Neonatal Calf Diarrhea

Transcription

1 Vet. Path. 10: (1973) Neonatal Calf Diarrhea Electron Microscopy of Intestines Infected with a Reovirus-Like Agent E. L. STAIR, C. A. MEBUS, M. J. TWIEHAUS and N. R. UNDERDAHL Department of Veterinary Science, University of Nebraska, Lincoln, Nebr. Abstract. Neonatal calf diarrhea induced by a reovirus-like agent was studied by electron microscopy. Gnotobiotic calves were inoculated with virulent and cell-cultureadapted isolates of viruses, and similar results were obtained with both isolates. The virus infected mature villous epithelial cells of the small intestine and replicated predominantly within cisternae of the endoplasmic reticulum. Macrophages within small intestinal villi also contained viral particles. Shedding of virus into the feces apparently resulted from desquaniation of infected epithelial cells. Replacement epithelium did not contain the reovirus-like agent, was of cuboidal or squamous type, and ultrastructurally resembled immature epithelium normally found in the crypts. Escherichia coli was nonpathogenic when inoculated alone and did not adhere to the epithelium when inoculated with the virus. The reovirus-like agent was a primary pathogen since it caused diarrhea and lesions when inoculated alone or with E. coli. Diarrhea of neonatal calves has been a serious and persistent problem of the dairy industry for many years. This disease has become a major problem on ranches with cow-calf operations. Apparently an increased frequency of neonatal calf diarrhea in beef-type calves has been caused by calving during a relatively short period of time in the late winter and early spring when inclement weather causes stress. Cows and their offspring have been concentrated into small areas during the calving season, and transmission of infectious diseases to susceptible calves has been favored. A virus designated initially as neonatal calf diarrhea virus was isolated in 1968 and reported as an etiologic agent in field cases of neonatal calf diarrhea [9]. The disease was reproduced experimentally in specific pathogen-free and gnotobiotic calves inoculated with bacteria-free filtrates obtained from infected calves. Fecal material purified by ultracentrifugation and examined by

2 156 STAIR/MEBUS/TWIEHAUS/UNDERDAHL Table I. Experimental protocol and results of electron microscopic examination Calf Treatment Age at Age at onset Duration Virions in segment of small inoculation of diarrhea of diarrhea intestine h h h jejunum middle ileum 1 control not no no neg. neg. neg. 2 E. coli inoculated 27 diarrhea no diarrhea no neg. neg. neg. 3 E. coli 29.5 diarrhea no diarrhea diarrhea no diarrhea neg. neg. neg neg neg virus virus virus E. coli and virus E. coli and virus virus virus neg neg. neg. neg. neg. neg. neg. electron microscopy had viral particles with a diameter of approximately 65 nm [9]. Further studies on morphology, biochemical and biophysical properties of neonatal calf diarrhea virusindicated that it was reovirus-like [I 51. Viral antigen was present in epithelial cells of the small intestine when frozen sections or fecal smears were stained by immunofluorescent procedures. Viral infections were widespread in Nebraska as determined by immunofluorescent staining of fecal smears [16]. This investigation was undertaken to determine the cell types affected, the intracellular sites of viral replication and the nature of the host response to viral injury, including the ultrastructural changes occurring in cells infected with virulent and cell-culture-adapted virus. Materials and Methods Experimental Calves Ten gnotobiotic calves were used. These calves were delivered by cesarotomy and maintained in isolation units as previously described [8]. Treatments are indicated in table I.

3 Neonatal Calf Diarrhea 157 Viruses The viruses used were obtained originally from field outbreaks of neonatal calf diarrhea in herds near Cody and Lincoln, Nebr. The Cody isolate had been passaged as follows: seven calf passages, two passages in embryonic bovine trachea cell line, one calf passage, three passages in primary fetal bovine lung cell culture, and one calf passage. Calves were 52 & 5 h of age when inoculated orally with the Cody isolate. The Lincoln isolate represented two passages in calves with 14 subsequent passages in fetal bovine kidney cell cultures. Calves 9 and 10 were inoculated orally with 10 nil of lo8 titer Lincoln isolate when they were 7 and 4 h of age, respectively. E. coli A 24-hour nutrient broth culture of E. coli isolated from the brain of a calf moribund with neonatal diarrhea was used as inoculuni. Calves were inoculated orally with 10 ml of broth culture at 28 i 5 h of age (table I). Inocirlafion of Calves All calves were inoculated orally at times indicated in table I. The inoculuni consisted of 10 ml of a 1 :3 dilution of bacteria-free fecal filtrate (Seitz 0.5 pni), 10 ml of cell culture virus, or a 24-hour broth culture of E. coli. When gnotobiotic calves were inoculated with virus and E. coli, the bacteria were inoculated first and approximateiy 24 h was allowed for colonization of the intestine prior to inoculation of virus (table I). Necropsy A systematic necropsy was performed on each calf either after the onset of diarrhea or at comparable times for control calves (table 1). Euthanasia was by electrocution followed by exsanguination. Segments of small intestine designated as jejunal, middle and ileal were removed from the calves as soon after death as possible (see MEBUS et al. [XI for exact location). Segments were ligated at each end, and the lumen was injected with fixative at 4" C. Electron Microscopy The fixative consisted of 2.5% glutaraldehyde in phosphate buffer solution, ph 7.2 for 2 h. Blocks of glutaraldehyde-fixed tissue were trimmed to suitable size and shape for further fixation ( I h in 1.0% osmium tetroxide-phosphate buffer solution, ph 7.2 with 0.54% dextrose). The tissues were then dehydrated in a graded series (35-100%) of ethyl alcohol, passed through three changes of propylene oxide and embedded in epoxy resin (Araldite-502, Ladd Research Industries, Inc., Burlington, Vt.). Embedment was such that the villi were oriented for longitudinal sectioning[l I]. Survey sections, 1.0 pm thick, were cut from epoxy-embedded blocks and stained with a polychrome stain (C & C Paragon Co., Bronx, N. Y.) [7] for examination by light microscopy. Sections suitable for electron microscopy were cut on an ultramicrotonie (Ultratonie 111, LKB Instruments, Inc., Rockville, Md.), mounted on formvar-coated single slot grids (LKB Instruments), stained with alcoholic uranyl acetate and lead citrate [14], and examined with an electron microscope (Hitachi HU-I 1 E-I, Perkin-Elmer Inc., Instrument Marketing Division, Palo Alto, Calif.) operating at an accelerating potential of 100 kv. Tissues were taken also for histopathology and imrnunofluorescent studies. Results of these studies were reported previously [8].

4 158 STAIR/MEBUS/TWlEHAUS/uNDERDAHL Results Uninoculated Control Cay Intestinal epithelial cells from all intestinal segments from the uninoculated calf were tall columnar with a prominent striated border as viewed by light microscopy and low-magnification electron microscopy (fig. 1). These cells often contained a multiloculated pink-staining (semi-electron-lucent) material in the basal aspect. Nuclei varied in position within the epithelial cells, being basal, apical, or medial. Vacuolation of epithelium was sparse or absent in the jejunal segment but tended to increase in the middle and ileal segments so that nearly all epithelial cells of the ileal segments were markedly vacuolated. Vacuoles were variable in size and location, but the larger vacuoles tended to be basal. Vacuoles by light microscopy appeared completely clear, empty and membrane-bound in contrast to the multiloculated material that was at the base of the cells. The size of vacuoles generally increased with increasing distance from the pylorus. Vessels and lacteals other than the central lacteal within the lamina propria were inconspicuous. Reticuloendothelial cells were not numerous. Goblet cells were usually evenly dispersed among the epithelial cells and opened into contraction creases along the lateral aspects of the villi. The ultrastructural morphology of the villi from the uninoculated control calf was similar to that described for the pig [3, 131. Microvilli were long and of regular length, width, orientation and spacing over the tips of villi with a tendency to become shorter and more irregular in spacing and length as the epithelium approached the crypts. Rootlets of the microvilli projected into the terminal web running parallel to the outer surface of the cell. Invaginations of cell membranes, the apical tubular system, often formed pinocytotic vesicles. Some of these connected with smaller vacuoles. Large and small clear vacuoles were membrane-bound and contained sparse electron-dense granules (fig. 1). Mitochondria predominated in the subnuclear and perinuclear positions with granular (rough) endoplasmic reticulum being more abundant in the supranuclear location. Agranular (smooth) endoplasmic reticulum was present only to a limited extent. Polyribosomes were numerous in the cytoplasmic material, and their number appeared to account for a part of the normal variation of cytoplasmic density among epithelial cells. Distinct Golgi bodies were not seen. Lysosomes were seen infrequently. The multiloculated material at the bases of cells was enclosed within the cell membrane, was characterized by definitely decreased homogeneous electron density, and was not separated from the rest of the cytoplasmic matrix by a membrane.

5 Neonatal Calf Diarrhea 159 Fig. 1. Ileum from control calf 1. Extensive cytoplasmic vacuolation, apical and basal positions of nuclei, contraction creases and regularity of microvilli are present. This and subsequent electron micrographs stained with uranyl acetate-lead citrate.

6 160 Fig. 2. Middle small intestine of E. coli-inoculated calf 3. Tall columnar cells with basal nuclei and areas of decreased electron density (arrow). Notice microvillous rootlets, length and regularity of microvilli. A lacteal (L) is present adjacent to membrane-bound multiloculated material. Calves Inoculated with Escherichia coli Only Other than for the presence of an occasional bacterium within the lumen at some distance from the cellular surface there was no appreciable morphologic difference between tissue sections from these calves (fig. 2) and those from the gnotobiotic control calf.

7 Neonatal Calf Diarrhea 161 Calves Inoculated with Reovirus-Like Agent of Neonatal Calf Diarrhea The type of epitlielium in these calves varied according to the segment of intestine examined and the duration of diarrhea prior to euthanasia. Epithelium covering the jejunal and sometimes the middle segments was reduced to cuboidal or A attened to squamous when calves were killed at the onset of diarrhea, whereas the epithelium of the lower segment retained the normal tall columnar form and vacuolated cytoplasm. When cuboidal or squamous epithelium was present, the striated border could not be detected by light microscopy, and numerous pyknotic and karyorrhectic nuclei along with erythrocytes were present immediately below the basement membrane. The lacteals within the lamina propria were dilated, and vascular hyperemia was prominent. Villi were increased in diameter, and reticuloendothelial cells were more numerous than in comparable control sections. Virions were not seen in cuboidal or squamous villous epithelial cells but usually were present in endoplasmic cisternae (fig. 3, 4) of tall columnar epithelial cells from ileal segments (table I). Such cells sometimes contained an electron-dense viral precursor material enclosed by a single membrane (fig. 3, 4). Marked ultrastructural changes in infected epithelial cells were rare (fig. 5), and it was not evident whether these were caused by normal necrobiotic turnover or a lytic effect of the virus. Macrophages containing virions (fig. 6) were found only in the lamina propria of jejunal and middle segments from calves 5, 6, and 10. The upper segment of the intestine from calf 10 (killed 6 h after the onset of diarrhea) had cuboidal epithelium, and viral particles were not present by ultrastructural examination. Ultrastructurally, the microvilli were shortened and irregular in length, spacing, shape and orientation. The terminal web and microvillous rootlets were absent. The apical tubular system was almost nonexistent but was represented in some cells by sparse membranous material. Vacuolation of the epithelium of the lower segment was not prominent in calf 10 killed 6 h after the onset of diarrhea; the cytoplasm of the epithelium was more electron dense and resembled more closely crypt epithelium of the control calves. Goblet cells were less numerous than in the control calf, although they were irregularly spaced and mucus was adherent to tips of villi. Virions were present in ileal villous epithelial cells of calves 9 and 10; both had been inoculated with cell-culture-adapted virus. Calves Inoculated with E. coli and Reovirus-Like Agent Calves inoculated with E. coli and virus had cellular lesions at the lightand electron-microscopic levels that were similar to those in calves inoculat-

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9 Neonatal Calf Diarrhea 163 ed with virus only. Adherence or close apposition of E. coli to epithelium did not occur. Bacteria were not found within the cytoplasm of epithelial cells. Presence or absence of viral particles in cells depended on the time at which tissues were collected after the onset of diarrhea (table I). Degenerating cells within the lumen sometimes contained cytoplasmic virus that had threads connecting adjacent nucleoids. The microvilli of the epithelium that remained attached to the basement membrane were shortened, club-shaped, disoriented in relation to the cell and other microvilli, and irregular in spacing and length. Mucus often covered tips of villi where the epithelium was denuded. General Observations Viral particles were seen within the cisternae of rough endoplasmic reticulum of intestinal epithelial cells. That the particles were within cisternae of the rough endoplasmic reticulum was determined by the presence of ribosomes along the membranes (fig. 7). Cisternae also contained an amorphous electron-dense material that was considered to be precursor viroplasm since particles, often in the beginning of a crystalline array, were detected within such material. Complete viral particles were about 65 nm in diameter and had a dense nucleoid about 25 nm in diameter surrounded by a less dense outer coat. Sharply defined membranes were not seen at the periphery of complete viral particles or the nucleoid (fig. 4,5,7). Nevertheless, complete viral particles and nucleoids were distinguishable from one another and precursor viroplasm or cellular structures. Viral particles were not seen in the process of extrusion from infected cells but a few were seen both within and on the surface of microvilli. Viral particles were not found within the nucleus, and no tubular structure had penetrated the nuclear membrane. Nuclear changes in infected cells consisted of decreased density of interchromatic material and irregular separation of the layers of the nuclear membrane. Fig. 3. Oblique section through ileal epithelial cells of virus-inoculated calf 6. Microvilli and apical tubular system at upper left. Viral precursor material (VPM) within membrane-bound structure along with a small viral crystal and virions in endoplasmic cisternae (area outlined presented in figure 4). Virions are present in adjacent cell (arrows). Nucleus (N) of adjacent cell present for comparison with viral precursor material.

10 164 Fig. 4. Outlined area of figure 3 rotated 90 deg. Viral precursor material (VPM) enclosed by a single membrane; ribosomes are present along membranes enclosing virions ; viral nucleoids can be distinguished from rest of viral particles at arrows.

11 Fig. 5. IIeal epithelial cells, calf 6. Microvilli are shortened, irregular, or missing from vacuolated degenerating cell at top, and only remnants of apical tubular system remain. Virions are present within cisternae of endoplasmic reticuium in both cells (arrows).

12 I66 Fig. 6,Virions(arrow)inrnacrophage with pyknotic nucleus ; jejunal lamina propria ofcalf 5. Discussion The location of neonatal caif diarrhea virus within the cisternae oithe endoplasmic reticulum closely parallels epizootic diarrhea of infant mice virus [ 1 1. The size of neonatal calf diarrhea and epizootic diarrhea viruses is similar, being about nm [15, 171. Replication of both viruses [ 1 ] within membrane-bound structures is a feature that has been reported for the reovirus group and biuetongue virus [5]. Calf diarrhea virus was not observed in a pinching off process from tubular structures, as has been reported for epizootic virus. Calf diarrhea virus was not seen in association with tubular structures that penetrate the nuclear membrane. Filamentous strands extending from one viral nucleoid to another have been reported for epizootic diarrhea of infant mice and bluetongue [ 1, 51.

13 Neonatal Calf Diarrhea 167 Fig. 7. Virions in cisternae of endoplasmic reticulum of ileal epithelial cell, calf 5. Nucleoid distinguishable from rest of viral particle between arrows. The authors of the two publications [I, 51 disagree on the significance of this finding, but it is probably a result of cellular and viral breakdown with the formation of ribonucleoprotein threads. Cells in the present study and the epizootic diarrhea study had sloughed into the lumen of the intestine and were in a state of degeneration, so this work is similar to that done with epizootic diarrhea virus. Vacuolation of intestinal epithelial cells could be misleading in the absence of adequate controls or prior knowledge of this normal alteration in structure, which was described in 1925 [I21 as a hydropic stage in the intestinal epithelium of newborn calves. Hydropic epithelium in the above-mentioned study was related to the age of the calves since it was found: (1) in an aborted fetus, (2) in 27 of 34 calves less than 24 h postpartum, (3) in 8 of 16 calves from 2 to 3 days postpartum, and (4) was not found in 18 calves from 4 to 9 days of age. Similar findings have been observed in the newborn pig [2]. The hydropic change may be related to the absorption of colostral globulins since there is ultrastructural evidence that the vacuoles connect with the cellular exterior via the apical tubular system [lo]. Hydropic change had no influence on the viral infection in this study since hydropic cells contained viral particles. Ultrastructural changes in the intestinal epithelium were difficult to evaluate when sections were mounted on mesh-type grids because the mesh inter-

14 168 STAlR/MEBUS/TWIEHAUS/UNDERDAHL fered with the observation of anatomical relationships. For this reason it was necessary to use formvar-coated single-slot grids and to orient longitudinal sections of villi parallel to the slot in the grid. An example of the type of problem encountered with mesh-type grids was that contraction creases on the villi could be confused with crypts. Thus, pathologic alterations of the microvilli would be confused with the normal sparseness, spacing, and shortness that the microvilli have in the crypts [lo]. Additionally, neonatal calf diarrhea viral antigens that were localized by immunofluorescent techniques have been described as being present primarily in the epithelial cells covering the apical two thirds of the villi [8]. Thus, it was necessary to determine what part of the villus was being examined before conclusions could be drawn as to the presence or absence of the virus and effects of the virus on cells. Ultrastructurally, differences in staining density between adjacent epithelial cells were seen in uninoculated as well as inoculated calves. These were usually single cells that stained either more lightly or darkly in contrastto adjacent cells, and it was concluded that this was a nonspecific change and represented necrobiosis regardless of the cause. Therefore, specific changes related to the presence of viral particles within cells were only those that differed from the necrobiotic changes. Since viral particles were not seen in the process of budding from cell membranes, it was assumed that the virus was shed to the exterior by sloughing of entire intestinal epithelial cells into the lumen with passage in the feces. This concept was further supported by (1) the presence of viral particles and antigens in sloughed epithelial cells in the feces, and (2) the rapid loss of highly vacuolated epithelium and its replacement by uninfected cuboidal or squamous type epithelium. Not explained by this hypothesis was the presence of viral particles within macrophages of the lamina propria. Possibly the macrophages engulfed particles during a short period when the denuded villus was exposed to contents of the lumen or passage of virus through the cell via the endoplasmic reticulum placed it in contact with phagocytic cells of the lamina propria. The results obtained by immunofluorescent staining [8] and electron microscopy (table I) were well correlated. The small sample size that can be evaluated by electron microscopy was probably the reason for failure to observe viral particles in some instances where positive immunofluorescence had been observed previously. Immunofluorescent [8] and ultrastructural results relating to the cytoplasmic location of antigens and viral particles were in agreement. The granular appearance of cytoplasmic immunofluorescence correlated with the presence of virions within discrete membrane-bound intracytoplasmic structures.

15 Neonatal Calf Diarrhea 169 Desquamation of viral-infected cells into the lumen was followed by replacement with crypt epithelial cells and mucus. This protected the underlying lamina propria, which otherwise would have been completely denuded. The magnitude of cellular loss was indicated by (1) the large number of infected cells in the feces [ 191, and (2) the fact that the type of epithelium covering the villi was reduced to a cuboidal or squamous type so that more area was covered. The microvilli on these cells were altered also, being clubshaped, widely spread, irregular in length and spacing, and increased in diameter. It was not clear whether the increased diameter of the villi was a result of larger than normal numbers of reticuloendothelial cells or whether contraction of the longitudinal smooth muscle in the lamina propria shortened the villus and thus increased the diameter. Contraction of smooth muscle because of the irritation of the inflammatory process may have caused even more rapid cellular shedding than otherwise would have been the case. Proteins in the intestinal lumen [6] probably represented a serous exudate resulting in increased intraluminal osmotic force with further dehydration of calves. Even though the villi were covered with replacement epithelium, this epithelium was immature and its functions were, therefore, probably not fully developed. Lack of function in these cells would result in decreased absorption. The results of this experimentation and reports of other workers [4] have indicated that: (I) viruses can be primary pathogens in the enteritides of calves, (2) viral enteritides complicated by E. coli were more severe than viral infections alone, and (3) viruses may initiate an E. coli septicemia by breaking down the mechanical barrier present as intact epithelium. Acknowledgements Published with the approval of the Director as Paper No. 3185, Journal Series, Nebraska Agricultural Experiment Station. The work was conducted under Nebraska Agricultural Experiment Station Project No Supported in part by a grant from the U. S. Department of Agriculture; Agricultural Research Service (No , 610 [45]). The authors are grateful for the technical assistance of Mrs. ARDELL BODEN. References 1 BANFIELD,W.G.; KASNIC, G., and BLACKWELL, J.H.: Further observations on the virus of epizootic diarrhea of infant mice. Virology 36: (1968).

16 170 STAIR/MEBUS/TWlEHAUS/~NDERDAHL 2 DJURICKOVIC, S. ; THORSEN, J.; DUNCAN, J. R., and ROE, C. K. : Transmissible gastroenteritis of swine in Ontario. Canad. J. comp. Med. 33: (1969). 3 DREES, D.T. and WAXLER, G.L.: Enteric colibacillosis in gnotobiotic swine: an electron microscopic study. Anier J. vet. Res. 31: (1970). 4 LAMBERT, G. and FERNELIUS, A.L.: Bovine viral diarrhea virus and Escherichiu coli in neonatal calf enteritis. Canad. J. comp. Med. 32: (1968). 5 LECATSAS, G.: Electron microscopic study of the formation of bluetongue virus. Onderstepoort J. vet. Res. 35: (1968). 6 MARSH, C.L.; MEBUS, C.A., and UNDERDAHL, N.R.: Loss of serum proteins via the intestinal tract in calves with infectious diarrhea. Amer. J. vet. Res. 30: (1969). 7 MARTIN, J.H.; LYNN, J.A., and NICKEY, W. M.: A rapid polychrome stain for epoxyembedded tissue. Amer. J. clin. Path. 46: (1966). 8 MEBUS, C.A.; STAIR, E.L.; UNDERDAHL, N. R., and TWIEHAUS, M. J.: Pathology of neonatal calf diarrhea induced by a reo-like virus. Vet. Path. 8: (1971). 9 MEBUS, C.A.; UNDERDAHL, N.R.; RHODES, M.B., and TWIEHAUS, M. J.: Calf diarrhea (scours) reproduced with a virus from a field outbreak. Nebraska Agric. Exp. Station, Univ. Nebraska, Lincoln, Nebr. Res. Bull. 233 (1969). 10 PINK, I. J.; CURRAN, R.C., and CREAMER, B.: The ultrastructure of the small intestine under normal and pathological conditions. Meth. Achieve. exp. Path. 3: 1-39 (1967). 11 PITMANN, F. E. and PITMANN, J. C.: Electron microscopy of intestinal mucosa. Arch. Path. 81: (1966). 12 SMITH, T.: Hydropic stages in the intestinal epithelium of newborn calves. J. exp. Med. 41: (1925). 13 THAKE, C.: Jejunal epithelium in transmissible gastroenteritis of swine. An electron microscopic and histochemical study. Amer. J. Path. 53: (1968). 14 VENABLE, J.H. and COCGESHALL, R.: A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25: (1965). 15 WELCH, A.B. : Purification, morphology and partial characterization of a reovirus-like agent associated with neonatal calf diarrhea. Canad. J. comp. Med. 35: (1971). 16 WHITE, R.G.; MEBUS, C.A., and TWIEHAUS, M. J.: Incidence of herds infected with a neonatal calf diarrhea virus (NCDV). Vet. Med. small anim. Clin. 65: ( 1970). 17 WILSNACK, R.E.; BLACKWELL, J.H., and PARKER, J. C.: Identification of an agent of epizootic diarrhea of infant mice by immunofluorescent and complement fixat ion tests. Amer. J. vet. Res. 30: (1969). Request reprints from: Dr. E. L. STAIR, Department of Veterinary Science, University of Nebraska, Lincoln, NB (USA)