cellular growth and differentiation. 0 25 RAR RXR nonfunctional AP-l ‘ RRE GENEI ] —{TREH GENEII L——¢> Figure 2. Mode of action of retinoid receptors. RAR, RXR, RR, RRE and TRE are the respective abbreviations of retinoic acid receptors, retinoid X receptors, retinoid receptors, retinoid-response element and tissue plasminogen activator (TPA)-receptor element. Gene I refers to genes, whose expressions are regulated by retinoid receptors, involved in promoting cell differentiation. Gene II refers to genes, whose expressions are regulated by AP-l, involved in the control of cell division (modified from Desbois, 1993). 26 Although polyamine cellular concentrations are highly regulated, their exact role in cellular growth and differentiation is unknown. Generally, prokaryotes have a higher concentration of putrescine than spermidine, and lack spermine. Eukaryotes, on the other hand, have little putrescine but contain high concentrations of spermidine and spermine (Heby, 1981). The cellular concentration of polyamines can be artificially depleted by either selection of mutant bacteria or by the use of various enzyme inhibitors for polyamine biosynthesis in mammalian cells. Metabolism The pathway for polyamine biosynthesis and the enzymes involved in their synthesis are shown in Figure 3, and the structure of polyamines and one of their biosynthetic inhibitors, DL-d—difluoromethylornithine (DFMO), are shown in Figure 4 (Pegg and McCann, 1982; Heby, 1981; McCann et al, 1987). Putrescine is a precursor of polyamines. In mammalian cells, putrescine is formed from ornithine by the action of ornithine decarboxylase (ODC). Ornithine is available directly from plasma or it may be formed from arginine by the action of arginase. In many microorganisms, putrescine! can be formed from agmatine by the action of agmatinase. Agmatine is formed from arginine by the action of arginine decarboxylase, an enzyme that mammalian cells lack. Putrescine is then converted to spermidine with the 27 ORNITHINE 1 Methionine up c°2 p-Aianine P1+PPi Pmummnmz aqwflmu¢r , O (.02 91W “2 2 02 Docarbox- SAM SM! 3 Nl-Acetylspermidinc 5' -Mothyl- thioadonosino CoA Methionine 5 B-Alanine ATP AcetleoA / SPERMIDINE - P1+PP1 3 Ac. do co; 6 prepaid-.1120: 0: SAM Decal-box- 2 SAN Nl-Acotylsponnino 4 S ' -Methyl- thicadonooino . m 5 AcetleoA 3m Figure 3. Metabolic pathway of polyamines. Enzymes involved in the polyamine metabolism are ornithine decarboxylase (1), S—adenosyl-L-methionine decarboxylase (2), spermidine synthase (3), spermine synthase (4), acetleoAzspermidine/ spermine Nl-acetyltransferase (5) and polyamine oxidase (6) (modified from Seiler, 1989). COOH H2NCH2CH2CH2CHNH2 H2NCH2CH2CH2CH2NH2 H2N(CH2)3NH(CH2)4NH2 H2N(CH2)3NH(CH2)4NH(CH2)3NH2 CH3CONH(CH2)3NH(CH2)4NH2 NH2 N ’ N U‘> \ N N COOH CH3 H2NCHCH2CH23§ O OHCNi NH2 N" N I‘) K N W3 H2NCHCH2CHZE§ 0 (HIGH NH2 N" N |‘> K N CH S 3 o OHCNI CHFZ H2N(CH2)3c—NH2 COOH 28 Ornithine Putrescine Spermidine Spermine Nl-Acetylspermidine S-Adenosylmethionine (SAM) Decarboxylated S-Adenosylmethionine (SAM-DC) Methylthioadenosine a-Difluoromethylornithine (DEMO) Figure 4. Structure of polyamines and related compounds (Bitonti and McCann, 1987; Mondovi et al, 1989). 29 addition of an aminopropyl compound from the decarboxylated S-adenosylmethionine. The conversion of putrescine to spermidine is catalyzed by spermidine synthase. The formation of decarboxylated S- adenosyl methionine is catalyzed by S-adenosylmethionine decarboxylase (SAM-DC). The action of S-adenosylmethionine is activated by putrescine and repressed by spermidine. Spermidine is converted to spermine by the action of spermine synthase. The conversion of spermidine to spermine also needs an aminopropyl moiety from the decarboxylated S- adenosylmethionine. Spermidine and spermine synthase reactions are irreversible; interconversion between polyamine products can occur in vivo. The interconversion needs acetyl CoA and the action of spermidine N'-acetyltransferase and polyamine oxidase enzymes. Enzymes for polygfline bioevntheeie Inhibitors for CDC and SAM-DC enzymes have been widely used to study the effects of both in vivo and in vitro polyamine depletion in various cells or tissues. Many studies have indicated that the activity of enzymes involved in 'polyamine biosynthesis. and cellular concentrations of polyamines are associated with tissue growth, Luk Hand Baylin (1983) found increased activities of ODC and SAM-DC, as well as their synthetic products (putrescine and spermidine), in rat ileal mucosa after intestinal resection. 30 These increases were closely associated with crypt cell production rate, the rate of villous and crypt lengthening, mucosal thickening and mucosal DNA content and synthesis. Correlation coefficients between the ODC activity and crypt cell production rate, villous length and crypt lengthening rate were 0.97, 0.98 and 0.94, respectively. Except in the prostate, the ODC activity is extremely low in "nondividing" cells. In early phases of cellular response to a variety of hormones and other substances that stimulate growth and division, the ODC activity increases significantly up to a thousandfold (Heby, 1981). Studies using ODC inhibitors revealed that polyamines are important for cell proliferation and differentiation. Mamont et al (1976) suggested that continual synthesis of polyamines was necessary to maintain cell division processes. Addition of the ODC inhibitor DL-a-methyl ornithine to rat hepatoma tissue culture (HTC) cells reduced the concentration of putrescine and spermidine, parallel to the reduction of icell proliferation and thymidine incorporation into DNA. Addition of exogenous putrescine, spermidine or spermine restores the proliferation of the inhibited HTC. Although the concentration of spermine was not decreased by the presence of DL-a-methyl ornithine, addition of spermine restored cell proliferation, probably due to its conversion to spermidine. 31 Intestinal adaptation, a proliferative response of the small intestine to a variety of stimuli, such as intestinal resection, cold exposure and lactation, has been shown to be affected by ODC activity and the concentration of intestinal mucosal polyamines. Yang et al (1984) showed that an increase in the ODC activity is not the only phenomenon observed during intestinal adaptation in rats but that it plays an important role in mucosal hyperplasia during lactation“ During the adaptation, increases in mucosal weight and thickness were accompanied by an increase in ODC and SAM-DC activities and polyamine content. Addition of DFMO, resulted in the suppression of both ODC activity and the adaptive response of the intestine during lactation. Using DFMO on the jejunectomized rat, the suppression of polyamine synthesis was associated with the suppression of DNA synthesis and resulted in no increases in intestinal weight, mucosal thickness or crypt cell production (Luk and Baylin, 1984). Accumulation of putrescine rather than spermidine- is needed for DNA synthesis ‘after partial jejunectomy in the rat (P656 and Pegg, 1982). An increase in total RNA, DNA and protein concentrations was also observed in the enterocytes proximal to the ligated intestine. Increases in total RNA, DNA and protein concentrations were lower in the DFMO treated rats (Seidel et al, 1984). Also, DFMO prevented intestinal repair and the recovery of DNA, RNA and protein concentrations in the duodenal mucosa of the rat following 32 stress (Wang and Johnson, 1991), and resulted in delayed intestinal mucosal maturation. and recovery subsequent to injury (Luk et a1 1980). A similar phenomenon was also observed. in. rats infected. with the nematode Trichinella spiralis (Wang et al, 1991). As an ODC inhibitor, DFMO, has an effect on intestinal mucosal cell proliferation in young animals. Addition of DFMO to the diet of weaning rats causes villous atrophy and significant decreases in small intestinal mucosal weight, total protein, DNA and various intestinal enzymes (Alarcon et a1 1987). Holland et a1 (1992) have also observed that the addition of DFMOI to Cryptosporidium parvum infected calves resulted in more severe villous atrophy. Addition of exogenous polyamines bypasses some of the effects of the inhibited ODC enzyme in various organs. Luk (1986) showed that the addition of putrescine to DFMO + hepatectomy treated rats reversed the inhibitory effect of DFMO on ODC activity. Protein synthesis, DNA synthesis and 'liver‘ weights in hepatectomized 'rats receiving DFMO and putrescine reached similar concentrations of those in hepatectomized rats without DFMO. However, the addition of putrescine had no effect on hepatectomized rats which did not receive DFMO. In addition, putrescine or diethylamine stimulate enterocyte proliferation and partially prevent the reduction of small intestinal absorption in calves caused by a soybean protein diet (Grant et a1, 1989). 33 Role of polyamines ineeell proliferation Spermidine plays an essential role in DNA synthesis by stabilizing DNA folds (Flink and Pettijohn, 1975), activating DNA-dependent DNA polymerase (Chiu and Sung, 1972), and. stimulating’ the :replication of single strand phage DNA (Wickner et al, .1973). Mannen and Russell (1977a,b) indicated that ODC increases the rate of initiation of the RNA polymerase I reaction. Putrescine, on the other hand, has no effect on the RNA polymerase I activity. Young and Srinivasan (1972) showed that the addition of putrescine into a polyamine deficient E. coli mutant resulted in the resumption of protein synthesis before the synthesis of DNA and RNA. Furthermore, Geiger and Morris (1980) showed that polyamines affect the DNA replication fork movement rather than the initiation of DNA synthesis. Shinki et a1 (1991) suggested a possible association between putrescine and vitamin D3 action. Administration of vitamin D to vitamin D deficient chicks significantly increased intestinal calcium transport activity. Administration of polyamine synthesis inhibitors, DFMO or N1,N4-bis(2,3-butadienyl)-1,4-butanediamine, to tflua chicks decreased the calcium transport activity induced by vitamin D3. The decrease in the calcium transport can be completely restored by supplementation with putrescine. 34 ASSOCIATION OF VITAMIN A AND POLYAMINE BIOSYNTHESIS Several studies on the interaction of vitamin A and polyamine ibiosynthesis during' cell proliferation and differentiation have been done. Studies where various retinoids have been given in association with other substances have focused on the effect of the combined substances on the CDC activity and the synthesis of polyamines, RNA and DNA. A study on ODC activity using Chinese hamster ovary (CHO) cells revealed that retinoids inhibit ODC induction during G1 progression (Russell and Haddox, 1981). In the G1 phase, ODC synthesis is transcriptionally regulated by the activation of a cAMP-dependent protein kinase. Retinoid did not affect the production of the cAMP-dependent protein kinase nor the production of other proteins required for ODC synthesis. However, in the retinol treated cells, there was no increase in RNA synthesis during G1 phase. Furthermore, they suggested that the specific effect of retinol was on the inhibition of messenger RNA synthesis for ODC prOduction during the G1 phase. The ODC activity appeared to increase again when the cells progressed to the S phase. Using an initiation-promotion ‘model system” a study involving the effect of retinoids on skin carcinogenesis in mice (Boutwell and Verma, 1981) revealed that retinoic acid increased ODC activity and tumor formation when applied with dimethylbenzanthracene (DMBA), a known tumor initiator. 35 However, when retinoic acid was applied in conjunction with phorbol ester, a cancer promoter agent, it significantly reduced ODC activity and tumor formation. The DNA synthesis, measured by thymidine incorporation, was significantly reduced initially but increased above normal 42 hours after phorbol ester treatment. In addition, since the phorbol ester acts through stimulation of the AP—l complex, inactivation of AP-l by RARs (retinoic acid receptors) might provide an explanation for the reduction of tumor formation (Desbois, 1993). Daliam et al (1988) showed that vitamin A deficiency increased the basal activity of ODC in the lung, esophagus and liver of rats. Addition of 100 ug'of retinoic acid significantly reduced or normalized the ODC basal activity in the lung and esophagus, but increased the ODC activity in the liver. ROTAVIRUS Rotaviruses are major viral agents causing acute gastroenteritis and diarrhea in young children (Banatvala et al, 1978) and a wide variety of young animals including calves (Mebus et al, 1969; Hall et al, 1993), piglets (Bohl, 1979), foals (Traub-Dargatz, 1988), suckling mice (Ijaz et al, 1989), and birds (McNulty et al, 1978). Rotavirus may also cause severe diarrhea in adult human beings (Bridger, 1987). 36 Rotaviruses, member of the genus Reoviridae, are nonenveloped double-shelled . infectious particles that possess 11 segments of double-stranded RNA genome. The complete virion has an average diameter of 60-70 nm (Estes et a1 1983). The name rotavirus was suggested due to the morphological resemblance of the virus to small wheels (Flewett and Woode, 1978). Three types of particles, including double-shelled, single-shelled and core particles, can be seen ultrastructurallyu Only the double-shelled particles are infectious (Estes et a1 1989). Rotaviruses are extremely infectious. As low as 90 OSU strain porcine rotavirus particles may induce infection in piglets (Payment and Morin, 1990). Graham et a1 (1987) indicated that the lowest dose for the same strain of rotavirus was 1 PFU. Classification end eerotvping Initial serological studies on rotaviruses isolated from cases of gastroenteritis in children, calves, piglets, mice and foals have indicated that the viruses share common group antigens located within the inner capsid of the virus, irrespective of their species of origin. The common group specific antigens were demonstrable with a variety of techniques including complement fixation (CF), immunofluorescence (FA), gel-diffusion (GD) and immunoelectron microscopic (IEM) tests (Woode et al, 1976; Flewett and Woode, 1978). 37 Further findings of viruses that are morphologically identical to but antigenically distinct from conventional rotaviruses have led investigators to classify the viruses into 6 groups designated as group A, B, C, D, E and F, based on the presence of group specific antigens (Bridger, 1987; Pedley et al, 1983, 1986). Reference virus isolates for grouping were the Ohio State University (OSU) strain for group A, NIRD-l for group B, Cowden and S strain for group C, strain D/132 for group D and strain E/DC-9 for group E. Bohl et al (1984) have Ifurther classified porcine rotaviruses using a plaque reduction neutralization (PRN) test (n1 MA-104 cells, cross protection studies in gnotobiotic piglets and electropherotype'migration of the RNA genome. Porcine rotaviruses can be divided into 2 subgroups on the basis of the ability to produce plaques on initial culture isolation. Subgroup or biogroup 1, which produces plaques, contains the OSU, EE and A-580 strains. Subgroup or biogroup 2, which is unable to produce plaques, consists of Gottfried (G), SB-lb, SB-3 and 88-5 strains. Although serotyping was done based on the antigenic differences and similarities found in the PRN test and cross protection studies, classification of the rotaviruses based on serotyping has been somewhat variable. For example, Bohl et a1 (1984) classify the OSU strain as serotype 1 and the G strain as serotype 2, whereas Hoshino et a1 (1984) classify these strains as serotype 5 and 4, respectively. In addition to the neutralization test, Sato et a1 (1982) used 38 an immunofluorescence technique for serotyping rotavirus isolates. Genome and virel proteine Electropherotype studies indicate that rotavirus contains an 11—segmented genome. Each segment encodes either a structural or nonstructural viral protein. Nine structural proteins (VP1-VP9) and 4 nonstructural viral proteins have been identified in rotavirus SA11. Inner capsid structural proteins, VP1, VP2 and VP6 are coded by gene segments 1, 2 and 6, respectively. Outer capsid protein VP3 is coded by gene segment 4. Precursors for outer capsid viral proteins, VP7 and VP9, are encoded by gene segments 9 and 11, respectively. Gene segments 5, 7, 8 and 10 code for :non-structural 'viral. proteins (Estes et .al, 1933). Biological properties of both structural and nonstructural glycoproteins of some rotaviruses have been studied. The outer capsid protein VP4 has been implicated in several important functions such as cell penetration, hemagglutination activity, generation of neutralizing antibody, and virulence (Burns et a1, 1988). Prasad et a1 (1990) showed that the surface spikes on rotavirus particles were made up of VP4. Ball et a1 (1996) showed that the rotavirus nonstructural glycoprotein NSP4 acts as a viral enterotoxin. by jpotentiating’ chloride secretion through a calcium-dependent signaling pathway. 39 Serotype specificity has been attributed to the outer capsid protein VP7 (Kalica et al, 1981). Studies on bovine rotavirus (neonatal calf diarrhea virus, NCDV) and simian rotavirus (SA11) have revealed that two genes encoding viral proteins VP3 and VP7 - gene segments 4 and 9, respectively - cosegregate with neutralization specificity (Offit and Blavat, 1986). Reassortant (recombinant) rotaviruses containing VP3 and VP7 from different parent rotaviruses induce a protective immune response against both parental serotypes (Offit et al, 1986a,b). There is also evidence that interaction among viral proteins and calcium availability is required for the virus to function. In calcium-deprived‘ cultures, the concentration of the outer capsid protein (VP7) decreases due to degradation (Shahrabadi et al, 1987). One study has determined that neutralization of VP7 requires interaction of VP7 with other rotaviral proteins (Dormitzer et al, 1992). Viral entry and growth Results of most rotaviral studies indicate that oral transmission is the common method of infection. Experimentally, the infection can also be transmitted via aerosol droplets in suckling rats (Prince et al, 1986). In a study using MA104 cells, Suzuki et a1 (1985) indicated that the KUN strain of human rotaviruses entered the cells by direct penetration of the nucleoid through the cell 40 membrane, and not by endocytosis as previously believed (Petrie et al, 1982). Furthermore, Kaljot .et a1 (1988) found that addition of endocytosis inhibitors, such as sodium azide and dinitrophenol, had only a limited effect on viral entry and that neutralizing antibodies can inhibit direct membrane penetration. Pretreatment with trypsin resulted in cleavage of VP3 and dissolution of the virus capsid within the cell membrane, followed by direct penetration of the viral nucleoid into the cytoplasm, probably through a protease-dependent mechanism. The non- trypsin treated virus (with intact VP3) enters the cell through the relatively slower endocytic pathway and is ultimately degraded in lysosomes (Kaljot et al, 1988). Virus replication has been studied with bovine and simian rotaviruses. Maximal protein synthesis of simian rotavirus SAll was noted. within 3-5 hours postinfection (Ericson et al, 1982). A review by Estes et a1 (1983) indicated that animal rotaviruses have fairly rapid generation cycles. A complete replication Cycle was achieved within 12 hours, with a 2- to 3-hour viral eclipse phase. In experimentally infected suckling rats, rotavirus antigen was excreted in a biphasic pattern. Following oral inoculation, the antigen was detected at 1, 4 and 5 days postinfection in 100% of the experimental animals, whereas, at 2 and 3 DPI, antigen excretion was only detected in 70 and 20% of the experimental animals, respectively (Vonderfect et al., 1988). am 90: Bat beg Sm Snc Egg 41 In addition to enterocytes, viral antigen can also be found in other tissues. Following infection, rotavirus has been found in the lungs of rats (Prince et al, 1986) and in the brain and spleen of pigs (Shaw et al, 1989b). Factors affecting infection Rotaviruses cause severe disease in young animals and human beings. Age-related resistance has been reported in mice (Patterson, 1987; Ball et al, 1996), but not in pugs (Gelberg, 1992), human beings and cattle (McNulty, 1978). Breast-feeding or continual administration of colostrum is an important factor in protection against enteric pathogens. Total protection against rotavirus can not be achieved with breast-feeding, but breast-feeding lessens the severity of diarrhea and vomiting associated with a rotavirus infection. Immune colostrum protects or lessens the severity of rotavirus infection in young animals and human beings. Using cow milk collected at various times, ranging from 8 hours to 10 days postparturition, colostral antibody titers against rotavirus ranged from a high 8 hours postpartum to no titer on day 10 postpartum (Acres and Babiuk, 1978). Administration of colostral supplements have been shown to have a protective effect or lessen the severity of rotavirus infection in calves (Saif et al, 1983; Snodgrass and Wells, 1978). Antibody directed against rotavirus present in chicken eggs is capable of inhibiting the growth of some rotavirus 42 strains in tissue culture (Yolken et al, 1988). Antibody levels can be increased by immunizing hens with rotavirus. The antibody can then be used to prevent the development of rotaviral enteritis in experimentally infected animals (Yolken et al, 1988). In, addition, Archambault et a1 (1988) have shown that colostral antibodies and colostral lymphocytes from immune cows have a protective effect on calves infected with rotavirus. The continual presence of antibody in the small intestinal lumen appears to be the prime mediator of protection against rotavirus infection (Woode et al, 1975; McNulty et al, 1976). In addition, virus-neutralizing antibody present in the intestinal lumen is predominantly in the form of IgA (Chanock, 1978; Burns, 1996). Recently, Burns et a1 (1996) indicated that rotavirus VP6—specific IgA has the capability to inactivate rotavirus intracellularly. Besides antibodies and lymphocytes present in the milk, another substance has been shown to have a protective effect against rotavirus infection. Mucin, a macromolecule found in human milk, directly binds to rotavirus and inhibits viral replication both in vivo and in vitro (Yolken et al, 1992). Polyions having negative charges, such as mucin, d- 1-acid glycoprotein heparin, heparan sulphate and dextran sulphate, inhibit rotaviral infectivity and replication, whereas polymers having positive charges, such as protamine, protamine sulphate, DEAR-dextran, histone and poly-L-lysine 43 hydrobromide, enhance viral infectivity (Superti et al 1993). A trypsin inhibitor present in human milk has also been shown to have a protective effect against rotavirus infection in infants (McLean and Holmes, 1978). The presence of rotavirus severely enhanced the lesions caused by enterotoxigenic E. coli in neonatal gnotobiotic calves (Torres-Medina, 1984). Similarly, Hall and Parsons (1989) showed that the combined effect of weaning and rotavirus infection in gnotobiotic piglets led to more severe damage in the distal part of the small intestine, than rotavirus infection alone. Vitamin A deficiency caused dramatic lesions in rotavirus-infected mice (Ahmed et al, 1990). In the small intestine of ‘vitamin Ix deficient mice, rotavirus caused almost complete destruction of the tips of the villi compared to the control animals. They concluded that although rotavirus infection and vitamin A deficiency caused few changes alone, their combined effect caused significant destruction of the mucosal barrier of the small intestine. Many authors have described the clinical signs associated with rotavirus infection, both experimentally and under natural conditions, in infants and animals. Among the clinical signs, diarrhea is the most prevalent. Vomiting is inconsistently seen in piglets. In infants, fever and vomiting also quite often accompany the diarrhea. 44 Pigs develop clinical signs, including depression, anorexia, vomiting and diarrhea, starting from 12-18 hours postinfection (Theil et a1, 1978). In a study by Svensmark et al (1989a,b), profuse watery diarrhea, weakness and dehydration were observed 1-2 days after inoculation and the surviving pigs recovered in 1—2 weeks. Later, McAdaragh et a1 (1980) reported that diarrhea in gnotobiotic piglets caused by rotavirus was usually observed within 12 to 96 hours postinfection. ffiua fecal consistency varied from a mixture of solids and liquid to a liquid with no detectable solids. The fecal consistency returned to normal 96 hours later. Diarrhea, indicated by increased quantities of bright yellow feces, and anorexia were noted in calves experimentally infected with a virulent strain of bovine rotavirus (Hall. et al, 1993). ‘Woode and. Crouch (1978) reported that calves either infected with human or bovine rotaviruses showed clinical signs of depression, weight loss and diarrhea. The diarrhea consisted of watery light green feces with mucus and fresh blood or yellow-white feces. Some of the experimental calves took 28 days to regain their birth body weight. Lesions The virus causes lesions restricted to the small intestine. The lesion usually is comprised of detached absorptive epithelial cells leading to villous atrophy. In 45 piglets, the earliest evidence of epithelial detachment and villous atrophy was noted 12 hours postinfection in the proximal jejunum and by 24 hours, the lesion extended to the distal jejunum and proximal ileum (McAdaragh et al, 1980). Villous atrophy in the caudal two-thirds of the small intestine is the consistent lesion caused by the OSU strain of porcine rotavirus (Theil et al 1978a). Villi were shortened, blunted and covered with cuboidal epithelial cells. McAdaragh et al (1980) on the other hand, found that the lesion was present throughout, but variably distributed in, the small intestine and frequently, both atrophic and normal villi were present within the same intestinal section. The presence of detectable rotavirus antigen in the small intestinal mucosal cells always precedes the onset of diarrhea and declines after the onset of diarrhea. With an immunofluorescence technique, Theil et al (1978a) reported that the OSU strain of porcine rotavirus antigen was (demonstrable throughout the small intestine but was detected most consistently in the jejunum and ileum as early as 4 flours postinfection. Within 16-24 hours postinfection almost all columnar epithelial cells covering the apical Ilalf of the jejunal and ileal villi were infected. After 24 llours, the number of infected cells became fewer, but the (:ells still remained positive up until 96 hours Ipostinfection. Rotavirus antigen was not detected after 168 ‘hours. These results differ from the finding of McAdaragh 46 et a1 (1980). They found that the rotavirus antigen was first detected in the duodenum and progressed to the distal part of the small intestine. In a study in calves experimentally infected with either one of two bovine rotaviruses of different virulence, Hall et a1 (1993) reported that even though both strains produced small intestinal lesions, villous atrophy and fusion were more consistently observed in calves infected with the virulent strain. Immunohistochemical staining revealed that the virulent strain was primarily found in the mid and distal small intestine whereas the low-virulent strain was mostly found in the proximal and mid small intestine. Diagnosis A definitive diagnosis of rotavirus infection is based on the finding of rotaviral antigen in the diarrheic specimen and its correlation with histological lesions. The presence of _ rotaviral antigen can be demonstrated, with a variety of methods including electron microscopy (Flewett, 1 9 78) , immunoelectron microscopy, complement fixation, Counter-immunoelectroosmophoresis , immunof luorescence , en zyme-l inked immunosorbent assay , radioimmunoassay , hemagglutination assay, cytopathic effect and peroxidase- antiperoxidase technique (Estes et al, 1983) . A solid—phase iI'l'ul‘lune electron microscopy double-antibody colloidal-gold technique has been recently developed. The technique is 800 47 times more sensitive than direct EM technique (Wu et al, 1990). CHAPTER 1 EFFECT OF DAILY DIETARY ADMINISTRATION OF RETINYL PALMITATE AND/OR PUTRESCINE ON THE CLINICAL SIGNS, LESIONS AND ROTAVIRAL ANTIGENIC EXPRESSION IN ROTAVIRUS-INFECTED PIGLETS ABSTRACT Experiments were conducted to determine the effects of daily, dietary administration of vitamin A and/or putrescine on the clinical signs, lesions and rotaviral antigenic expression in rotavirus-infected piglets. One hundred and twenty-eight piglets were removed from the sows at two days of age. Starting on day 3, piglets were randomly divided into 4 groups of 32 piglets. Three of the groups received milk containing either retinyl palmitate, putrescine or a combination of retinyl palmitate and putrescine. One group received milk only. On day 5, half of each group was infected with porcine rotavirus. Piglets were euthanatized on days 6, 7, 8 and 13 (1, 2, 3 and 8 days postinoculation). Results of this experiment indicate that treatment failed to produce any significant effect on the course of rotaviral infection in piglets, including clinical signs, gross and histologic lesions, and the expression of rotavirus antigen in the small intestinal mucosa. 48 49 INTRODUCTION Rotavirus is the most- frequent cause of viral gastroenteritis in young children (Estes, 1983) and animals (Holland, 1990). Rotaviral infections may be asymptomatic (Banatvala and Chrystie, 1978), or cause mild to severe disease which may result in death (Rocchi et al, 1981; Carlson et a1, 1978). Common clinical signs of rotaviral infection in young animals are diarrhea, vomiting and dehydration (Mebus et al, 1969; Collins et al, 1989; Theil et al, 1978). Lesions induced by rotavirus are limited to the small intestine. Rotavirus infects mature enterocytes, resulting in intestinal epithelial cell desquamation followed by ‘villous atrophy (McAdaragh et a1, 1980; Theil et a1, 1978; Woode and Crouch, 1978). The desquamated epithelial cells are replaced by immature squamous to cuboidal cells which lack both digestive and absorptive capacities. The loss of digestive and absorptive capacities leads to an osmotic ciiarrhea due to nutrient (primarily carbohydrate) malabsorption (Graham et al, 1984). Severity of rotaviral infections, as indicated by the number of deaths, slowed weight gain and the severity of diarrhea, may be influenced by pre- and postnatal dietary effects. Low caloric and/or low protein diets increased the SeVerity of rotaviral infections in suckling mice (Noble et al I 1983) . Also, malnourishment increased the 50 susceptibility and the severity of rotaviral infection (Offor et al, 1985), as well as rotaviral antigenic expression in small intestinal enterocytes (Riepenhoff-Talty et a1, 1985, 1989) when compared to well-nourished suckling mice. Additionally, vitamin A deficiency caused more severe clinical signs in rotavirus-infected mice than in mice with adequate vitamin A status (Ahmed et al, 1990). Vitamin A and putrescine have received much attention because of their potential beneficial effects in mucosal cell regeneration. Vitamin A affects the growth and differentiation of various epithelial tissues, including small intestinal epithelial cells (Wolf, 1984). Supplementation of vitamin A to children living in vitamin A deficient populations in many developing countries has been reported to reduce the morbidity and mortality rate associated with diarrheal and respiratory diseases (Sommer est al, 1984, 1986; Bloom et al, 1990). Ahmed et al (1990) .indicated that the degree of destruction of small intestinal epithelium in rotavirus-infected mice was negatively associated with the concentration of vitamin A. Rotaviral .irifection in vitamin A deficient mice caused severe (Esaithelial damage when compared to the infection in normal nnj.ce. Putrescine has been reported to improve small intestinal functions and maintain normal morphology in Calves fed soy protein. Villous atrophy occurs in calves fed diets containing soy protein (Kilshaw and Slade, 1982). 31 SC at ti us re in pi if in. ad: 51 Supplementation with putrescine in calves' diets containing soy protein prevented a reduction in small intestinal absorption and enhanced enterocyte proliferation caused by the soy protein diet (Grant et a1, 1989). The purpose of this study was to evaluate the usefulness of vitamin A and/or putrescine in preventing or reducing rotaviral infectivity and the clinical signs and intestinal mucosal damage caused by a rotaviral infection in piglets. The objectives of this study were 1) to determine if the clinical signs and lesions produced by a rotaviral infection in piglets could be altered by the daily dietary administration of vitamin A Land/or putrescine and 2) to determine if rotaviral antigenic expression in the small intestinal mucosa of piglets could be altered by the daily dietary administration of vitamin A and/or putrescine. MATERIALS AND METHODS Experimental animals One hundred and twenty eight, five-day-old piglets from 115 litters were obtained from the Michigan State University swine farm. All piglets received colostrum from the sows for two days. Experimental design is shown in Figure 1.1. I>ingets within the same litter were allocated for a variety Of treatments. Piglets were reared in individual cages in a temperature controlled room (30-320C). Each piglet was 552 128 newborn piglets received colostrum for 48 hours l Day 3, piglets caged individually Daily Vitamin A (30,000 IU/kg) 32 piglets Daily Daily . Putrescine Vitamin A (30,000 IU/kg) ‘Daily (25 g/kg dm) Putrescine (25 g/kg dm) Milk. °nly 32 piglets 32 piglets 32 piglets l l l r l Day 5, half of each group orally inoculated with rotavirus [ INFECTED l - H l I | NON I NFECT ED Vit Put 16 16 piglets piglets Vit—Put Milk 16 16 piglets piglets Vit Put 16 16 piglets piglets Vithut Milk 16 16 piglets piglets l I l l {Daily clinical observation] l l J 4 piglets from each group necropsied at 6,7,8 and 13 days of age l 6 segments of small intestine for histopathology, morphometric, mitotic indices, and goblet cell numbers 6 segments of small intestine for immuno- histopathology Intestinal mucosal scrapings for protein and nucleic acid analyses Liver for vitamin A analysis Figure 1.1. Experimental design. 53 given a total of 300 ml evaporated milk1 (14% dry matter) daily. The nutritional composition of the milk is shown in Table 1.1. Milk was offered three times a day at 8:00 a.m., 12:00 p.m. and 5:00 p.m. Starting from day 3, subgroup 1 (RP) was given milk containing vitamin A in the form of retinyl palmitate3 (30,000 IU/kg of milk) daily. Subgroup 2 (P) received putrescine3 (25 g/kg dry matter of milk) in the milk. Subgroup 3 (RPP) was fed milk containing retinyl palmitate (30,000 IU/kg of milk) and putrescine (25 g/kg dry matter of milk). Subgroup 4 (MO) received milk only. Addition of vitamin A and putrescine in the milk was done just prior to feeding by diluting each of the substances in approximately two ml of distilled water; On day 5, the rotavirus-infected piglet group was orally inoculated with 1 ml 10'10 dilution of the stock intestinal homogenate. Following inoculation, all animals were observed three times a day during the feeding for clinical signs of diarrhea. Body weight, as well as combined fecal and urinary output weight, were determinedi. "Weighing was performed daily prior to the morning feeding. Four piglets from each group were euthanatized on days 6, 7, 8 and 12. The time for necropsy was scheduled so that only five to six animals were euthanatized in a day. 1 Carnation Evaporated Milk, Nestle Food Co., Glendale, CA. 2 Sigma Chem. Co., St. Louis, MO. 3 Sigma Chem. Co., St. Louis, MO. 4 Mettler, Type BD6000, Mettler Instrument Corp., Highstown, NJ. 54 Table 1.1. Nutritional composition of milk used in the experiments Nutrients 100 grams of milk Calories 134 Protein, g 6.81 Carbohydrate, g 10.00 Fat, g 7.56 Ash, 9 1.55 Crude fiber, g 0 Vitamin A, IU 159 PD and 55 Necropsy and specimen collection Pigs were euthanatized by an overdose of sodium pentobarbital5 administered intravenously. Three-cm segments from six sites of the small intestine were removed immediately and placed in buffered neutral formalin for histopathologic anxi immunohistochemical examinations. The first segment was removed from the duodenum approximately 5- 10 cm distal to the pyloric junction and the last segment was obtained from the lower part of the ileum, approximately 5 cm proximal to the ileocecal junction. The remaining segments were obtained at approximately a sixth, third, half and two-thirds of the distance from the pylorus to the ileocecal junction. These segments were designated as duodenum (DU), upper jejunum (UJ), lower jejunum (LJ), mid- intestine (MI), upper ileum (UI) and lower ileum (LI). The lumen of the segments were rinsed with 10% neutral buffered formalin and the segments were then immersed in the fixative. Histopathology Formalin-fixed, small intestinal samples were routinely processed for histopathologic examination. The tissues were oriented in cross sections (ring shaped) and embedded in paraffin. Tissues were cut by a microtome into 6-um thick sections. One slide contained DU, UJ, LJ segments and the 5 The Butler Co., Columbus, OH. (n in mix A 1 int USE 'I] 56 second slide contained MI, UI and LI segments. The sections were stained with hematoxylin and eosin (H&E) and examined by light microscopy for lesions. Qualitative ELISA for rotevirus antigen in feces Pre-infected and post-infected feces of experimental pigs were assayed for the presence of rotavirus antigen using a commercially available qualitative ELISA kité. To a polystyrene tube coated with rabbit anti-rotavirus IgG, 100 pl horseradish peroxidase (HRP)-conjugated monoclonal antibody and 300 pl of 10% raw stool was added. Three hundred pl samples of an inactivated, simian rotavirus (SA- 11) suspension and the sample diluent were used as positive and negative controls. After gentle mixing, the suspension was incubated at room temperature for 60 minutes. The suspension was then discarded and the tube was washed 5-7 times with 2—4 ml distilled water per wash. To each tube a 0.5 ml chromogen substrate solution of tetramethylbenzidine was added. The tube was then covered and incubated for 15 minutes at room temperature to allow for color development. A blue color indicated the presence of rotavirus antigen. Immunohiepochemical assay Unstained 4 pm sections from six parts of the small intestine, designated as DU, UJ, LJ, MI, UI and LI, were used in an avidin-biotin-based immunohistochemical assay for 6 PathfinderTM®, Kallestad Diagnostics, Austin, TX. 57 the presence of rotavirus antigen in enterocytes. Two intestinal slides from known rotavirus-infected piglets and two intestinal slides from uninfected piglets were used as controls. The immunohistochemical assay kit and its protocol were obtained from SIGMA7. The protocol for the standard procedure outlined in the kit was followed with some modification (Parsons et al, 1984). The unstained sections were deparaffinized in two consecutive xylene baths of 10 minutes each. The sections were rehydrated by immersing the slides for 2-3 dips in consecutive baths of 100, 100, 100, 95, 95, 80, 70, 50 percent ethanol and distilled water. Then the slides were washed in PBS for 5 minutes. The slides were then placed for 10 minutes in 3% hydrogen peroxide, 10 minutes in tap water and 5 minutes in a phosphate-buffered saline (PBS) wash. The slides were then incubated with diluted goat serum (blocking reagent) for 10 minutes at room temperature. After wiping the slides and removing the excess reagent, the slides were incubated with the primary antibody (1:100 rabbit antihuman rotavirus‘ antibody8 in PBS), for 24 hours at 4°C in a humid chamber. In this step, normal rabbit antiserum containing the same amount of protein as the primary antibody was added to one positive control and one negative control slide (DeLellis et al, 1979). After a 5 minute PBS wash, the slides were incubated with the secondary biotinylated antibody 7 Sigma Chemical Co., St. Louis, MO. 8 DAKO Corp., Santa Barbara, CA. 58 (biotinylated goat-antirabbit IgG). After another 5 minute PBS wash, the slides were incubated with peroxidase reagent (ExtrAvidin®-conjugated peroxidase in buffered saline) at room temperature for 20 minutes. After a 5 minute wash in PBS, the slides were incubated with the substrate reagent (0.1% 3,3'-diaminobenzidine in PBS with an addition of a drop of 3% hydrogen peroxide per 10 m1 of substrate solution) at room temperature for 10 minutes. The slides were then washed in deionized water for 5 minutes, counterstained with Mayer's hematoxylin and mounted with a coverslip. The cytoplasm of the infected epithelial cells developed a golden-brown color. The severity of infection in each segment of the intestine was sCored as 0 if no rotavirus antigen was found in any enterocyte, 1+ if less than 5 percent of the villi contained positive enterocytes, 2+ if between 5 to 25 percent of the villi contained positive enterocytes, 3+ if between 25 to 50 percent of the villi contained positive enterocytes and 4+ if more than 50 percent of the-villi contained positive enterocytes. Statistical analysis Data were grouped based on the infection status, treatment and time. Each group consisted of four infected and four control animals as in the experimental design. Statistical analyses were performed using three-factor analysis of variance (three-way ANOVA) with main effects of treatment, infection and time (Devore, 1991). The numbers IO 5‘9 di YE daj di. Cl al‘ 91‘: 59 of levels of the three factors were denoted as 2, 4 and 4 for the infection status, treatment and time factors, respectively. Differences between group means within each factor were analyzed by one-way ANOVA followed by Tukey's procedure (Fowler and Cohen, 1990). Differences between groups were considered significant at the level of P50.05. Statistical analyses were done using SPSS Computer Softwareg. RESULTS Clinical signs Before inoculation with the SDSU strain of porcine rotavirus, piglets were active, alert and healthy. Their appetites were usually very good, indicated by the disappearance of milk from the pan. Their feces were yellowish to tan with a firm to pasty texture. Clinical signs of diarrhea were observed from day 1 to day 8 postinoculation (Table 1.2). The highest incidence of diarrhea occurred 2, 3 and 4 days postinoculation (DPI). Clinical signs in infected piglets were similar and were not altered by treatment. Diarrhea was determined by the presence of yellowish watery feces on the floor pan and hindquarters of the piglets. Occasionally, the diarrheic feces contained curds of undigested milk. Also, vomitus containing curds of undigested milk was observed with some 9 SPSS Inc., Chicago, IL. 60 Table 1.2. Number of diarrheic piglets l — 8 days postinoculation with the SDSU strain of porcine rotavirus Days postinoculation Treatment gr0u91 1 2 3 4 5 6 7 8 RP 5/162 8/12 2/8 2/4 1/4 1/4 0/4 0/4 9 5/16 6/12 2/8 1/4 0/4 0/4 0/4 0/4 RPP 7/16 6/12 5/8 3/4 2/4 2/4 1/4 1/4 MO 2/16 6/12 3/8 2/4 2/4 3/4 2/4 1/4 Group RP: 9,000 IU retinyl palmitate/day. Group P: 1,050 mg putrescine/day. Group RPP: 9,000 IU retinyl palmitate and 1,050 mg putrescine/day. Group MO: milk only. Number of diarrheic piglets/observed piglets. On days 1, 2 anui 3 postinoculation, four piglets were euthanatized, thus the decline in animal numbers. 61 infected piglets. Vomiting was observed in two piglets from the P group, one piglet from the RPP group and two piglets from the MO group. Most piglets, especially those which had diarrhea, became anorectic, indicated by their reluctance to drink milk at the time of feeding or the presence of milk remaining in the pan at the time of the next feeding. Also, diarrheic animals were mildly dehydrated. At 5 DPI, signs of diarrhea began to subside and animals regained their appetites. Most piglets recovered by 7 DPI. All piglets survived until the time of necropsy. Combined fecal and urinary weights between treatment groups of animals were not significantly different (Table 1.3). Piglets that were not inoculated‘ with the virus remained active and alert throughout the experiment. They rapidly consumed their milk and had firm to pasty, golden yellow feces. Body weight On 1, 2 and 3 DPI, infected piglets in the MO group had average body weight and daily weight gains similar to the treated groups (Tables 1.4 and 1.5). Body weights and weight gains were not significantly different. All piglets, irrespective of the treatment group, had highly variable body weights and daily body weight gains, as reflected by high standard deviations (data not shown). 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Similarly, on 2 DPI, one piglet in the RPP group had a weight loss of 1% whereas another piglet in the same group had a weight gain of 18%. Although differences were not statistically significant, all infected piglets in the supplemented groups 8 DPI had higher body weights and body weight gains than the MO group of infected piglets 8 DPI. Gross lesions The external appearance of some of the diarrheic piglets necropsied on 1, 2 and 3 DPI was: thin, dehydrated, as judged by a decrease in skin pliability and the prominence: of ibony' processes, rough. hair' coat. and fecal staining on the hindquarters. Some diarrheic piglets, especially those euthanatized 8 DPI, appeared in good condition, except for the presence of fecal staining on the hindquarters. Internal lesions in the infected piglets, regardless of treatment, were very similar. Most diarrheic piglets that were euthanatized 1, 2 and 3 DPI had minimal to no chyle in lymphatics. Piglets euthanatized 8 DPI, regardless of the state of diarrhea, had normal amounts of chyle in the lymphatics. Stomachs of all piglets contained various amounts of curdled milk. Small intestines of diarrheic piglets euthanatized 1, 2 and 3 DPI were pale, flaccid, thin-walled and contained large amounts of watery, yellow fluid. The ceca and colons of the diarrheic piglets 66 euthanatized 1, 2 and 3 DPI were distended with a large amount of watery yellow fluid which occasionally contained a small amount of curdled, undigested milk. Other organs were grossly normal. Histologic lesions Enterocyte vacuolation was seen in all of the experimental piglets (Figures 1.2 and Table 1.6), but the number of vacuolated enterocytes was higher in the infected group than in the control piglets (P<0.01). Vacuolated enterocytes contained either a single large vacuole or multiple small vacuoles. Piglets 1 DPI had less vacuolated enterocytes than piglets 8 DPI (P<0.01). The number of vacuolated enterocytes did not differ between treatment groups. Vacuolar degeneration of enterocytes (VDE) in the villi was present in almost all infected groups (Figure 1.3 and Table 1.7). The VDE was indicated by separation and clumping of epithelial cells, lack of icolumnar form, irregular appearance of the cell walls and pleomorphism. Cells had distended cytoplasms and the nuclei were displaced centrallyu The numbers of degenerated enterocytes were highest in animals 1 to 3 DPI, and lowest in animals 8 DPI (P<0.01). No VDE was observed in the control group. No differences existed in the number of degenerated enterocytes between treatment groups. 67 fl“ ‘ 7'.€r'i:_ figgggi I I 'I: ’ . ‘iifl. J‘ . . V i I. ~ 1: H a'. .r . ”Ti ’- o . - ...,- ymmwwzixu , raw/...." '9. - _. .,_ .: I“, f' ”In“ I‘M " .9M3....‘1'1/Im.m., .. ‘ "~‘ :3. ‘1 .Figure 1.2. Photomicrograph of a section of ileum from a rotavirus-infected piglet in the retinyl palmitate 'and putrescine group, 2 DPI. Normal enterocyte vacuolation. Each epithelial cell contains a single, large, clear, cytoplasmic vacuole (Hematoxylin and eosin, 120X). 68 Table 1.6. Enterocyte vacuolation1 of control piglets vs. piglets infected with the SDSU strain of rotavirus DP12 Treatment Intestinal sites4 3 gmup DU UJ LJ MI UI LI 1 Infected RP 1.25 1.00 1.25 2.75 2.75 1.75 P 2.75 3.25 3.25 3.00 2.50 2.25 RPP 3.25 2.50 2.50 2.50 1.00 1.00 MO 1.50 1.75 1.25 3.25 4.00 0.75 Control RP 3.25 3.00 3.25 3.00 2.25 1.00 P 0.75 1.25 1.25 1.25 1.25 1.00 RPP 1.50 2.00 1.00 2.00 1.50 1.00 MO - 1.00 2.75 4.00 4.00 4.00 2 Infected RP 0.75 1.50 2.50 1.75 2.00 1.25 P 0.25 0.75 1.75 3.25 3.25 1.50 RPP 2.50 2.75 3.25 4.00 3.25 1.00 MO 2.00 1.50 2.50 3.00 3.00 1.00 Control RP 0.25 - 1.00 1.00 1.00 1.00 P 1.25 1.00 2.00 1.00 1.00 1.00 RPP - - 0.75 1.75 1.50 - MO 1.00 2.25 1.75 2.00 1.00 1.50 3 InfeCted RP 0.25 1.00 1.00 2.00 2.00 1.00 P 0.50 1.00 0.50 3.25 2.25 1.25 RPP 0.50 0.50 0.50 2.00 2.00 2.00 PK) 0.75 1.50 2.25 3.50 3.50 1.00 Control RP 1.00 - - 1.50 2.00 1.00 P 0.75 -. - - - 0.75 RPP - 1.00 0.50 - 1.00 2.00 MO - - - 2.00 2.00 3.00 8 Infected A — - - 0.75 0 50 B 0 25 0 50 1.50 1.75 2.25 2 00 C - - l 75 0.50 2.00 D _ _ _ _ Control A - - - — 1.00 — B 0 50 - 0.50 - — C — - — — 2.00 1.00 D 0.25 - - - 1.00 0.50 1 Data are presented as average values (n=4). Values expressed as -: no enterocyte vacuolation, 1: < 25% villi contain vacuolated enterocytes, 2: between 26-50% villi contain vacuolated enterocytes, 3: between 51-75% villi contain vacuolated enterocytes, 4: > 75% villi contain vacuolated enterocytes. N DPI: days postinoculation. L» RP: 9,000 IU retinyl palmitate/day, P: 1,050 mg putrescine/day, RPP: 9,000 IU retinyl palmitate and 1,050 mg putrescine/day, MO: milk only. . DU: duodenum, UJ: upper jejunum, LJ: lower jejunum, MI: mid intestine, UI: upper ileum, LI: lower ileum. 69 Figure 1.3. Photomicrograph of a section of jejunum from a rotavirus-infected piglet in the putrescine group, 2 DPI. Notice enterocyte vacuolar degeneration (arrow), especially in the tips of the villi (Hematoxylin and eosin, 120X). 70 Table 2157. vacuolar“ degeneration (If enterocytes1 1J1 piglets infected with the SDSU strain of rotavirus Intestinal sites‘ DU UJ LJ MI UI LI 1 RP 0 5 0 25 1 1 5 1 1 P 2 5 3 2.25 2 1 5 0.75 RPP 1.5 1 0.75 1 5 - - MO 1.25 0.5 — 2 1.5 - 2 RP 0.75 1 5 2 1 0 5 - P 0.25 0 75 0.75 1 25 1.25 — RPP 2 2.5 2.5 1 1.5 0 75 MO 1.25 1.5 2.25 1 1 - 3 RP 0.25 1 1 2 1 - P 0.5 1 0.5 2.25 2.25 RPP 0.5 0.5 — 1 1 1 MO 0.75 1.5 2.25 2.5 2 5 — 8 RP - - - - 0.75 0.5 P 0.25 0.25 0.25 0.5 0.25 - RPP — — 0.25 - - - MO - — — - - - 1Data are presented as average values (n=4). Values expressed as -: no vacuolar degeneration of enterocyte (VDE), 1: < 25% villi contain VDE, 2: between 26-50% villi contain VDE, 3: between 51- 758 villi contain VDE, 4: > 75% villi contain VDE. 2DPI: days postinoculation. 3RP: 9,000 IU retinyl palmitate/day. P: 1,050 mg putrescine/day. RPP: 9,000 IU retinyl palmitate and 1,050 mg putrescine/day. MO: milk only. 4DU: duodenum, UJ: upper jejunum, LJ: lower jejunum, MI: mid— intestine, UI: upper ileum, LI: lower ileum. 71 Immature enterocytes (Figure 1.4), indicated by the presence of cuboidal and squamous epithelial cells, were seen in the infected group 1, 2, 3 and 8 DPI (Table 1.8). The infected group had significantly higher numbers of immature enterocytes than the control group (P<0.01). There were no statistical differences between treatment groups and the time after inoculation. Dilatation of villous lymphatic vessels was occasionally observed in both infected and control piglets. Desquamated cells, either from degenerated or infected enterocytes, were not detected in the intestinal lumen. Immunohistochemistry Immunohistochemical staining was used to indicate the presence of rotaviral antigen in small intestinal enterocytes. Villous enterocytes containing a fine to 'globular intracytoplasmic, golden-brown precipitate were considered positive cells. Enterocytes containing a golden— brown precipitate were seen lining the sides and the tips of small intestinal ‘villi. of some, but. not all, rotavirus— infected piglets euthanatized on 1, 2, 3 and 8 DPI (Figure 1.5, Table 1.9). When present, the predominant site of rotaviral antigenic expression 'was either the middle or posterior part of the small intestine (Table 1.10). (No rotavirus antigen was detected-in the intestinal lumen. The statistical differences between treatment groups were not significant. 72 Figure 1.4. Photomicrograph of a section of jejunum from a rotavirus-infected piglet in the milk-only group, 8 DPI. Notice the atrophic villi covered by squamous to cuboidal enterocytes (Hematoxylin and eosin, 220X). 73 Table 1.8. Immature enterocytes1 in piglets infected with the SDSU strain of rotavirus Intestinal sites4 DPI2 Group3 DU UJ LJ MI UI LI 1 RP - 0.75 0.25 - P 1.25 1.5 1 1.75 1 1 RPP — 2 1.5 0 5 o 75 M0 0.5 — - - — — 2 RP o 5 - - 1 1 1.25 P 2 2 2 o 75 o 75 0.25 RPP 0.75 - 0.25 — - 0.5 MO 1 1 - — — - 3 RP 2 1 0.5 - - — P 1.5 0.5 — ~ 0.5 1 RPP 1 1 1 1 1 — MO — — - — — — 8 RP 1 — 0.5 1 _ 1 1 P .. ._ _ _ _ _ RPP — - - - - - MO 1 1 1 1 1 1 1Data are presented as average values (n=4). Values expressed as -: no immature enterocytes, 1: < 25% villi contain immature enterocytes, 2: between 26-50% villi contain immature enterocytes, 3: between 51-75% villi contain immature enterocytes, 4: > 75% villi contain immature enterocytes. 2DPI: days postinoculation. 3RP: 9,000 IU retinyl palmitate/day. P: 1,050 mg putrescine/day. RPP: 9,000 IU retinyl palmitate and 1,050 mg putrescine/day. MO: milk only. 4DU: duodenum, UJ: upper jejunum, LJ: lower jejunum, MI: mid- intestine, UI: upper ileum, LI: lower ileum. 74 Figure 1.5. Photomicrograph of a section of duodenum showing the presence of rotaviral antigen. The section was taken from a rotavirus-infected piglet in the retinol palmitate group, :1 DPI. Notice the presence of rotaviral-antigen- positive enterocytes as indicated by the golden brown pigment. Immunohistochemical staining based on avidin- biotin binding with DAB as the chromogen, counterstained with Mayer's hematoxylin, 440x. 75 Table 1.9. Number of piglets with rotavirus-antigen— positive enterocytes, based on an immunohistochemistry Treatment Days Postinoculation Groups1 1 2 3 8 RP 2/412 1/4 1/4 0/4 P 3/4 1/4 1/4 5 1/4 RPP 2/4 1/4 4/4 2/4 MO 1/4 0/4 3/4 2/4 1 Group RP: 9,000 IU retinyl palmitate/day. Group P: 1,050 mg putrescine/day. Group RPP: 9,000 IU retinyl palmitate and 1,050 mg putrescine/day. Group MO: milk only. 2 Number 50% villi contain positive enterocytes. 2 DPI: day postinoculation. 3 Each group consisted of 4 piglets. RP: 9,000 IU retinyl palmitate/day, P: 1,050 mg putrescine/day, RPP: 9,000 IU retinyl palmitate and 1,050 mg putrescine/day, MO: milk only. 4 DU: duodenum, UJ: upper jejunum, LJ: lower jejunum, MI: mid intestine, UI: upper ileum, LI: lower ileum, Average: average from all intestinal sites. 77 DISCUSSION Findings in this study indicated that supplementation with vitamin A and/or putrescine did not cause differences in. clinical signs, histopathologic lesions and rotavirus antigenic expression in the small intestinal enterocytes of piglets infected with the SDSU strain of porcine rotavirus. Most inoculated piglets became infected, as indicated by clinical signs of diarrhea or the demonstration of rotaviral antigen in enterocytes with immunohistochemistry. Although most of the piglets in the study were euthanatized in the acute stage of disease (1, 2 and 3 DPI), those euthanatized 8 DPI were in good condition. or had recovered, indicating that the rotavirus infection was self-limiting. Generally, clinical signs observed in this study agree with those previously reported (McNulty et al, 1976; Theil et al, 1978). All piglets survived until the time of necropsy. The lack of mortality caused by the SDSU strain of porcine rotavirus infection agrees with the findings of other investigators (Theil et a1, 1978; McAdaragh et al, 1980). Mortality associated with rotavirus infection has been reported in gnotobiotic piglets (Tzipori and Williams, 1978), in colostrum-deprived piglets (McNulty et al, 1976) and in conventional piglets (Bohl et a1, 1978). ‘ Supplementation with retinyl palmitate and/or putrescine did not cause any significant differences in the 78 body weights and body weight gains of both rotavirus- infected and control piglets. Body weights between piglets varied greatly, as can be seen from the high standard deviations. Piglet—to-piglet variation in body weight and body weight gain associated with rotavirus infection was also reported by Woode et al (1979). The reason for piglet- to-piglet variation is not known, but may be associated with host physiological factors. However, even though not significantly different, at 8 DPI, body weights and body weight gains of infected piglets given RP and/or P were higher than infected piglets given M0. Similarly, body weights and body weight gains of the control piglets given RP were higher than control piglets given M0. This result may indicate the potential of long term beneficial effects of vitamin A and/or putrescine supplementation. Enterocyte replacement time in young pigs is about 7-10 days (Moon, 1971), therefore, the effects of treatment might have been significant if the time for terminating the experiment was ' extended. Clinical signs of diarrhea observed in this study corresponded with findings by other investigators (McNulty et al, 1976; Theil et a1, 1978; McAdaragh et a1, 1980). In the present study, diarrhea was based on clinical signs only and could not be judged based on the fecal output weight due to an inability to separate the fecal and urinary output. No differences were observed between treatment groups in the 79 severity of the diarrhea and the number of animals with diarrhea. In the present study, vacuolated enterocytes were observed in both infected and control piglets. Vacuolation is normally present in the jejunal and ileal epithelial cells of young, especially gnotobiotic, piglets. Most vacuolated enterocytes contain a single large vacuole which distends the cytoplasm and displaces the nucleus towards the base or apex of the cell (Moon, 1972). Vacuolated ileal epithelium_ is 'present. up to ‘the third. week. of age and vacuolated jejunal epithelium is present up to eleven days of age (Moon, 1972; Moon et al, 1973). In the first few days of age, the vacuole does not contain lysosomal enzymes (Kraehenbuhl and Campiche, 1969) which enable the cells to actively transport intact antibody (Staley et al, 1968). No reports regarding the enzyme content in the ileal vacuoles after cessation of intact antibody transport were found in the literature. Moon et al (1973) speculated that ileal vacuoles in pigs after antibody transport cessation might be lysosomes 'which transport. or: process antigen for active immunization. Infected piglets had higher numbers of vacuolated enterocytes than control piglets. This finding was in contrast to the findings of Theil et a1 (1978), McAdaragh et a1 (1980) and Collins et a1 (1989) who found that the vacuolation in the small intestine of rotavirus-infected piglets was less conspicuous than in control piglets. 80 However, some investigators reported the presence of numerous vacuoles, (Pearson and McNulty, 1977) containing neutral fat (Hall et a1, 1976) in rotavirus-infected enterocytes. The increased number of vacuolated enterocytes in the present study was probably a result of a mixture of both normal enterocyte vacuolation present in young pigs and pathologic vacuolation occurring in association with rotaviral infection. Vacuolar degeneration of villous enterocytes was observed only in infected piglets. Increased numbers of degenerated enterocytes in infected piglets 1 to 3 DPI corresponded with the onset of diarrhea, indicating that degenerated enterocytes may ‘have lost 'their absorptive function. No differences were observed between treatment groups based on the number of degenerated enterocytes in rotavirus-infected piglets. The preference for rotavirus replication in the mature epithelial cells on the tips of the villi has been demonstrated. uSing' a variety of methods, 'including immunofluorescence and immunohistochemistry (McAdaragh et al, 1980; Gelberg, 1992). As a result of desquamation and lysis of infected epithelial cells, free or cell-associated rotaviral antigen can also be demonstrated in the intestinal lumen (McAdaragh et al, 1980: Theil et al, 1978). In the present study, rotaviral antigenic expression was only observed in enterocytes that were still attached to intestinal villi. No rotaviral antigen was detected in the 81 intestinal lumen. The inability to detect antigenic expression in the intestinal lumen was probably due to the flushing of fixative during tissue preparation. Although not statistically significant, the number of piglets with rotaviral antigen positive enterocytes at 1 and 3 DPI tended to be higher in piglets fed diets containing putrescine than in piglets fed diets not containing putrescine. Polyamines, because of their multiple amino groups, are known to have a strong positive charge at cellular pH, which makes them highly reactive with a variety of cellular molecules, including DNA and RNA (Wolfe, 1993). Polymers having positive charges, such as protamine, protamine sulphate, DEAR-dextran, histone and poly-L-lysine hydrobromide, enhance rotaviral infectivity, whereas polymers having negative charges such as mucin, a-l-acid glycoprotein heparin, heparan'sulphate and dextran sulphate inhibit rotaviral infectivity and replication (Superti et al, 1993). Since polyamines are known to have a strong positive charge, polyamines may also enhance rotaviral infectivity. In the present study, at 1 DPI, the P group had the highest number of piglets showing rotaviral antigenic expression in enterocytes and at 3 DPI, the RPP group had the highest number of positive animals. Based on the results of this study the daily dietary administration of vitamin A and/or putrescine was ineffective in the prevention or alleviation of rotaviral infectivity, and the clinical signs and intestinal mucosal 82 damage caused by rotaviral infection in piglets. Putrescine appeared to increase rotaviral infectivity as indicated by the increased number of infected piglets and a greater distribution of enterocytes showing rotaviral antigenic expression. CHAPTER 2 EFFECT OF DAILY DIETARY ADMINISTRATION OF RETINYL PALMITATE AND/OR PUTRESCINE ON THE SMALL INTESTINAL MUCOSAL MORPHOMETRY OF ROTAVIRUS-INFECTED PIGLETS ABSTRACT Experiments were conducted to determine the effects of the daily dietary administration of retinyl palmitate and/or putrescine on the small intestinal morphometry of rotavirus- infected piglets. One hundred and twenty-eight piglets were removed from sows at two days of age. On day 3, piglets were divided into four groups of 32 piglets. Each group received either milk only, ‘or retinyl palmitate and/or putrescine. On day 5, half of each group was infected with porcine rotavirus. Piglets were euthanatized on days 6, 7, 8 and 13 (1, 2, 3 and 8 days postinoculation, DPI). Complexity indices. did. not. differ between the treatment groups. Infected animals had greater complexity indices than control animals (P<0.05). Piglets 1, 2 auui 3 DPI had greater complexity indices than. piglets 8 DPI (P<0.01). Relative mucosal volume of the small intestine was less in infected than control piglets (P<0.01). Crypt depth of piglets 8 DPI was greater than the crypt depth of piglets 1, 2 and 3 DPI (P<0.05). Average mitotic indices of supplemented piglets were higher than piglets given milk 83 84 onLy. Supplementation significantly increased the average mitotic indices of the infected piglets 3 DPI and control piglets 21 DPI (P<0.01). Rotaviral infection ix: piglets reduced the relative small intestinal mucosal volume. Supplementation with retinyl palmitate and/or putrescine increased mitotic indices in the small intestinal crypts of both rotavirus-infected and control piglets. INTRODUCTION Small intestinal mucosal regeneration is essential for animals and human beings, especially for recovery from intestinal injury. Intestinal injury with mucosal damage can be caused by viral pathogens, including rotavirus. Rotavirus is one of the most important viral agents causing destruction of absorptive enterocytes in young animals (Holland, 1990) and children (Estes, 1983). The virus replicates within mature enterocytes located at the tips of villi, causing cell detachment and villous atrophy (McAdaragh et a1, 1980). Vitamin A and putrescine have received much attention because of their potential beneficial effects in mucosal cell regeneration. Vitamin A deficiency affects the growth and differentiation of various epithelial tissues, including small intestinal epithelial cells (Wolf, 1984). Also, vitamin A can protect rats from intestinal damage caused by 85 the antitumor drug, methotrexate (Tsurui et a1, 1990). Methotrexate, as an Iantitumor' drug, inhibits mitosis of small intestinal epithelial cells in the crypts, disrupts the steady state system of the epithelium and progressively reduces the size of crypts and villi (Jeynes and Altmann, 1978). Putrescine affects small intestinal functions and morphology in calves fed soy protein. Villous atrophy may occur in calves (Kilshaw and Slade, 1982) and in early- weaned pigs (Dunsford et al, 1989) fed diets containing soy protein. Addition of putrescine to the soy protein containing diet of calves enhanced enterocyte proliferation (Grant et al, 1989). The purpose of this study was to evaluate the usefulness of vitamin A and/or putrescine on intestinal mucosal regeneration in piglets during and after the onset of rotaviral infection. The objectives of this study were to determine the effect of daily dietary administration of vitamin A and/or putrescine to rotavirus-infected piglets on the following: 1) small intestinal mucosal villous height and crypt depth, 2) small intestinal mucosal complexity indices, and 3) small intestinal mitotic indices and goblet cell numbers. 86 MATERIALS AND METHODS Experimental animals One hundred and twenty eight, five-day-old piglets from 16 litters were obtained from the Michigan State University swine farm. All piglets received colostrum from the sows for two days. Experimental design is shown in Figure 1.1. Piglets within the same litter were allocated for a variety of treatments. Piglets were reared in individual cages in a temperature controlled room (BO-32°C). Each piglet was given a total of 300 ml evaporated ndikfo (14% dry matter) daily. The nutritional composition of the milk is shown in Table 1.1. Milk was offered three times a.day at 8:00 a.m., 12:00 p.m. and 5:00 p.m. Starting from day 3, subgroup 1 (RP) was given milk containing vitamin A in the form of retinyl palmitatell (30,000 IU/kg of milk) daily. Subgroup 2 (P) received putrescine12 (25 g/kg dry matter of milk) in the milk. Subgroup 3 (RPP) was fed milk containing retinyl palmitate (30,000 IU/kg of milk) and putrescine (25 g/kg dry matter of milk). Subgroup ’4 (MO) received milk only. Addition of vitamin A and putrescine was done just prior to feeding by diluting each of the substances in approximately two ml of distilled water. On day 5, the rotavirus-infected -10 piglet group was orally inoculated with 1 ml 10 dilution of the stock intestinal homogenate. Four piglets from each w Carnation Evaporated Milk, Nestle Food Co., Glendale, CA. “ Sigma Chem. Co., St. Louis, MO. 12 Sigma Chem. Co., St. Louis, MO. 87 group were euthanatized on days 6, 7, 8 and 12. The time for necropsy was scheduled so that only five to six animals were euthanatized in a day. Necropsy and specimen collection Pigs were euthanatized with an overdose of intravenous injection of sodium pentobarbitali3. Three-cm segments from six sites of the small intestine were removed immediately and placed in buffered neutral formalin for histopathologic and immunohistochemical examinations. The first segment was removed from the duodenum approximately 5-10 cm distal to the pyloric junction and the last segment was obtained from the lower part of the ileum, approximately 5 cm proximal to the ileocecal junction. . The remaining segments were obtained at approximately a sixth, third, half and two- thirds of the distance from the pylorus to the ileocecal junction. These segments were designated as duodenum (DU), upper jejunum (UJ), lower jejunum (LJ), mid-intestine (MI), upper ileum (UI) and lower ileum (LI). The lumen of the segments were rinsed with 10% neutral buffered formalin and the segments were then immersed in the fixative. Morphometric measurements Formalin-fixed small intestines were routinely processed for histopathologic examination. The tissues were oriented in cross sections (ring shaped), embedded in U BUTLER®, The Butler Co., Columbus, OH- 88 paraffin and marked accordingly. Tissues were cut by a microtome into 6-um thick sections. One slide contained DU, UJ, LJ segments and the second slide contained MI, UI and LI segments. Sections were stained with hematoxylin and eosin (H&E) and subsequently, morphometric assessments were made, including mucosal complexity index, villous height, crypt depth, mitotic index and goblet cell number. Mucosal complexity index was determined using six segments_of intestine. Complexity of the intestinal mucosal surface (intestinal mucosal surface to volume ratio) was measured using a Weibel graticule mounted in the microscope eyepiece (Weibel, 1963; Dunnill and Whitehead, 1972). A Weibel graticule, consisting of 15 equal length lines ('1'), was superimposed on the intestinal section so that the most lateral graticule line fell on the border of the lamina propria and the muscularis mucosa (Figure 2.1). The magnification (10 X objective and 10 X ocular lenses) was kept constant throughout the experimental studies. With this magnification, the length of '1' was 1.8 X 10'2 cm. Lines cutting the mucosal surface were counted as 'cuts' ('c') and the end points of the lines falling in the tissue between the mucosal cells and the muscularis mucosae were counted as 'hits' ('h'). The complexity index of the villous pattern was obtained by using c:lh ratio. 'The number of 'h' per field also represented the relative mucosal volume. 89 Figure 2.1. Photomicrograph of a section of ileum with a Weible graticule superimposed on the image to illustrate the use of a Weible graticule. The lines cutting the mucosal surface are counted as 'cuts' ('c') and the end points of the lines falling in the tissue between the mucosal cells and the muscularis mucosae are counted as 'hits' ('h'). The length of the line ('1') is measured with a micrometer. The complexity index is obtained by calculating the c:lh ratio. In this example, the number of cuts is 23 and the number of hits is 26. 90 The villous height and crypt depth of 5 well-oriented villi and crypts were measured using a computer-assisted video image analyzer. All segments of intestinal sections were viewed with an MTI series 68 video camera mounted on a Nikon Microphot-FX microscope”. ‘Villous length and cmypt depth were measured with the aid of JAVA Image Analysis Software”. Counting epithelial cells from approximately 30 crypts, the mitotic index and goblet cell number was determined using a light microscope. The mitotic index was calculated as the total number of mitotic figures in any stage of mitosis divided by the number of crypts. statistical analysis Morphological measurement data were grouped based on the infection status, treatment and time. Each group consisted of 4 animals, as in the experimental design. Statistical analyses were performed using three-factor analysis of variance (three-way ANOVA) with main effects of treatment, infection and time (Devore, 1991). The numbers of levels of the three factors were denoted as 2, 4 and 4 for the infection status, treatment and time factors, respectively. Differences between group means within each factor were analyzed by one—way ANOVA followed by Tukey's procedure (Fowler anui Cohen, 1990). Differences ‘between groups were considered significant at the level of P50.05. ” Nikon Inc., Instrument Group, Garden City, NY. 6 Jandel Scientific, Corte Madera, CA. 91 Statistical analyses were done using SPSS Computer Software”.. RESULTS Mucpgpl complexity The average values of mucosal complexity indices as measured by using a Weibel graticule (Table 2.1 and Figure 2.2), ranged from 45.48 (i 4.66) to 68.58 (i 8.82). Three— way ANOVA results indicated that the complexity indices were influenced by infection status and time after infection, but not by treatment. There were no interaction existed between the main effects. The infected group had higher average complexity indices than the control group (P<0.05). ‘When each intestinal segment was examined, the DU, UI and LI of the infected group had higher complexity indices than the control group. Regardless of the infection status, piglets 1, 2 anui 3 DPI had higher complexity indices than piglets 8 DPI. Three-way .ANOVA results indicated that the relative volume of the small intestinal mucosa (average number 'of ‘hits' per field) of the control group was higher than the infected group. Piglets 8 DPI had higher average relative mucosal volumes than piglets 1, 2 and 3 DPI, but the average relative volumes were not significantly different. However, the relative volumes of the duodenum and upper jejunum of m SPSS Inc., Chicago, IL. 92 .Amo.ovmv mumamwo Houucoo Mo Ho A mumaoflm couomwca mo Ho 0 Ham 8 60 Ho A newsman Hmo m 888 N .H no lHoc meannee suuxmaesou 8 .umHOHQ umo coauomm HMCflummucH Hanan mo mucmfiomm me .mumaowm v Eouw mommm><_o .coaumadoocaumoo >60 "Hmo Q .xaco xHHE "02 .>mp\0CHommuusm o8 omo.H can unmeasame escape“ 5H ooo.m "mam .smn\mcnommuuse as omo.H “a .smv\mumuasamm escape“ 6H ooo.m "mm m I 1. NH NM NM. NM HM NM NM NM NM. .mm H cams H + an em am pm mm an em em om om me pewsummue am an em me me on em mm me mm mm em Hmo m Nu.mm mm so an em em om om an me He Hmo m HH.H6 . mm so H6 mm em m6 om me we be Hue N HH.m6 me me mm mm me we so me me 0 mm nsHmo H I 02 max 8 mm or 8mm m 88 0mm + G008 G005 GMOE M Ham Ham Hmo Hd< Houucoo Houucoo UmuommcH UmuommcH msuw>muou mafiuuom mo cwmuum DQO on» nufl3 owuomucfl muoHOHo .mb mumamflo Houucou mo museummuca Hanan ecu ca mwoflcca auwxmaofioo .H.N «Home 93 l Infected 1:) Control 1 l l l I l ‘11 .4 8 8 8 8 Complexity Index 8 _L o i l t l l 1 O 2 _ , 3 Day Postinoculation Figure 2.2. Pooled small intestinal mucosal complexity indices (SIMCI) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus (mean i standard error). SIMCI of infected piglets > SIMCI control piglets (P<0.05). SIMCI of 1, 2 and 3 DPI piglets > SIMCI of 8 DPI piglets (P<0.01). 94 piglets 8 DPI were significantly higher than those of _piglets 1, 2 and 3 DPI (P<0.05). Villous height and crypt depth Results from a three-way ANOVA indicated that villous height (Table 2.2 and Figure 2.3) was not influenced by time, treatment nor infection. Also, crypt depth (Table 2.3 and Figure 2.4) was not influenced by treatment nor infection. However, crypt depth was influenced by time. No interaction was found between the main effects On both villous height and crypt depth. The average crypt depth 8 DPI, was significantly higher than the average crypt depth 1, 2 and 3 DPI (P<0.05). The average crypt depth 3 DPI was significantly higher than the average crypt depth 1 and 2 DPI, but significantly lower than the crypt depth 8 DPI (P<0.05). When each intestinal segment was examined, the crypt depth values of the DU on 3 and 8 DPI were significantly higher than the values on 1 and 2 DPI (P<0.05). The crypt depth values of the UJ, LJ, MI, UI and LI 8 DPI were significantly higher than the values 1, 2 and 3 DPI (P<0.05). In addition, the crypt depth value of the LJ 3 DPI was higher than the value 1 DPI. Ratios of villous height to crypt depth were influenced by time, but were not influenced by treatment nor infection status. The average villous height to crypt depth ratio from all parts of the small intestine was higher on 1 DPI than cn1£3 DPI (P<0.05). When each individual segment 95 .coauuoucw no: ucofiumouu .Hmo uo uommwo 02m”v .uwaoflo Moo c0fip00m Hmcwummucfl Hamfim mo mucofioom me .mumamfim v Souu ommum>¢nv .GOHOMHSUOGHUmoo >60 "Home. .aaco xHflE "oz .hmp\mcHUmouusm me omo.H can oomoHsHme HHcHumu DH ooo.m "mam .>88\mcHomouusa ms omo.H “m .Hmo\oumuHsHmo HscHuou 5H ooo.m "mm.. eH H 036 NNH emu emu emu men GNH HmH man can hen. mm H same b mew mew hoe mew mew mme mam mHe Hmm mam ocusumoue omn_ome mmo 0mm new New mme mHe 6mm «em H66 Hmo Ham 8 mNmane NHe new eem 8H6 moo mHe emm 6mm mmo «we Hoe m mmn.meo mme New own one 6mm owe eve Hme men ems Hoe m pen bee owe Nem mam mew 666 mme mHe mom mme numme .HHao H mm + some Ham Ham Ham Ham HOHUGOU HOHUGOU UOpUOHCH UOUUQMCH msufl>muou 0GHUHOQ mo awmuum Dmom onu cue: Umuoowcfl muoaoam .m> muoamfim Houucoo mo mmcflumoucfl Hanan 0:» Ga A513 mnuocoa msoaafl> .N.N magma 96 I lnfeded [3 Control Figure 2.3. Pooled small intestinal mucosal villous length (SIMVL) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus (mean : standard error). No significant differences were found between groups. 97 .Amo.ovmc mooHoHo Hoe m 8:8 N .H yo no A muoHoHo Hon 8 No loos spoon nosuo b .uoaoflo Hon cofluuom Hmcflumoucfl Hanan mo mucoEoom xflm .mumaowo e Eoum oomuPBmo .coflumHsoogHumoo >mp "Hon Q .>Hco xHHE "oz .>mp\oCHOmmHusm o8 omo.a pcm mumpHEHmo H>cfluou DH ooo.m “mom .>mp\ocflomouudo me omo.H "m .>mp\oumufifiamo H>cfiuou DH ooo.m “mmm. e H omH mu New HHH HHN. ow m“ NHH HHN RH HHH mm H some emH emH mON GON ooH mmH meH omH meH NmH poosumone pH.eNN mNN NeN mmN NmN mmH mNN meN emN emH oeN Hmo m em HmH mON NmH VHN mHN HHN meH 88H eNH mmH eoH Hoe m oH.meH emH mnH mmH NON mmH NeH moH mmH mmH moH Hoe N mH.moH meH mmH HoH oeH NeH mmH eeH meH HoH “oHoH nHHNo H I oz Nam m mm oz 92 N 8 mm CMOE Emma now + some Ham Ham Hmo Had Houucoo Houpcoo couoowcH pouooucH nuHB popoowcfl mumaoflo .m> muoaofio Honucoo Mo mocflumoucfl Hanan 0:» CH msufl>muou ocfiumom mo cannum Dmom ecu 1215 msuooo posuo .m.N oHome 98 I Infected CI Control Figure 2.4. Pooled crypt depths for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus (mean 1 standard error). Crypt depth of 8 DPI piglets > crypt depth of 3 DPI piglets > crypt depth of 1 and 2 DPI piglets (P<0.05). 99 was examined, the villous height to crypt depth ratios of DU, LJ, MI and UI were higher on 1 DPI than on 8 DPI (P<.05). The villous height to crypt depth ratio of the LJ 1 DPI was also higher than the villous height to crypt depth ratio 3 DPI. Mitotic index and goblet cell number Results from a three-way ANOVA indicated that the mitotic indices in the small intestinal mucosa (Figures 2.5, 2.6, Table 2.4 and Figure 2.7) were significantly influenced by treatment and in some portions were also significantly influenced by time. However, mitotic indices were not influenced by infection. At 2, 3 and 8 DPI, the average value of mitotic indices of the small intestine of infected piglets given RP and/or P were higher than the mitotic indices of the infected piglets given MO. However, results from a one-way ANOVA showed that only those in the 3 DPI groups were significantly different (P<0.05). The average value of mitotic indices of the control piglets given RP and/or P were also higher than the mitotic indices of the control piglets given MO. Statistical differences were noted in piglets 1 and 3 DPI. Results from a one-way ANOVA showed that at 1 DPI, all the control piglets given RP and/or P had significantly higher mitotic indices than the control piglets given MO (P<0.05). However, at 3 DPI, only the control piglets given P had a 100 Figure 2.5. Photomicrograph of a section of jejunum with a high number of mitotic figures. The section was taken from a rotavirus-infected piglet, 3 days postinoculation in the putrescine group (Hematoxylin and eosin, 440X). Figure 2.6. low number of mitotic figures. The section was taken from a rotavirus-infected piglet, 3 days postinoculation in the milk only group (Hematoxylin and eosin, 440X). 101 Photomicrograph of a section of jejunum with a 102 .188.8V88 muoHoHo H88 8 0: 8o H: A 888H8H8 H88 8 888 888 8.88 80 HHze mooHocH oHooonflo .uonHo mom COHuoom Honeumouca Hamfim mo mucofioom me .muoamam v Eouu oomum>¢ o .coauMHSUOCHumom amp "Hmo a .haco xHHE “oz .>MU\QCHOmouuSQ me omo.H poo oumuHsHmo HchuoH 8H 888.8 “888 .88o\oeHomouu:8 ms 888.H "8 .amo\oumuHsH88 HscHuoH 5H 888.8 "88 8 I 88.8“ 88.8“. 88.8“. 88.8“ 88.8w 88.88 88.8“. 8848“. 88.8“ 88.8“. 88 H came 88 8 + 88 8 88.8 88.8 88.8 88.H 88.8 88.8 H8.8 88.8 H8.8 88.8 888288888 88.88 88.8 88.8 H8.8 88.8 88.8 88.8 88.8 N8.8 NH.H 88.8 8H.H H88 8 88.8w 88.8 88.8 88.8 88.8 NN.H N8.8 H8.8 88.8 88.H 8H.H 88.8 8H88 m 88.88.88.8 88.8 88.8 88.8 88.8 H8.8 88.8 88.8 88.8 88.8 H8.H H88 N 88.8H.88.8 N8.8 N8.8 88.8 88.8 88.8 88.8 88.8 88.8 88.8 oNe.8 AHH88 H mm + come H88 HH< H88 H88 Houucoo Houucoo UmuoomcH oouoowcH msufl>muou ocfiouom mo camuum DQO on» cuHZ pmuoowcw muoamfla .m> muoaoflo Houucoo mo mocflumouCH HHmEm on» :8 mooHUcH Ufluouflz .v.N manme 103 Putrescine I Vitamin A and Putrescine [I Milk Only I Vitamin A 2%- 2 / 4. 2. 0. 8. 6. 4. 2. 0. 00000000 5E3: 238 288.5. Pooled mitotic indices of control and infected piglets (mean 1 standard error). * Significantly different from milk only groups (P<0.01). Figure 2.7. 104 significantly higher mitotic indices than the control piglets given MO (P<0.05). When the data were analyzed from individual segments of the small intestine, results of the effect of treatment were similar to the results from the average values. However, results on 'the effect. of time after infection 'were not consistent. Time had no effect on mitotic indices in the upper part of small intestine, but it had a significant effect in the lower portions of the intestine. Mitotic indices in the UI and LI of piglets 3 and 8 DPI were higher than those of piglets 1 DPI (P<0.05). No statistical differences existed in average goblet cell numbers among the RP, P, RPP and MO groups and also, no differences existed between the infected and control groups (Table 2.5). However, the goblet cell number at 8 DPI was higher than the number of goblet cells at 1,2 and 3 DPI (P<0.05). DISCUSSION Results of morphometric studies using a Weibel graticule indicated that control piglets had lower complexity indices than the rotavirus-infected piglets (57.19 vs. 59.78), but no differences existed between the treatment groups. However, linear measurement results indicated that treatment, infection status and 1 time postinoculation did not have an effect on villous height. .1H8.8V88 muoHoHo H88 8 888 N .H 80 zoo A 888H8H8 H88 8 80 12888 888588 HHoo uoHnoo 8 .uoHoHQ Moo mafiuoom Hmcflumoucfl Hamfim mo mucofioom me .muoamflo v Scum ommuo>¢ o .GOHuMHDUOGHumOQ moo "Hmo Q .>Hco xHHE "OZ .xmv\ocaomouusm me omo.H 0cm 888888H88 HmaHuoH 8H 888.8 "888 .888\88H088Hus8 ms 888.H "8 .888\8888H2H88 HscHuoH 8H 888.8 "88 m 105 . I . 88.8w 8H.8H. 8N.8H 8H.8H 8H.8H 8H.8H 8H.8H 8N.8H 8H.8H 8N.8H. mm H 8888 88 8 + 88 N 88.N 88.N 88.N 88.N N8.N 88.N 88.N 8H.m 88.N 88.N neosumoue 8H.8H.N8.m NH.8 88.8 8H.8 HN.8 88.8 H8.8 No.8 N8.m 88.8 88.8 H88 8 8H.8H.88.N 88.N 8N.N N8.N 88.N 88.N N8.N N8.N N8.N 88.8 88.N H88 8 HH.8H_N8.N 88.N 88.N 88.N 8H.N 88.N H8.N 88.N 88.N 88.N. 88.N H88 N 8H.8H.88.N 88.N 88.N 88.N 88.8 88.N 88.N 8H.N 88.8 88.N o88.N 8.H88 H I o: 888 8 88 oz 888 8 88 pmm + GMOE GMOE CMOE M H88 H88 H89 HH< Houucoo Houucoo pouomwcH pmuoomcH msua>muou ocaouoo mo cflmuum Dmnm men eufiz pouoomcfl mumamam .m> muoaofio Houucoo mo moCHumoucH Hanan on» CH HQ>HU Hon mumnszc Hfimo umfinom .m.N wfinmh 106 In the following discussion, unless specifically mentioned, there were no interaction existed between the main effects. Normal porcine complexity index values for comparison were not available. However, reduced complexity index values indicate the presence of villous atrophy (Dunnill and Whitehead, 1972; Wright and Tomkins, 1978). Thus, results from the present experiment indicate that the control group had more mucosal atrophy than the infected group. However, this is probably not the case, since histopathologic results indicated that the infected animals had more villous atrophy than the control animals. Since the complexity indices were calculated from the formula 'c:lh?, any increase of 'c' and/or decrease of 'h' would result ’in an increased complexity indices. The average value of 'c' per field reflects the surface area and the value of 'h' per field reflects the relative volume of the small intestinal mucosa (Dunnill and Whitehead, 1972; Wright and Tomkins, 1978; Hart and Kidder, 1978). For example, the complexity index Value in normal, human jejunum is 46.0 i 12.0 (Dunnill and Whitehead, 1972) and ranged between 40 to 60 (Wright and T<31't'lkins, 1978), whereas in healthy dogs, ‘which have a thicker mucosa than human beings, complexity indices ranged be‘CWeen 22.1 to 31.3 for young dogs and 22.8 to 28.1 for acil—‘llt dogs (Hart and Kidder, 1978). In the present e)(EDIeriment, no differences were found in the 'c' values. when the value of 'h' in the present experiment was calculated, the infected group had a smaller 'h' value than 107 the control group, indicating that the infected group had thinner intestinal walls than the control group. The thinning of the intestinal wall in the rotavirus-infected group was consistent with the thin-walled gross appearance of the intestine. The thinning of the intestinal wall may have been caused by reduced crypt depth or villous atrophy. In this experiment, the histological appearance of the crypts was uniform and no differences were noted based on the linear measurement. Thus, the volume or thickness of the crypts was not affected by the rotaviral infection. However, the histological appearance of the villi was not uniform. This agrees with a previous investigator who observed the presence of atrophic and normal villi within the same intestinal section (McAdaragh, 1980). Linear measurements of the villous lengths in the present study were obtained from the longest remaining villi, resulting in an inability to demonstrate villous atrophy. Although not statistically Significant, the average villous length of the infected piglets was lower than the control piglets (632 j; 208 mm for ir‘fected piglets versus 649 i 178 pm for control piglets). By evaluating both the results from the Weible graticule and the linear measurement of the crypt depth, it was concluded that the reduction in mucosal volume was caused by a re(auction of villous volume, which was to be expected in a r Qtaviral infection . 108 Complexity indices of piglets 8 DPI were less than in piglets 1, 2 and 3 DPI. The 'h' value and the linear measurement of the crypt depth from piglets 8 DPI were greater than piglets 1, 2 and 3 DPI (P<0.05). This may be associated with the increased thickness of the mucosa as the piglets grew (Moon, 1971). The number of mitotic figures in the crypts appeared to be influenced by vitamin A and/or putrescine supplementation. In response to villous atrophy caused by a rotaviral infection, crypt hyperplasia is usually present (Dchdaragh et al, 1980; Theil et al, 1978; Woode and Crouch, 1978) . However, results of the present experiment indicate that the number of mitotic figures per crypt in infected and control groups was not significantly differentf Treatment With either vitamin A and/or putrescine significantly increased the number of mitotic figures both in the infected and control piglets. Piglets fed milk supplemented with ei‘ther RP, P or RPP had greater mitotic indices than those fEECi with milk only. Retinyl palmitate may exert its effect in increasing mitotic figures by its association with TGF B. Transforming growth factor B stimulates proliferation of meSeenchymal cells (Lamprecht et al, 1989; Blachowski et al, 1994.), but in contrast, inhibits the growth of a large VaI‘llety of epithelial cells, including actively dividing e1‘1t—erocytes (Lampert et al, 1989; Potten et al, 1995; Booth et al, 1995). Expression of TGF B-l is largely governed by :-1__ — ' ‘- 109 AP-l promoter (Salbert et al, 1993). The activity of AP-l is inhibited by RAR and RXR (Salbert et a1, 1993; Chen et al, 1995; Desbois, 1993; Fanjul et al, 1994). Thus, inhibition of AP-l activity by administration of vitamin A would result in the suppression of TGF B, which would consequently increase cell proliferation. In the present experiment, the increase in cell proliferation may have been reflected by an increased number of mitotic figures. An increase in mitotic figures was also observed in piglets treated with putrescine. Putrescine, as a polyamine, is required for cell growth and differentiation (Heby, 1981). The finding in the present study supports the suggestion that exogenous polyamines are needed for maximal cell growth (Pegg and Cann, 1982). Data from the present study indicate that the number of goblet cells per crypt was not influenced by infection and treatment. A reduced number of goblet cells in the small intestine of rats has been associated with a vitamin A deficiency (De Luca et al, 1969.; Rojanapo et al, 1980). Thus the result of the present study may indicate that all piglets, regardless of the infection status and treatment, still had adequate concentrations of vitamin A. However, goblet cell numbers were influenced by intestinal site and time. Goblet cell numbers in the LI of piglets 1, 2, 3 and 8 DPI were significantly greater than those of other segments of intestine (P<0.01). Also, the average goblet 110 cell numbers in piglets 8 DPI was higher than in piglets 1, 2 and 3 DPI. The finding of a greater number goblet cells in the LI was consistent with the normally high population of goblet cells in the posterior part of the small intestine (van-Kruinigen, 1988). The greater numbers of goblet cells in piglets 8 DPI were probably related to the increased size of the crypts. In summary, this study indicated that rotaviral infection in piglets reduced small intestinal mucosal volume. The volume reduction occurred in the villous portion of the intestine which is consistent with the pathogenesis of rotaviral infection. Daily dietary administration of retinyl palmitate or putrescine alone or in combination increased the mitotic index in small intestinal crypts, suggesting a possible beneficial effect of retinyl palmitate or putrescine on the regeneration of the intestinal mucosa. CHAPTER 3 EFFECT OF DAILY DIETARY ADMINISTRATION OF RETINYL PALMITATE AND/OR PUTRESCINE ON INTESTINAL MUCOSAL NUCLEIC ACID AND PROTEIN, AND ON HEPATIC VITAMIN A CONCENTRATIONS OF ROTAVIRUS-INFECTED PIGLETS ABSTRACT Experiments were conducted to determine the effects of the daily dietary administration of retinyl palmitate and/or putrescine on the concentrations of small intestinal nucleic acids and protein, and the concentration of hepatic vitamin A in rotavirus-infected piglets. One hundred and twenty- eight piglets were removed from the sows at two days of age. On day' 3, piglets were divided into four groups of 32 piglets. Three of the groups received milk containing either retinyl palmitate, putrescine, or retinyl palmitate and putrescine combined. One group received milk only. On day 5, half of each group was infected with porcine rotavirus. Piglets were euthanatized on days 6, 7, 8 and 13 (1, 2, 3 and 8 days postinoculation, DPI). Results of this experiment indicated that mucosal protein and RNA concentrations were higher in piglets 8 DPI than in piglets 1 and 2 DPI. Mucosal protein and RNA concentrations were not affected by infection status and treatments. Mucosal DNA concentrations were higher in piglets 8 DPI than in piglets 1 DPI. Mucosal DNA concentrations were decreased in 111 112 not affected by treatment. The hepatic vitamin A concentration was influenced by treatment and time, but not by infection status. Daily dietary vitamin A administration increased hepatic concentrations of vitamin A” Putrescine tended to decrease the concentration of hepatic vitamin A in the infected animals. INTRODUCTION Vitamin A is necessary for the maintenance, growth and differentiation of 'various epithelial cells (Wolf, 1984; Zile and Cullum, 1983). Many factors, such as dietary intake, absorption and tissue utilization of vitamin A, affect the availability of this vitamin in the liver (Herdt and Stowe, 1991). Malabsorption of vitamin A may occur in association. 'with. diarrhea, intestinal infections and intestinal parasitic infestations (Holland, 1992; Rosenberg et al, 1977; Mansour et al, 1979). Increased utilization of vitamin A may occur in animals with increased demand for cell renewal (Herdt and Stowe, 1991). Vitamin A and putrescine have received much attention because of their potential beneficial effects in mucosal cell regeneration. Intestinal mucosal growth is associated with increased intestinal mucosal synthesis of DNA, RNA and protein (Dembinski et al, 1984). Several reports indicate that. in ‘the small intestinal mucosa of rats, vitamin. A deficiency reduces protein synthesis (Rojanapo et al, 1980; De Luca et a1 1969; Olson, et al, 1981)), lengthens the DNA 113 synthesis phase (Zile et al, 1977) and reduces RNA synthesis (Johnson et al, 1969). Administration of vitamin A to vitamin A deficient animals reverses the effects. Several reports also indicate that putrescine affects the growth of intestinal mucosal cells. When putrescine was infused into the rat small intestine, small intestinal mucosal growth was stimulated (Seidel et al, 1985). Also, 30 hours after the oral administration of spermine in rats, increased concentrations. of DNA. in ‘the small intestinal mucosa were noted (Wery and Dandrifosse, 1993). The objectives of this study were the following: 1) to determine the effect of the daily dietary adminiStration of vitamin A and/or putrescine on_small intestinal nucleic acid and protein concentrations of rotavirus-infected piglets and 2) to determine the effect of the daily dietary administration of vitamin A and/or putrescine on the hepatic vitamin A concentration of rotavirus-infected piglets. MATERIALS AND METHODS Experimental animals One hundred and twenty eight, 5-day-old piglets which had received colostrum from the sow for two days were used for the study. The experimental design is illustrated in Figure 1818 Piglets were reared in individual cages in a temperature controlled room (30-320C). Each piglet was 114 given a total of 300 ml evaporated milk17 (14% dry matter) daily. The nutritional composition of the milk is shown in Table 1.1. Milk was served three times a day at 8:00 a.m., 12:00 p.m. and 5:00 p.m. Starting from day 3, subgroup 1 (RP) was given milk containing vitamin A in the form of retinyl palmitate18 (30,000 IU/kg). Subgroup 2 (P) received putrescine19 (25 g/kg dry matter) in the milk. Subgroup 3 (RPP) was fed. milk containing retinyl palmitate (30,000 IU/kg) and putrescine (25 g/kg dry matter). Subgroup 4 (MO) received milk-only. Addition of vitamin A and putrescine was done just prior to feeding by diluting each of the substances in approximately two ml of distilled water. On day 5, the rotavirus-infected group was ‘orally inoculated with 1 nfl.]1fo dilution of the stock intestinal homogenate. Four piglets from each group were euthanatized on days 6, 7, 8 and 12. Necropsies were scheduled so that only five to six animals were euthanatized in a day. Necropsy and specimen collection Pigs were euthanatized with an overdose of intravenous injection of sodium pentobarbitalm. Three-cm segments from six small intestinal sites were removed immediately and placed in buffered neutral formalin for histopathologic and immunohistochemical examinations. Intestinal mucosal Carnation Evaporated Milk, Nestle Food Co., Glendale, CA. Sigma Chem. Co., St. Louis, MO. Sigma Chem. Co., St. Louis, MO. m BUTLER®, The Butler Co., Columbus, OH. 19 115 scrapings were obtained from the remaining small intestine. The intestinal lumen was rinsed with cold saline and then the intestine was incised longitudinally along the mesenteric attachment. The intestine was opened, and placed on a cold glass surface, blotted with a paper towel and then scraped with a blunt glass slide. Mucosal scrapings were stored at -20°C in jplastic microtubes prior to further testing to determine mucosal nucleic acid and protein concentrations. .Approximately five. grams of liver were collected from each pig, placed in a plastic container, and stored at -80°C for vitamin A analysis. Nucleic acid analyses A modified Fleck and Munro procedure for nucleic acid estimation (Munro and Fleck, 1966) was performed to determine the amount of DNA and RNA present in the intestinal mucosal preparations (Figure 3.1). Approximately 200 mg (i 20 mg) of thawed mucosa was homogenized in 5 Rd distilled water with a motor-driven Teflon homogenizerz1 at 4°C. One ml of the homogenate was used for protein analysis and 4 ml was used for RNA and DNA analyses. The homogenate for protein analysis was then diluted with 4 ml of distilled water. One-hundred ul of the solution (in duplicate) were transferred to test tubes and stored at -20°C for subsequent protein analysis. m THOMAS® Teflon pestle Tissue Homogenizer, Thomas ScientificTM USA, Philadelphia, PA. 116 Tissue (200 mg _+_ 20 mg) -— homogenize in 5 ml distilled water or buffer at 0-4°C (motor- driven Teflon homogenizer) Homogenate (aliquot for protein determination) — add PCA (70%) to final concentration of 5% -— overnight cold storage —-— centrifuge at 5,000 x g, 15 minutes I l Supernatant Pellet (discard) —- dissolve in 4 ml of 0.3M NaOH - incubate at 37°C, 60 minutes — cool in ice and add ‘Iz volume of 15% PCA —— cool in ice for 30 minutes — centrifuge at 5,000 x g, 15 minutes at 24°C F l Supernatant Pellet (hydrolyzed RNA) suspend in 4ml 5% PCA, Determined by UV absorption centrifuge, add the supemetant 1 mg/ml of hydrolyzed RNA to the RNA fraction gives A260 of 34.5 ‘ — suspend in 4 ml of 5% PCA — heat in boiling water, 10 minutes — cool in ice — centrifuge at 5,000 x g, 15 minutes at 2-4°C F l Supernatant Pellet (hydrolyzed DNA) (Residual protein) Determined by UV absorption 1 mg/ml of hydrolyzed DNA gives A260 of 28.0 Figure 3.1. Outline of nucleic acid and protein analyses (Munro and Fleck, 1962, with some modification). 117 9». ...“ A 70% solution of perchloric acid (PCA) was added to 4 ml of homogenate for RNA and.DNA analyses to make a final 5% PCA concentration. The solution was vortexed for 5 seconds. After keeping the solution overnight at 4°C, the solution was centrifuged“)3 at.5,000 g for 15 minutes at 4°C and the supernatant was discarded. The pellet was dissolved in 4 ml 0.3M NaOH and incubated at 37°C for 60 minutes with multiple agitations during the incubation. Two ml of 15% PCA were added to the sample which was then cooled on ice for 30 minutes. Next, the sample was centrifuged at 5,000 g for 15 minutes at 4°C and the supernatant was saved for RNA analysis. The pellet was washed with 4 ml of 5% cold PCA, centrifuged at 5,000 g for 15 ‘minutes at 4°C, and the supernatant added to the initial RNA fraction. The remaining pellet was used for DNA analysis. One ml of the collected supernatant was diluted with 5% PCA to a final volume of 5 ml and the optical density was measured in a spectrophotometer“ set at 260 nm. One mg/ml of hydrolyzed RNA gives an optical density of 34.5. DNA was analyzed by resuspending the pellet in 4 ml of 5% PCA. The suspension was heated in boiling water for 10 minutes. After cooling on ice for 10 minutes, the sample was centrifuged at 5,000 g for 15 minutes at 4°C and the n Baker Analyzed®, Phillipsburg, NJ. “ Refrigerated Centrifuge, Model PR-7000, International Equipment Co., Needham, MA. Shimadzu. UV-Visible Recording Spectrophotometer, Model UV- 260, Shimadzu Co., Kyoto, Japan. 24 118 optical density of the supernatant was measured in a spectrophotometer set at 260 nm. One mg/ml of hydrolyzed DNA gives an optical density of 28.0. Protein analysis Protein. concentrations ‘were: determined. by 'using the Bradford. method (Bradford, 1976). The dye reagent was prepared by diluting 1 part dye reagent concentrate25 with 4 parts distilled water. The solution was then filtered using a Whatman #1 filter”\ Protein standards were prepared by diluting bovine serum albumin (BSA);7 to the following concentrations: 0.194, 0.388, 0.583, 0.777 and 0.971 mg/ml. Standards and samples, 100 pl of each in duplicate, were pipetted into test tubes, and 5 m1 of diluted dye reagent was added to each tube. The mixtures were vortexed for 5 seconds. After a 5 minute incubation at room temperature, the absorbance was read in a spectrophotometer at 595 nm. Vitamin A analysis The determination of vitamin A in the liver was performed in the Nutrition Laboratory of the Animal Health Diagnostic Laboratory, Michigan State University. Vitamin A was quantitated by the method established by Stowe (1982) using a modification of the high performance liquid chromatography procedure described by Dennison and. Kirk ” Bio-Rad Protein Assay, BIO-RAD Lab., Inc., Richmond, CA. N Whatman® Int., Ltd., Maidstone, England. N Bio-Rad Protein Assay, BIO—RAD Lab., Inc., Richmond, CA. 119 (1977). One gram of liver tissue was placed in a tube and up to 5 ml of distilled water was added. The mixture was then homogenized and from this homogenate, 0.5 ml was pipetted into a disposable test tube. An equal amount of absolute ethanol was added to the homogenate (0.5 ml) and the mixture was vortexed for 5 seconds. After the addition of 5 ml hexane (68°—69°C b.p.), the mixture was vortexed for 1 minute and centrifuged at 3000 rpm for 10 minutes. The hexane supernatant was removed and passed through a 0.45 Millipore filter in a Swinny-type filter holder. The aliquot (100ul) was then injected into a chromatograph. Separation was isocratic in a Microporasil column (3.9 mm ID x 30 cm long) with a 60:40 mixture of degassed and filtered hexane. The mixture was pumped through the HPLC unit at 2.5 ml/minute at 63.4 kg/cmz. Forms of vitamin A were detected with a 35 ul flow cell in a spectrofluorometerw set at 330 and 470 nm for the excitation and emission wave lengths, respectively. Liver vitamin A content was calculated from the value derived from the chromatogram, the wet weight and the dry weight of the tissue prepared for the assay. The dry weight of the liver was determined by placing 2 grams of the tissue in an aluminum pan with subsequent drying in an oven at 56°C for 24 hours. ” Waters Assoc., Inc., Milford, MA. 120 statistical analysis Analytical data were grouped based on the infection status, treatment and time. Each group consisted of 4 animals as in the experimental design. Statistical analyses were performed using three-factor analysis of variance (three-way ANOVA) with main effects of treatment, infection and time (Devore, 1991). For intestinal mucosal protein and nucleic acid concentrations, the numbers of levels of the three factors were denoted as 2, 4 and 4 for the infection status, treatment and time factors, respectively. For hepatic vitamin A concentration, the numbers of levels of the three factors were denoted as 2, 4 and 3 for the infection status, treatment and time factors, respectively. Differences between group 'means within each factor were analyzed by one-way ANOVA followed by Tukey's procedure (Fowler and Cohen, 1990). Differences between groups were considered significant at the level of P50.05. Statistical analyses were done using SPSS Computer Softwaresi RESULTS Nucleic acid.and protein conggntrations Intestinal mucosal. protein and nucleic acid concentration results (dry matter weight) are presented in Tables 3.1, 3.2, 3.3, Figures 3.2, 3.3 and 3.4. Intestinal mucosal RNA concentrations of piglets 3 and 8 DPI were m SPSS Inc., Chicago, IL. 121 .1H8.8V8 muonHo HmQ H H0 omZHm A mumeHQ Hmn m Cam M No AUmZHmc mGOHuoHHCOOGOO camuoum HmmOUQE Hmcflummucfl Hamfiw D .muuHmHQ v.50um omouo>¢ U .coaumasoocfiumoa moo “Hmo Q .haco xaafi "oz .>oo\ocflomouudm we omo.H can ououflfiaoo Hmcauou DH ooo.m "mom t>mc\ocflomouusm o8 omo.H um s>mn\ououflfiaom ahcwuou DH ooo.m “mm m 8 H 888 8H HHH 8H 8H“. 8HH mu HHH. 8H8 8H 8H 88 H smog 888 888 NH8 H88 H88 . 888 888 N88 888 888 usgsomoue 8H 8N8 8N8 888 8N8 888 8H8 8H8 8N8 888 8N8 8H8 H88 8 88 888 8H8 888 888 HH8 888 N88 888 N88 H88 NN8 H88 8 8H“ N88 N88 8N8 888 8N8 888 N88 N88 888 888 H88 H88 N 88.888 888 888 888 H88 888 888 888 888 888 e888 AHH88 H or 888 8 88 oz 888 8 88 I GMQE Smog m mm + some 8 H88 H88 HmQ H2 HOHHGOO HOHHGOU COUOOMGH UOOUQMGH A a msuwwwwow ocaouom Mo caouum Dmommueu euflz pouoomsfi muwHOfiQ .m> mumfiuwm 80.3800 80 Hobbes .8 H8882: \ E meoaumuucoocoo cfiwuou H3835 .35..qu . 85 2888 4.8 8388 122 I infected G Control 420 - mglg dry matter Day Pownoculation Figure 3.2. Pooled small intestinal mucosal protein concentration (SIMPC) for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus (mean -_I-_ standard error). SIMPC of 3 and 8 DPI piglets > SIMPC of 1 and 2 DPI piglets (P<0.01). .188.8VNV 888H8H8 Houusoo 80 oosz v muoHoHo eouooHsH 80 oosz .1H8.8V88 mnoHoHa H88 N 888 H 80 oosz A muoHoHa H88 8 80 oosz 8888H8H8 H88 H 80 oosz A moonHo H88 8 No looszc coHumuusoosou 828 Homouse HmsHumousH HHmsm "momxamco HobaumaumumNV .muoaoflm 8 Sony oomum>¢o .coauoasoocaumom amp "Home. .>aco xHHE 802 .hoo\og80mouusm o5 omo.H Ugo oomuasHmo HssHowu 5H 888.8 ”888 .smo\ocHomouoso me 888.H "8 .smo\oumuasH88 HscHuou 8H 888.8 ”888. 123 8H.8 H 8H 8H 8HH 8H 88“ 8H“ 88 NHH 8HH 8H 8H 88 H 8888 8 888 888 NH8 H88 H88 888 888 N88 888 888 888588888 8N.8H 8H 8H 8H 8H 8H 8H 8H 8H 8H 8H 8H H88 8 NN.88.8H 8H 8H 8H 8H 8H 8H 8H 8H 8H 8H H88 8 88.8“.8H 8H 8H 8H NH 8H 8H 8H 8H 8H 8H H88 N 8N.88.8H 8H 8H 8H 8H 8H NH 8H NH NH 8 8H .HH88 H HmQ H2 HmQ HmQ Houucoo Houucoo oouooucH OouooucH msua>muou ocaouom no Gammon DQO on“ sue: o 00 ca . Homecoo nHo Homoode mo 8.58.03 map o\95 mcowumuucoocou 429 ammoozfi aflcuaummmueamumHOfiQ «S mumfimflm . . HHmsm .N.m 8H888 124 I infected 16 8777777 1,, e eel! l Figure 3.3. Pooled small intestinal mucosal DNA concentrations for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus (mean i standard error). DNA of 8 DPI piglets > DNA of 1 DPI piglets; DNA 3 DPI piglets > DNA 1 and 2 DPI piglets (P<0.01). 125 .188.8V88 80HuomooosH H88 8 soHoooHsH .HH8.8V8V 888H8H8 H88 N 888 H No 882H8 A 888H8H8 H88 8 888 8 80 1882H8c msoHumnosmosoo <28 Hmmooss HmsHumousH HHmem “momhamcm Hooaumauoummo .muoaoam v Eouw momuo><88 .COHHMHsoocHumom mop "HmQHQ .>Hco xaefi "OE .mmo\ocaomouuso oE omo.H Ugo despafiaoo chaumu DH ooo.m “mom .>mc\ocaomouusm OE omo.a Hm .mmo\ououflfiaoa cheuou DH ooo.m "mm M 88.88 88.88. 88.88 88.HH. 88.8w 88.8“ 88.88 88.8“. 88.8“ 88.88. 88 H 8888 88N.8+ 8N 8N 8N 8N 8N 8N 8N 8N 8N 8N 8N coagumoue 88.88.8N 8N 8N 8N 8N 8N 8N 8N 8N 8N 8N H88 8 88.88.8N 8N 8N 8N 8N 8N 8N 8N 8N 8N 8N H88 8 88.8“.8N NN NN 8N 8H 8N 8N 8N 8N 8N 8N H88 N 88.88 NN 8N NN 8N 8N 8N NN NN NN NN o 8N .HH88 H I o: 888 8 88 oz 888 8 88 8mm + smog come cmoE m H88 H88 HmQ Hd< HOHUCOU HOHDGOU DOUOOHCH UOUUOHCH msHflsmpo8 mafiouoq c8 cflmuum mem 828 cu“; nmuomuefi 888Q8Ho .8> mumHmHQ Houucoo Ho Aueafloz eun o\oe c mQOHumuucmucou mam ammooss chHummbcH HHmsm .m.m 8H888 126 I Infected Ci Control 15- W9 dry matter 10» Figure 3.4. Pooled small intestinal mucosal RNA concentrations for control piglets vs. piglets infected with the SDSU strain of porcine rotavirus (mean i standard error). RNA of 3 and 8 DPI piglets > RNA of 1 and 2 DPI piglets (P<0.01). Infection X DPI interaction existed (P<0.05). 127 higher than in piglets 1 and 2 DPI (P<0.01). A significant infection X time interaction existed .in the mucosal RNA concentration. Intestinal mucosal DNA concentrations of infected piglets were lower than those in control piglets (P<0.01). Mucosal DNA concentrations of piglets 8 DPI were higher than in piglets 1 DPI. Mucosal DNA concentrations of piglets 3 DPI were higher than in piglets 1 and 2 DPI (P<0.01). Protein concentrations of piglets 3 and 8 DPI were higher than in piglets 1 DPI (P<0.01). The results of protein and nucleic acid ratios are presented in Table 3.4. There were no main effect interactions on intestinal mucosal protein and DNA concentrations. The ratio of protein:RNA between groups was not significantly different. The ratios of RNA:DNA and protein:DNA in the infected group were significantly higher than those in the control group (P<0.01) . Vitamin A concentration Total hepatic vitamin A concentrations (Table 3.5, Figure 3.5) ranged from 108 ug/g dry weight in a control piglet 1 DPI treated with putrescine to 1433 ug/g dry weight in an infected piglet 8 DPI treated with retinyl palmitate. Total liver vitamin A, retinyl palmitate and retinol cOncentrations in RP and RPP groups were significantly higher than those in P and MO groups (P<0.01). Piglets 8 DPI had higher concentrations of liver retinol than piglets 1 DPI. Livers from piglets 3 and 8 DPI had higher Table 3.4. protein:RNA ratios1 128 Protein:DNA, RNA:DNA and from control piglets VS. piglets infected with the SDSU strain of porcine rotavirus 2 Treatment DPI 3 PROT:DNA RNA:DNA PROT:RNA group 1 Infected RP 29 i 1.42 1.70 1 0.09 173;1.70 P 32 1 2.13 1.81 1 0.09 183:1.84 RPP 31 1 1.80 1.92 1 0.07 161:0.37 MO 29 1 2.21 1.72 1 0.14 171:1.12 Control RP 27 1 1.65 1.69 1 0.06 16:;l.35 P 29 1 1.34 1.70 1 0.09 171:1.43 RPP 28 1 1.13 1.66 1 0.09 17311.18 MO 26 1 2.44 1.56 1 0.06 173:1.08 2 Infected RP 30 1 1.68 1.67 1 0.09 183:0.96 P 29 1 1.73 1.75 1 0.09 16: 1.42 RPP 27 1 2.35 1.74 1 0.11 15:;0.73 MO 30 1 3.86 1.87 1 0.20 161:0.74 Control RP 27 1 1.18 1.58 1 0.06 171:0.39 P 27 1 0.84 1.56 1 0.02 171:0.46 RPP 28 1 1.26 1.66 i 0.05 173:0.72 MO 32 1 1.64 1.70 1 0.09 191:1.81 3 Infected RP 29 1 2.56 1.71 1 0.05 173:1.16 P 29 1 1.30 1.79 1 0.07 163:0.17 RPP 27 1 1.36 1.79 1 0.06 153:0.87 MO 28 1 2.15 1.69 i 0.08 163:0.80 Control RP 27 1 1.25 1.63 1 0.05 161:0.52 P 29 1 1.30 1.75 1 0.06 171;0.96 RPP 29 1 0.69 1.78 1 0.08 163:0.68 MO 28 1 1.68 1.64 1 0.14 173:1.78 8 Infected RP 32 1 2.47 1.85 1 0.13 17:_0.15 P 32 1 2.08 1.80 1 0.05 17__0.73 RPP 29 1 1.62 1.79 1 0.07 161:0.98 MO 31 1 1.28 1.84 1 0.01 171;°°71 Control RP 29 1 0.85 1.81 1 0.05 163:0.83 P 29 1 0.41 1.96 1 0.10 151:0.67 RPP 29 1 1.82 1.67 1 0.10 173:0.65 MO 27 1 1.41 1.76 1 0.10 153:0.29 1Values are presented as Mean : Standard Error. 2DPI: day postinoculation. 3Each group consisted of 4 piglets. Group RP: 9,000 IU retinol palmitate/day, Group P: 1,050 mg putrescine/day, 9,000 IU retinol palmitate and 1,050 mg putrescine/day, Group MO: milk only. Group RPP: '1 129 .HHo.ovmc coauoououca Hmo x ucofiumoue .HH8.8V88 mooHoHo oz 888 8 mo oases A muoH8H8 888 888 88 mo 88>88 .HHo.ovmc muoamao HmQ H No U¢>Bm A woodman Hmo m can m mo HU¢>Bmc mcoeumuucuocoo (.cflfiou8> Hmuou venomom "momaamco Hooaumwuoumo .muoaoam 8.80Hw moouo>¢88 .QOHumasoocHumom amp "HmoHQ .haco xaflfi "oz .aoo\ocaomonusm OE omo.H new oomuHsHmo HosHooH 8H 888.8 .888 .888\88H8888888 88 888.H .8 .888\oumuHsH88 HosHuoH 8H 888.8 "88.. I 88“ 8N“ 88H 8HN 88“ 88M 8H8. 888 8H8 88H 88 H 8885 888 + 888 H88 88N 888 88H 888 888 88H 888 88H 8N8 888888888 888 8N8 888 88H H88 88H N88 888 8HN 888 88H 888H H88 8 88M 8N8 888 88N 888 88N 888 HN8 88H 888 88H 8N8 H88 8 NNH.88N 88N 88H N88 88H N88 88N 88H 8N8 88H oN88 .HH88 H um + some H88 H88 H88 H88 Houucou Honucoo pouooucH pouooucH msufl>muou ocflouoo mo cflmuum mem 888 88H: 8888888H 888H8H8 .88 888H8H8 Hososoo 8o 888H8888888888 4 8HE88H> H8888 8H88Q8m .n.m 8Hn88 130 IthmA aHMawn I Vitamin A and Putrescine CI Milk Only Iomwvmmm Figure 3.5. Pooled' hepatic total vitamin A concentration of control and infected piglets (mean 1 standard error). * Significantly different from putrescine and milk only groups (P<0.01). 131 concentrations of total vitamin A and retinyl palmitate than the livers from piglets 1 DPI (P<0.01). _A significant interaction of treatment X time effect existed (P<0.01). Livers from infected piglets 3 DPI which were treated with vitamin A only had higher concentrations of total vitamin A than those from piglets that were treated with vitamin A and putrescine combined (P<0.01). DISCUSSION The results of this experiment indicated that intestinal mucosal protein, DNA and RNA concentrations in dry matter weight were significantly influenced by time. Older piglets (13-day-old) had significantly higher RNA concentrations than younger piglets (six-day-old). These results are similar to the findings of Grant et a1 (1990). They found that the intestinal mucosa of 14-day-old piglets fed with milk only, contained higher protein, DNA and RNA than_the intestinal mucosa of seven-day-old piglets. Higher concentrations of protein and nucleic acids were probably associated ‘with the ‘thickness of 'the intestinal mucosal crypts. Moon (1971) indicated that small intestinal mucosal cell replacement time in young piglets was slower than in older piglets. The shorter replacement time in older piglets was caused by a reduction in villous length and an increase in crypt depth (Moon, 1971). In the present study, crypt depths of older piglets were significantly higher than 132 those in younger piglets. As a proliferation compartment, crypts contain higher concentrations of protein, DNA and RNA. Thus the greater concentrations of protein, DNA and RNA in older piglets may have occured because they have larger crypts. Results of the present study also indicated that protein. and DNA. concentrations of infected piglets were lower than control piglets. Except in piglets 2 DPI, RNA concentrations of the infected piglets were also lower than the control group. Several investigators have shown that the concentrations of protein and nucleic acids in the intestine are influenced by the level of nutrition and the degree of intestinal damage. lProtein 'and nucleic acid concentrations in the intestine of sheep fed ad libitum were higher than in sheep with restricted feed intake (Burrin et al, 1991). Intestinal. mucosal. protein and. nucleic racid concentrations of rats with intestinal mucosal atrophy were lower than those in normal rats (Wang and Johnson, 1991). In the present study, rotavirus infection reduced intestinal mucosal volume and caused a malabsorptive diarrhea. This might explain the decreased concentrations of protein and nucleic acids in the small intestinal mucosa of the infected piglets. The ratios of RNA:DNA and protein:DNA in the intestinal mucosa were significantly influenced by infection status but not by treatment nor time. The ratios of RNA:DNA and protein:DNA in the infected piglets were significantly 133 higher than in the control piglets (P<0.01). Protein:DNA and RNA:DNA ratios are indicators of intestinal epithelial cell size (Allison et al, 1963, Robinson 1969, Winich and Noble 1965), therefore the results of this experiment indicate that the intestinal epithelial cell size was greater in the infected than in the control piglets. However, this is probably not the case, since histopathologic results indicated that the infected animals had higher numbers of squamous and cuboidal cells. Thus, the infected piglets had more attenuated or smaller cells than the control piglets. In addition, results also indicated that infected piglets had lower DNA concentrations without any increase in protein concentration. Thus, the increased ratios of protein:DNA and RNA:DNA in the infected animals may not indicate larger cell volume, but may be the result of a reduction in DNA concentration. Daily dietary administration of retinyl palmitate and/or putrescine did not have any effect on the mucosal protein and nucleic 'acid concentrations in infected’ or control piglets. A deficiency of vitamin A reduced protein synthesis in the rat intestinal mucosa (De Luca, 1969). Administration of vitamin A to vitamin A deficient animals increased RNA synthesis in the small intestinal mucosa (Johnson et al, 1969; Zachman, 1967). The absence of vitamin A effects in the present study compared to the previous study was probably because the piglets were not in a deficient status. .ql» —— 134 Also, putrescine did not have any effect on the intestinal mucosal protein and nucleic acid concentrations. Reduced concentrations of intestinal mucosal protein and nucleic acids were seen in animals treated with DMFO, an inhibitor of the polyamine biosynthesis enzyme (Wang and Johnson, 1991; Seidel et al, 1984). In hepatectomized rats, exogenous putrescine reversed the reduction of protein and nucleic acid synthesis caused by DMFO. Exogenous putrescine did not have any effects on hepatectomized rats without DMFO (Luk, 1986). Since putrescine did not have any effect on the protein and nucleic acid concentrations in both infected and control piglets, rotaviral infections may not interfere with polyamine biosynthesis. Increased DNA synthesis and content in intestinal mucosa were shown to be associated with increased cellular proliferation in the crypts (Luk and Baylin, 1984; Tsujikawa et al, 1990). Results of the present study indicate that administration of vitamin A and/or putrescine increased cellular proliferation in the crypts. However, the data regarding intestinal. mucosal. DNA. concentrations ‘were not significantly Iiifferent. The inability' to show' a correlation between cellular proliferation and increased DNA concentrations in the present study may have been due to technical problems. The amount of DNA produced by the proliferative cells may have been too small to be detected adequately by the present method of analysis. In addition, the proliferating cells are located in the crypts, whereas 135 the specimens for the analyses were obtained from both crypt and villous portions of the intestinal mucosa, thus affecting the present results. Hepatic vitamin A concentrations, including total vitamin A, retinyl palmitate and retinol, were significantly influenced by treatment and time, but not influenced by infection status. Reduced vitamin A absorption, as indicated by a reduced serum retinol and retinyl palmitate concentrations, was seen in calves infected with by Cryptosporidium parvum (Holland et a1, 1990). In the present study, no analysis was done on serum vitamin A concentrations. The concentrations of hepatic vitamin A observed in this study appeared to be associated with the amount of vitamin A intake which corresponded with findings by other investigators (Hoppe et al, 1992; Wellenreiter et al, 1969; Pryor et al, 1969). Vitamin A concentrations of RP and RPP groups increased steadily, whereas the concentrations of vitamin A in P and MO groups remained at ‘the 'base concentration. This‘ pattern resulted in a significant interaction of treatment X time effect. Reference values for normal concentrations of vitamin A in piglets were not available. Compilation of data and references generated. by 'the HNutrition Laboratory' in the Department of Animal Science, Michigan State University indicated that the normal concentration of liver total vitamin A in young piglets varied between 125 to 200 IIg/g 136 dry weight, which agrees with a variety of sources (Steinhardt. et al, 1985; Hennig’ et al, 1985). In the present study, all piglets (1, 3 and 8 DPI) given P and M0 had normal concentrations of vitamin A. Vitamin A concentrations tended to be lower in the liver of animals treated with putrescine. The infected piglets 1 and 8 DPI in the P group had lower total hepatic vitamin A concentrations than infected piglets in the MO group. Also, the infected piglets given RPP had lower hepatic total vitamin A concentrations than infected piglets given RP. The difference between infected piglets given RPP or RP was statistically significant 8 DPI. The reason for this trend is not known, and warrants further investigation. In the present study, vitamin A was given in the form of retinyl palmitate, stabilized in an acacia-starch matrix with antioxidants. The stability of vitamin A in dry feeds or premixes is reduced by hygroscopic agents such as salt and urea (Mitchell, 1967; Shields et al, 1982). Putrescine is a highly reactive sUbstance, especially with a variety of cellular molecules (Wolfe, 1993). Also, putrescine is a strong hygroscopic substance, but this hygroscopicity was unlikely to affect the stability of vitamin A since in the present experiment both substances were given in liquid milk. Some concern has been expressed about the instability of vitamin A in the retinol form. Retinyl palmitate is converted to retinol prior Ito absorption by intestinal mucosal cells. Thus, retinol may be present in the 137 intestinal lumen or within the intestinal mucosal cells. Since putrescine is also present both in the intestinal lumen and within the intestinal mucosal cells, the degradation of retinol may occur intraluminally or intracellularly. In conclusion, the results of this study indicate that 13—day-old piglets had higher protein and nucleic acid concentrations than six-day-old piglets. Rotavirus infection reduced intestinal mucosal protein and DNA concentrations in piglets. Supplementation with vitamin A and/or putrescine did not have any effect on the intestinal protein and nucleic acid concentrations. Rotavirus infection did not have any effect on the hepatic vitamin A concentrations. Daily administration of vitamin A increased hepatic vitamin A concentrations in piglets. CONCLUSIONS The results from the study presented in this dissertation indicate that: 1. Daily dietary administration of vitamin A (30,000 IU/kg of milk) and/or putrescine (25g/kg dry matter of milk) was ineffective. in the prevention or attenuation of rotaviral infectivity, and the clinical signs and intestinal mucosal damage caused by rotaviral infection in piglets. Furthermore, putrescine appeared to increase rotaviral infectivity. Rotaviral infection in piglets caused villous atrophy resulting in a reduction of the small intestinal mucosal volume. Daily dietary administration (M? retinyl palmitate or putrescine alone or in combination increased the mitotic index in small intestinal crypts, suggesting a possible beneficial effect of retinyl palmitate or putrescine on the regeneration of intestinal mucosa. Rotavirus infection reduced intestinal mucosal protein and DNA concentrations in piglets. Supplementation with vitamin A and/or putrescine did not have any effect on the intestinal protein and nucleic acid concentrations. 138 139 Thirteen-day-old piglets had higher protein and nucleic acid concentrations than six-day-old piglets. ( Rotavirus infection did not have any effect on the hepatic vitamin A concentrations. Hepatic vitamin A concentrations in piglets correlated with the amount of vitamin A consumed. APPENDIX Information regarding the dose of rotavirus inocula is generally unavailable and varies depending on the host and environmental factors. It was therefore necessary to perform a series of studies to propagate the rotavirus and determine the pig-infectious-dose fifty (PIDEU, dilution of the virus pool that caused diarrhea in 50% of the piglets) and the dose of inocula allowing infected piglets to survive until eight days postinoculation (the end of the observation period). MATERIALS AND METHODS Two four-day-old, naturally born, colostrum-deprived piglets were used for virus propagation. Each piglet was orally inoculated with 1 ml of a filtered intestinal homogenate containing a South Dakota State University (SDSU) strain of porcine rotavirus, obtained from fun i). Benfield (South Dakota State University, Brookings, SD). Piglets were placed in individual cages in a temperature-controlled room (30-32°C) and fed 100 ml of evaporated milk three times a day. Forty-eight hours after oral inoculation with rotavirus, piglets were euthanatized with sodium pentobarbitalw. Immediately after opening the carcasses, 3° The Butler Co., Columbus, OH. 140 141 the small intestine was placed on a cold glass surface. The intestine ‘was opened longitudinally’ along’ the :mesenteric attachment and the mucosa was scraped. Small intestinal mucosal scrapings and intestinal contents were collected and frozen at -20°C until further processing. A 10% suspension of the intestinal mucosa and its contents in Hank's balanced salt solution (HBSS)31 was prepared. The suspension was homogenized with a Teflon pestle tissue homogenizerk and then frozen and thawed twice to rupture the infected epithelial cells. The homogenate was centrifuged33 20 minutes at 5,000 x. g at 4°C. The supernatant was collected and then filtered through a series 4 of filters3 with average pore-diameter of 0.80 pm, 0.45 pm and 0.22 pm. The filtrate (10 ml) was then divided into 0.5 ml plastic tubes and frozen at -70°C for stock virus. Fecal samples from the two piglets were also collected and tested using an ELISA kitfl for the presence of rotavirus antigen. Virus infectivity titration was done on eight four-day- old, caesarean-derived, colostrum-deprived (CDCD) piglets. One tube containing 0.5 ml of virus suspension was thawed “ Sigma Chemical Co., St. Louis, MO. ” Thomas® Teflon pestle tissue homogenizers, Thomas ScientificTM USA, Philadelphia, PA. ” Refrigerated Centrifuge, Model.PR-7000, International Equipment Co., Needham, MA. M Nalgene® Disposable Filterware, Nalge Comp., Rochester, NY. ” PathfinderTM®, Kallestad Diagnostics, Austin, TX. 142 and resuspended with H888 to dilutions of lofifl JIY7 and 10”. Two piglets were each orally inoculated with 1 ml of 10'” dilution of the viral suspension, three piglets with 1 ml of the 10'7 dilution and three piglets with 1 ml of the 10'8 dilution. Piglets were observed 3 times daily for signs of diarrhea. Fecal samples were collected just prior to inoculation and 48 hours after inoculation for ELISA testing. Piglets that had diarrhea and a positive ELISA test 48 hours after inoculation were considered infected. The pig infectious dose fifty (PIDSO) was defined as the dilution of ‘the stock ‘virus that caused diarrhea and a positive ELISA test in 50% of the piglets. Sixty-six CDCD and four naturally born (NB) piglets were used to determine the dose of virus inocula that would allow the infected piglets to survive until 8 DPI. The NB piglets used in this study were taken from the sows at two days of age.f All the CDCD and NB piglets were infected with a serial 10-fold dilution of stock rotavirus (10'8 to 10'10 dilutions) at five days of age. Piglets were observed for signs of diarrhea, vomiting and dehydration. Piglets were euthanatized at 1, 2, 3 or 8 DPI, and intestinal sections were collected and examined microscopically. The presence of rotaviral antigen was detected either by ELISA using the fecal material or by an immunohistochemistry technique on the intestinal sections. 143 RESULTS Virus infectivity titration All eight CDCD piglets inoculated with loflfi JIY7 or 10” dilutions of stock rotavirus showed clinical signs of diarrhea. Fecal samples collected at 2 DPI were positive for the presence of rotavirus using the ELISA. Since all of the piglets were positive for rotavirus infection, the PIDW of the stock rotavirus was not determined, but was considered to be less than 104. study using 10"8 dilution of stock rotavirus in CDCD piglets Of the 18 CDCD piglets that were inoculated with 10’8 dilution of stock rotavirus, 13 piglets were euthanatized as scheduled on 1, 2 or 3 DPI. Five piglets which were scheduled to be euthanatized 8 DPI did not survive. All piglets showed clinical signs of rotaviral infection, including diarrhea,. vomiting and severe dehydration. In addition to severe villous atrophy, some of the piglets also had colonies of rod-shaped bacteria adherent to intestinal villi and crypts. Rotaviral. antigen was detected in all piglets. 144 Study using 10"9 dilution of stock rotavirus in CDCD piglets Of the nine piglets that. were inoculated. with. 10'9 dilution of stock rotavirus, five piglets were euthanatized as scheduled On 1, 2 or 3 DPI. Four piglets did not survive until 8 DPI. The clinical signs and lesions were similar to those of CDCD piglets infected with 10"8 dilution of stock rotavirus. Study using 10'10 dilution of stock rotavirus in CDCD piglets Of the 14 piglets that were inoculated with 10'10 dilution of stock rotavirus, seven piglets were euthanatized as scheduled on 1, 2 or 3 DPI. Seven piglets did not survive until 8 Inn; The clinical signs and lesions were similar to those previously described. Study using CDCD piglets not infected with rotavirus Of the 25 piglets observed in this study, 10 piglets were euthanatized as scheduled when they were six, seven or eight days of age (1, 2 or 3 DPI). Fifteen piglets did not survive until 13 days of age (8 DPI). Clinical signs were mild to severe diarrhea and dehydration. Intestinal lesions in most of the piglets were severe villous atrophy with the presence of rod-shaped bacteria adherent to the intestinal villi and crypts. Both the ELISA with the fecal material and the immunohistochemistry technique with the intestinal 145 sections were negative for the presence of rotavirus antigen. study using 10'1'o dilution of stock rotavirus in NB piglets 0 dilution of the Four NB piglets were infected with 10'1 stock rotavirus. Diarrhea were observed in all piglets 2 and 3 run; Fecal samples from all piglets collected at 3 DPI were ELISA positive, indicating a rotaviral infection. The clinical signs of diarrhea disappeared at 5 [NHL All piglets looked healthy after 5 run; One piglet died at 7 DPI with a ruptured stomach. Rotaviral antigen was not detected in fecal material nor the intestinal section from this piglet. The remaining three piglets were euthanatized 8 DPI. Rotaviral antigen was not detected in piglets euthanatized 8 DPI. DISCUSSION All of the CDCD piglets, whether infected or not, did not survive until 8 DPI. It was very difficult to maintain piglets that had not consumed colostrum in a relatively germfree environment. Villous atrophy and the presence of rod-shaped bacteria adherent to the small intestinal villi and crypts were consistently seen in the CDCD piglets, indicating the possibility of a dual infection with E. coli. 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Nutr., 111: 778-788. VITA The author was born in Surabaya, Indonesia in June 20, 1953. He received the degree of Doctor of Veterinary Medicine from University of Airlangga, Indonesia in 1978. Since 1979, the author has worked at the Department of Pathology, Animal Disease Investigation Center' in. Maros, Indonesia. In 1985, the author received a scholarship from Australian International Development Assistance Bureau and spent 2 years (1985-1986) as a graduate student in the Department of Pathology, James Cook University, Australia. He received the degree of Masters of Science in 1989. In the fall of 1991, the author received a scholarship from Indonesian Development Planning Agency (BAPPENAS) and was admitted into a Ph.D. program in the Department of Pathology, Michigan State University.