A Scanning and Transmission Electron Microscopic Study of Rotavirus-Induced Intestinal Lesions in Neonatal Gnotobiotic Dogs

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1 Vet. Pathol. 23: (1986) A Scanning and Transmission Electron Microscopic Study of Rotavirus-Induced Intestinal Lesions in Neonatal Gnotobiotic Dogs C. A. JOHNSON, T. G. SNIDER, 111, W. G. HENK, AND R. W. FULTON Department of Veterinary Pathology, Department of Veterinary Microbiology and Parasitiology; and Department of Veterinary Anatomy and Fine Structure, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA Abstract. Neonatal gnotobiotic dogs orally inoculated with canine rotavirus had ultrastructural changes limited to the jejunal and ileal regions of the small intestine. Early scanning electron microscopic findings consisted of swollen villus epithelial cells, denuded foci on intestinal villi, and slight to moderate villus atrophy. Later changes were slight villus atrophy with no denuded intestinal villi. Transmission electron microscopic changes in villus epithelial cells from 1 2 to 48 hours post-inoculation included: rotavirus particles associated with intracytoplasmic vacuoles near the terminal web and apical tubules; viral particles in dilated cisternae of rough endoplasmic reticulum; and moderate numbers of necrotic cells having no microvilli, swollen mitochondria, membrane-bound lipid-like material in the cytoplasm, clumped chromatin around the periphery of the nucleus, and disruption of the cytoplasmic membrane. In jejunum and ileum at 72 to 154 hours postinoculation, there were fewer necrotic villus epithelial cells and fewer virus particles. In addition, the ultrastructural morphology of the majority of the villus epithelial cells was similar to crypt epithelium. These studies showed that rotavirus infected the villus epithelial cells with subsequent propagation of the rotavirus and destruction of villus epithelial cells. The changes seen by scanning and transmission elec- Materials and Methods tron microscopy in rotavirus-infected small intestines Experimental design have been well characterized in gnotobiotic piglets, 13,L5~18 gnotobiotic calves,i4j6 and suckling mi~e.',~,~ Twenty gnotobiotic puppies were derived by caesarean section from three mongrel bitches by methods previously The scanning electron microscope showed swelling and described. lo Eleven two-day-old puppies were inoculated loss of absorptive villus epithelial cells from the upper orally with 1 ml cell culture suspensions containing the 16th one-half of intestinal villi, denuded villi, villus atroand 17th passage of a canine rotavirus i~olate.~ The virus phy, and recovery of the denuded villi by epithelium titer was los TCIDSO/ml as determined by indirect immuin rotavirus-infected gnotobiotic piglets,13j8 gnoto- nofluorescence. Nine control gnotobiotic puppies were not biotic calves,14 and suckling mice.5 Transmission elec- inoculated. tron microscopic studies in these same species infected At least one inoculated and one control puppy were aneswith rotavirus have demonstrated replicative stages of thetized at 12, 18, 24, 48, 72, 96, 132, and 154 hours postthe rotavirus within villus epithelial cells, necrosis of inoculation. Samples were removed from three sites on the villus epithelial cells, and the presence of cuboidal ep- small intestine and one site on the colon and immediately ithelial cells on intestinal villi with ultrastructural mor- immersed in a 1.25% glutaraldehyde and 2% formaldehyde phology similar to immature crypt epithelium. solution with 0.1 M sodium cacodylate buffer (ph 7.4) for 1,3,15,18 scanning and transmission electron microscopy. The puppies The purpose of this study was to examine the scanwere subsequently sacrificed, and a complete necropsy was ning and transmission electron microscopic changes in performed. the small intestine of newborn gnotobiotic puppies infected with a canine rotavirus isolated from a neonatal Scanning electron microscopy puppy with fatal diarrhea.9 This canine rotavirus iso- Small (0.5 cmz) portions of tissue were cut from the samlate, LSU 79C-36, has been shown to cause clinical ples of gut previously fixed in glutaraldehyde-formaldehyde disease, small intestinal lesions, serum antibody re- fixative. These specimens were fixed for 1.5 hours, rinsed sponse, and shedding of rotavirus in the feces of orally twice with cold 0.1 M cacodylate buffer in 5% sucrose (ph inoculated gnotobiotic puppies.' 'J* 7.4), and stored in this buffer at 4 C. Glutaraldehyde, tannic 443

2 444 Johnson et al. Fig. 1. Scanning electron micrograph of duodenum from control puppy killed at 12 hours post-inoculation. Majority of the intestinal villi are long and slender with numerous transverse grooves. Sections of jejunum and ileum from control puppy similar in appearance. Fig. 2. Jejunum from an infected puppy killed at 12 hours post-inoculation. Numerous clumps of villus epithelial cells protrude from upper portion of villi. Fig. 3. Higher magnification of Fig. 2; clumps of rounded villus epithelial cells and area denude of epithelial cells (arrows) on villi. Fig. 4. Jejunum in infected puppy killed at 24 hours post-inoculation; short and fused (arrow) intestinal villi. Only a few epithelial cells protrude from surface of the villi. acid, and osmium tetraoxide (OsO,) were used to post-fix the tissue samples using a method previously described.17 The specimens were washed in distilled water and dehydrated through one change each of 50%, 70%, 8O%, 90%, and 95% ethanol for 10 minute intervals. The tissues went through three changes of 100% ethanol at 10 minute intervals. The dehydrated specimens were placed in a critical point drying apparatus and dried from liquid carbon dioxide. Dried sam-

3 Canine Rotavirus in Neonatal Gnotobiotic Dogs 445 Fig. 5. Ileum from infected puppy killed at 72 hc 3urs post-inoculation. Villus atrophy still evident. ples were attached with silver conducting paint to 12 mm aluminum scanning electron microscopy studs. The specimens were then sputter-coated with gold palladium alloy. Processed tissue samples were examined with a Cambridge Stereoscan 150 scanning electron microscope operated at an accelerating voltage of 20 kv and a working distance of 12 to 15 mm. Images were recorded on Polaroid@ film with a scan of 2,000 linedframe. Transmission electron microscopy Small (1 mm3) pieces of tissue were minced from the samples of gut. These specimens were fixed for 1.5 hours, rinsed twice with cold 0.1 M sodium cacodylate buffer in 5% sucrose (ph 7.4), and stored at 4 C. The tissue samples were postfixed in 1% OsO, in sodium cacodylate buffer (ph 7.4) for 1 hour, washed three times (5 minutes each wash) in 5% buffered sucrose (ph 7.4), placed in 1% tannic acid for 1 hour, and washed three times (5 minutes each wash) in distilled water. The specimens were then dehydrated in graded concentrations of ethanol and embedded in an epon-araldite mixture. Sections were cut with a diamond knife on an ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM- 10 transmission electron microscope. Results In sections of small intestine from the control puppies, the majority of villi were long and slender with numerous transverse grooves on the villus surfaces. The epithelial surface appeared smooth without distinct cell borders except on the tips of the villi (Fig. 1). The colon had a flattened, undulating surface with numerous goblet cells, and most of the mucosal surface was covered by a thick layer of mucus. Differences in appearance of small intestines between control puppies and infected puppies were found only in the jejunum and ileum. At 12 and 18 hours post-inoculation numerous single and small clusters of rounded epithelial cells on the upper one-third of several intestinal villi protruded from the surface (Fig. 2). A few focal sites on the villi were denude of absorptive intestinal epithelium (Fig. 3). By 24 and 48 hours postinoculation, mild to moderate villus atrophy and broadened intestinal villi were seen in the jejunum and some villi appear fused with only a few rounded epithelial cells and denuded areas (Fig. 4). In the ileum, moderate villus atrophy, numerous broadened, and several fused villi were present. A few sites denude of epithelium and small numbers of protruding intestinal epithelial cells were also seen. The jejunum from infected puppies killed from 72 to 154 hours post-inoculation had long, slender villi and did not appear different from the sections of control puppies sacrificed at these times. However, at 72 and 96 hours post-inoculation, mild villus atrophy was still evident in the ileum of the infected puppies (Fig. 5). In addition, the epithelial surface of the intestinal villi appeared roughened and irregular. No differences were seen in the ileum between infected and control puppies at 132 and 154 hours post-inoculation. In the small intestine of uninfected puppies, ultrastructural morphology of the villus epithelial cells in the duodenum did not change throughout the experiment. These cells were tall and columnar with many long, uniform, regularly spaced microvilli and a well developed terminal web. Mitochondria were numerous; most were oriented in a plane perpendicular to the lumen. The nucleus was located at the base of the cell. A moderate amount of rough endoplasmic reticulum and numerous polyribosomes were present in the cytoplasm. There were numerous small to moderatesized aggregates of electron-dense material in the cisternae of vacuolated endoplasmic reticulum. The morphology of the jejunal villus epithelial cells in control puppies was similar to the duodenal epithelium through 18 hours post-inoculation. However, at 24 through 154 hours post-inoculation, the villus epithelium in the jejunum contained a single large or

4 446 Johnson et al. Fig. 6. Jejunal epithelial cells from a control puppy killed at 24 hours post-inoculation. Supranuclear, membrane-bound, cytoplasmic vacuoles contain variable quantities of electron-dense material and lamellate bodies (arrow). Uranyl acetate and lead citrate stain. Bar = 2 pm. multiple moderate-sized, membrane-bound, supranuclear, cytoplasmic vacuoles (Fig. 6). The majority of the vacuoles contained variable quantities of homogenous membrane-bound electron-dense material and small to moderate-sized lamellate bodies. Numerous invaginations of the cell membrane on the luminal surface were seen, and several pinocytotic vesicles could be seen as well beneath the terminal web. There were dilatations of the lateral interdigital spaces which contained variable numbers of homogenous, electron-dense globules. The ultrastructural morphology of villus epithelial cells in the ileum of the control puppies was similar to the jejunum, except the supranuclear vacuoles were present at 12 hours postinoculation and persisted through 154 hours post-inoculation. The epithelial cells of the crypts at the base of the jejunal and ileal mucosa were cuboidal to columnar with a reduced number of shorter microvilli than absorptive villus epithelial cells. In addition, no large, clear, membrane-bound vacuoles were present

5 Canine Rotavirus in Neonatal Gnotobiotic Dogs 447 Fig. 7. Jejunal epithelial cells from infected puppy killed at 12 hours post-inoculation; rotavirus particles about 60 nm diameter in dilated cisternae of rough endoplasmic reticulum. Virus particles have electron-dense core about 30 nm in diameter surrounded by one or two membranes (arrows). Uranyl acetate and lead citrate stain. Bar = 100 nm. in the crypt epithelium, and there appeared to be reduced numbers of mitochondria. Polyribosomes were abundant in the cytoplasm of the crypt cells. The epithelial cells in the colon of control puppies were cuboidal to columnar with sparse, short, irregularly-spaced microvilli on the surface. The nuclei were located at the base of the cell, numerous polyribosomes were present throughout the cytoplasm, and the quantity of rough endoplasmic reticulum and the numbers of mitochondria appeared to be reduced when compared to the villus epithelium of the small intestine. Some epithelial cells in the colon contained moderatesized, clear, membrane-bound cytoplasmic vacuoles. Numerous goblet cells were present throughout the colonic mucosa. In the infected puppies, ultrastructural changes were limited to the absorptive villus epithelial cells of the jejunum and ileum. Changes in the epithelium of the jejunum and ileum were quite similar. At 12 hours post-inoculation, some villus epithelial cells had a less electron-dense cytoplasm than the majority of the others, and virus particles could usually be seen in these cells. In these cells, virus particles had a centrallylocated, round, electron-dense core about 25 to 30 nm in diameter surrounded by one or two membranes. The virus particles were located in dilated cisternae of rough endoplasmic reticulum (Fig. 7). At 12 and 18 hours post-inoculation, virions were found on the luminal surface of villus epithelial cells in the jejunum and ileum (Fig. 8). Some virions appeared to be associated with intracytoplasmic vacuoles in the region of the apical tubules. From 18 through 48 hours post-inoculation, moderate numbers of necrotic villus epithelial cells were seen in the jejunum and ileum. Often, two to four necrotic cells were present at the same site, and the changes in the ultrastructure of the cells were characterized by loss of microvilli, swollen mitochondria, a moderate amount of membrane-bound lipid-like material in the cytoplasm, clumped chromatin around the periphery of the nucleus, indistinguishable cell boundaries between adjacent degenerative cells, and disruption of the cytoplasmic membrane with loss of cellular contents into the lumen (Fig. 9). Also, from 18 to 48 hours post-inoculation, rotavirus-like particles were seen in dilated cisternae of rough endoplasmic reticulum, and some virions appeared to be budding through the membrane into the dilated cisternae (Fig. 10). Two sizes of rotavirus-like particles were seen in these cisternae of rough endoplasmic reticulum, one approximately 75 to 80 nm in diameter, and the second, about 55 to 65 nm in diameter. In addition, small to moderate-sized, membrane-bound spaces in the cytoplasm were composed of varying quantities of a fine granular

6 448 Johnson et al. Fig. 8. Surface of a villus epithelial cell in ileum from infected puppy killed at 18 hours post-inoculation. Two rotavirus particles (arrows) on luminal surface. Uranyl acetate and lead citrate stain. Bar = 100 nm. material and round, electron-dense particles approximately 25 to 30 nm in diameter (Fig. 11). Large, clear, membrane-bound cytoplasmic vacuoles similar to the vacuoles seen in the jejunal and ileal villus epithelial cells of the control puppies were present in the jejunal and ileal villus epithelial cells of infected puppies from 12 to 18 hours post-inoculation. They were not seen from 24 through 154 hours postinoculation. In the jejunum and ileum at 72 to 154 hours postinoculation, there appeared to be fewer necrotic villus epithelial cells and fewer cells containing virus parti- cles. The ultrastructural morphology consisted of cuboidal to columnar cells with tall, regularly-spaced microvilli, and a nucleus located at the base of the cell (Fig. 12). There appeared to be fewer mitochondria and less rough endoplasmic reticulum than in the timematched control puppies jejunal and ileal villus epithelium. Discussion Ultrastructural changes in this study demonstrated that the rotavirus infected the villus epithelial cells with subsequent propagation of the rotavirus and de-

7 Canine Rotavirus in Neonatal Gnotobiotic Dogs 449 Fig. 9. Villus epithelial cells in ileum from infected puppy killed at 18 hours post-inoculation. Two degenerating villus epithelial cells (a & b); goblet cell (c). Swollen mitochondria (arrows), lipid-like material (arrowheads), loss of tight cellular junctions between adjacent degenerating epithelial cells (double arrows), loss of cellular contents from cell (b) into lumen. Aggregate of rotavirus particles in cell a (v). Uranyl acetate and lead citrate stain. Bar = 1 pm. struction of villus epithelial cells. These changes were similar to ultrastructural changes seen in gnotobiotic pigs, I 3 ~1 5, * gnotobiotic calves, * 4, and suckling mice 1,3~5 which were each inoculated with rotaviruses isolated from their own species. The early scanning electron microsopy findings in this study closely paralleled the scanning electron microscopy findings in rotavirus-infected gnotobiotic pigs.13j8 Though the same sequence oflesions was seen by scanning electron microscopy in the small intestine of rotavirus-infected gnotobiotic calves14 and suckling mice,5 the initial lesions were not seen until 20 to 40 hours post-inoculation. Changes in the infected puppies intestine suggested that in initial stages of infection the rotavirus did infect villus epithelial cells on the upper portion of the intestinal villi with subsequent cell swelling, degeneration of infected epithelial cells, and detachment of these epithelial cells from the villus.

8 450 Johnson et al. Fig. 10. Ileal villus epithelial cell from infected puppy killed at 48 hours post-inoculation. Rotavirus particle appears to be budding (arrows) into the dilated cisternae of rough endoplasmic reticulum. Two sizes of rotavirus particles in dilated spaces: 75 to 80 nm in diameter (arrowhead), and 55 to 65 nm in diameter (double arrowheads). Swollen mitochondria (m). Uranyl acetate and lead citrate stain. Bar = 1 pm. The partially denuded intestinal villi contracted resulting in villus atrophy. In the later stages of the infection, the denuded intestinal villi were covered by epithelial cells, and the length of the villi increased to near the same length as intestinal villi in the control puppies. Transmission electron microscopy findings in this study have shown that the canine rotavirus replicated within villus epithelial cells and provided an insight into the pathogenesis of rotaviral enteritis in the neonatal dog. The morphogenesis of the canine rotavirus isolate used to inoculate newborn gnotobiotic puppies in this study was similar to that reported for gnotobiotic piglets,ls,l8 gnotobiotic calves,16 and suckling mi~e.',~,~ The process by which the rotavirus enters the absorptive villus epithelial cells has not been clearly demonstrated. In this study, rotavirus particles were associated with intracytoplasmic vacuoles near the terminal web and apical tubules of villus epithelial cells in thin sections of the jejunum and ileum at 18 hours post-inoculation. It could have been possible that the virus gained entry into these villus epithelial cells by way of the pinocytotic activity of the cell. The presence of variable sizes of rotavirus particles within dilated cisternae of rough endoplasmic reticulum seen in this study has been documented in experimental inocula- tions in gnotobiotic piglet~,'~j~ gnotobiotic calves,16 and suckling mi~e.~,~j In addition, the budding of virions through the rough endoplasmic reticulum into the dilated space has also been seen in these three species. This finding has been interpreted as part of the

9 Canine Rotavirus in Neonatal Gnotobiotic Dogs 45 1 Fig. 11. Jejunal villus epithelial cell from infected puppy killed at 24 hours post-inoculation. Fine granular material (g) and numerous, small, round, electron-dense particles about 25 to 30 nm in diameter (arrows) encompassed by membrane within cytoplasm. Nucleus (n) of cell located in upper Y3 of the photograph. Uranyl acetate and lead citrate stain. Bar = 1 pm. maturation process of the rotavirus and represents acquisition of the outer capsid layer. However, the presence of the smaller rotavirus particles within these cisternae of rough endoplasmic reticulum has raised doubt about this hypothesis. In discussing the presence of these smaller virus particles, one author suggested that these virus particles were degenerating particles or the developmental process may have been altered. The dilated spaces filled with a fine granular material and small, electron-dense spheres seen within the cytoplasm of some villus epithelial cells in infected puppies was interpreted to represent the viroplasm and nucleocapsids reported in ultrastructural studies in pigs, calves, and mice. 1,3,5,15~16 In this study, virus-associated single or doublemembraned tubules were not seen in the nucleus or cytoplasm of the infected puppies intestinal villus ep- ithelial cells. The presence of intranuclear and intracytoplasmic tubules has been reported and associated with the morphogenesis of rotavirus in other studies. However, the presence of developmental stages of the rotavirus within infected villus epithelium, coupled with the shedding of rotavirus particles in the feces of the infected puppies, as previously reported, indicated that the canine rotavirus did infect and replicate in the villus epithelial cells of the inoculated puppies. Necrosis of rotavirus-infected villus epithelial cells was seen in the small intestine of the infected puppies. The majority of the necrotic villus epithelial cells were seen between 18 and 48 hours post-inoculation similar to reports in gnotobiotic piglets15 and suckling mice.3 At 24 through 154 hours post-inoculation, villus epithelial cells in the jejunum and ileum of rotavirusinfected puppies lacked the large, clear, membrane-

10 452 Johnson et al. Fig. 12. Villus of epithelial cells in ileum from infected puppy killed at 154 hours post-inoculation. Villus epithelial cells cuboidal with long, uniform microvilli, several mitochondria in apical position of the cell. No large, membrane-bound cytoplasmic vacuoles. Uranyl acetate and lead citrate. Bar = 2 pm. bound cytoplasmic vacuoles seen in the time-matched control puppies. A similar finding was reported in gnotobiotic calves.16 Since age-matched infected and control puppies were used in this study, the absence of these cytoplasmic vacuoles in the infected puppies was interpreted as a pathophysiologic change. This finding may have represented a decreased absorptive activity of the jejunal and ileal villus epithelium in the infected puppies. At 72 through 154 hours post-inoculation, many villus epithelial cells in the jejunum and ileum of the infected puppies were cuboidal to low columnar and had ultrastructural morphology similar to the crypt epithelium. Thus, evidence was provided to suggest that the damaged villus epithelium was replaced by the immature crypt epithelium as reported in other species infected with rotavirus.1,3,5,15,16 The ultrastructural changes of the intestinal villi and villus epithelial cells of the small intestine from rotavirus-infected puppies correlated well with the changes described in gnotobiotic piglets, gnotobiotic calves, and suckling mice. These findings demonstrated that the pathogenesis of canine rotavirus infection in newborn gnotobiotic puppies is similar.

11 Canine Rotavirus in Neonatal Gnotobiotic Dogs 453 Acknowledgements 10 This work supported by grants from the School of Veterinary Medicine Organized Research Fund, Louisiana State University. The authors thank Sherry Gibson, Lindsay Bullock, and Ross Payne for invaluable technical support. References Adams WR, Kraft LM: Electron-microscopic study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus). Am J Pathol 51:39, 1967 Banatvala JE, Chrystie IL, Totterdell BM: Rotaviral infections in human neonates. J Am Vet Med Assoc 173: 527, 1978 Banfield WG, Kasnic G, Blackwell JH: Further observations on the virus of epizootic diarrhea of infant mice: an electron microscopic study. Virology 36:4 1 1, 1968 Carmichael LE, Pollock RVH: Viral diseases of puppies. Gaines Progr p. 1, Fall 1979 Coelho KIR, Bryden AS, Hall C, Flewett TH: Animal model. Pathology of rotavirus infection in suckling mice: a study by conventional histology, immunofluorescence, ultrathin sections, and scanning electron microscopy. U1- trastruct Pathol 259, 1981 Dagenais L, Schwers A, Pastoret PP, Chappuis G: Propagation of bovine rotavirus by dogs [letter]. Vet Rec 109:187, 1981 England J J, Poston RP: Electron microscopic identification and subsequent isolation of a rotavirus from a dog with fatal neonatal diarrhea. Am J Vet Res 41:782, 1980 Eugster AK, Sidwa T: Rotaviruses in diarrheic feces of a dog. Vet Med/Sm Anim Clinic 74:817, 1979 Fulton RW, Johnson CA, Pearson NJ, Woode GN: Isolation of a rotavirus from a newborn dog with diarrhea. Am J Vet Res 42:841, Heneghan JB, Floyd CE, Cohn I: Gnotobiotic dogs for surgical research. J Surg Res 624, 1966 Johnson CA, Fulton RW, Henk WG, Snider TG: Inoculation of neonatal gnotobiotic dogs with a canine rotavirus. Am J Vet Res 44:1682, 1983 Johnson CA, Snider TG, Fulton RW, Cho D: Gross and light microscopic lesions in neonatal gnotobiotic dogs inoculated with a canine rotavirus. Am J Vet Res 44: 1687, 1983 McAdaragh JP, Bergeland ME, Meyer RC, Johnshoy MW, Stotz IJ, Benfield DA, Hammer R: Pathogenesis of rotaviral enteritis in gnotobiotic pigs: a microscopic study. Am J Vet Res 41:1572, 1980 Mebus CA, Newman LE: Scanning electron, light, and immunofluorescent microscopy of intestine of a gnotobiotic calf infected with reovirus-like agent. Am J Vet Res 38553, 1977 Pearson GR, McNulty MS: Ultrastructural changes in small intestinal epithelium of neonatal pigs infected with pig rotavirus. Arch Virol 59:127, 1979 Stair EL, Mebus CA, Twiehaus MJ, Underdahl NR: Neonatal calf diarrhea: electron microscopy of intestines infected with a rotavirus-like agent. Vet Pathol 10: 155, 1973 Sweeny LR, Shapiro BL: Rapid preparation of uncoated biological specimens for scanning electron microscopy. Stain Techol 52221, 1977 Torres-Medina A, Underdahl N R: Scanning electron microscopy of intestine of gnotobiotic piglets infected with porcine rotavirus. Can J Comp Med 44:403, 1980 Tzipori S, Makin T: Propagation of human rotavirus in young dogs. Vet Microbiol 355, 1978 Woode GN: Viral infections of the intestinal tract: pathological and clinical aspects. Les Colloques de 1 Inserm Enteritis Virales/Viral Enteritis Inst Natl Sante Rech Med 99:15, 1979 Request reprints from Dr. Charles A. Johnson, Department of Pathology, The Squibb Institute for Medical Research, PO Box 191, New Brunswick, NJ (USA).