Archives of Virology Ultrastruetural Chanties in the Small Intestinal Epithelium of Sueklinti Pitis Affected with a Transmissible Gastroenteritis (TGE)-Like Disease
The small intestine of piglets collected during a sudden outbreak of diarrhoeal disease resembling transmissible gastroenteritis (TGE) was examined by light and electron microscopy. The principal histopathological changes were moderate infiltration by mononuclear cells in the lamina propria of the villi and cytoplasmic vacuolation. These were most pronounced in the epithelial cells covering the villous tips. By scanning electron microscopy, the intestinal villi were swollen and the transverse furrows disappeared. Mierovilli were reduced in number leaving denuded areas on the brush border of the villous epithelial cells.
The ultrastructural changes were restricted to the cytoplasm of affected villous epithehal cells. The cell organelles were missing in rounded areas leaving cleared areas in the cytoplasm. Parallel fascicles and bundles were seen in these areas. Viral particles with an average diameter of 70 nm were found within the dilated apical tubulo-vesicular system, free in the cytoplasm, among the microvilli or lying free in the intestinal lumen. Viral particles surrounded a non-membrane bound viroplasm in some cases. The negatively stained particles showed a typical coronavirus morphology. These particles were found to be distinct from the known coronaviruses of swine, TGE virus and hemagglutinating encephalomyelitis virus by immune electron microscopy.
Transmissible gastroenteritis (TGE) is a highly contagious enteric disease of swine of all ages causing important economic losses, especially in dense swine populations. The disease was first described by DOYL]~ and HUTCHINGS (9) in the United States, in 1946. In Hungary, SZENT-IVXN¥I et a l . (20) reported the occurrence of TGE in 1964. Following the elaboration of methods for the routine 0304-8608/81/0068/0103/$ 02.20 I04 IR]~N I-IoRviTH and E. M o c s~I : laboratory diagnosis of the disease (7), CSO~TOS a n d BENYE;)A (6) t u r n e d their a t t e n t i o n in 1970 to a new TGE-like epizootic diarrhoea of swine in which the causative role of T G E virus was excluded on the basis of virological examinations. Clinical observations and the experimental reproduction of diarrhoea in colostrumdeprived pigs with bacteria-free intestinal material have also supported the viral etiology of this diarrhoeal disease (1, 11) .
I n the past few years, an increased frequency of newborn pig diarrhoea caused b y rotavirus was reported (3, 8, 12, 13, 16, 22, 24) . However, in a n u m b e r of instances, the significance of rotavirus could not be established (23) . Finally, in Belgium PE~SAE~T and DE BOUCK (15) and in E n g l a n d CHASEY and CART-WRIGHT (4) detected coronavirus-like particles in the feces and intestinal epithelium of pigs affected with a TGE-like epizootic diarrhoea.
The present report describes the ultrastructural changes in the small intestinal epithelium and the detection of a virus morphologically similar to the coronaviruses in the intestinM content a n d epithelium of pigs affected with a TGE-like disease, which has occurred periodically in H u n g a r y since 1970.
Three to seven days old piglets were collected from 3 large Hungarian swinebreeding farms during sudden outbreaks of diarrhoeal disease affecting animals of all ages. All the pigs showed a watery diarrhoea. The etiological rote of TGE coronavirus was excluded by the negative result of direct irnmunofluorescence tests carried out on serapings of the small intestinal mucosa, by unsuccessful viras isolation attempts on secondary swine thyroid cell cultures and by the absence of TGE virus-neutralizing antibodies in the acute~ and convalescent-phase sera (14) . Direct immunofluorescence (21 ) , counter-immunoelectrophoresis (19) , electron microscopic ( t 8) and immune electron microscopic examinations (17) for rotavirus were also negative and no rotavirus antibodies were found by counter-immunoelectrophoresis (5) in acute-and convalescent-phase sera from the recovered animals.
Intestinal segments were removed from six different regions of the small intestine of sacrificed piglets and fixed in buffered formalin. The sections were stained with hemotoxylin-eosin (HE).
Disc-shaped portions of intestinal wall, approximately 1.5 em in diameter were stretched on a dental wax sheet. They were fixed in 2.5 per cent glutaraldehyde at pH 7.3 and postfixed in 1 per cent phosphate-buffered osmium tetroxide, pH 7.3. They were dehydrated in graded acetone and vacuum-dried. Finally, tissue blocks were vapor coated with gold in an Edwards' vacuum-evaporator. The preparations were examined under a JEOL SM 15 scanning electron microscope using an acceleration potential of 21 kV.
Small segments were removed from six sites of the small intestine, adjacent to those taken for histopathology. The segments were fixed in Karnowsky's paraformaldehyde or in 2.5 per cent glutaraldehyde at pH 7.3 and post-fixed in 1 per cent phosphate-buffered osmium tetroxide at pH 7.3. Tissue blocks were dehydrated in graded ethanol and embedded in Durcupan ACM. The sections were cut with a Reichert Om U3 ultramierotome. Semi-thin sections were stained with toluidine blue and thin sections with uranyl acetate and lead citrate. I n t e s t i n a l c o n t e n t s were diluted 1 to 5 (v/v) in p h o s p h a t e -b u f f e r e d saline (PBS), p H 7.2 a n d clarified b y c e n t r i f u g a t i o n at 3000 × g for 30 m i n u t e s at 4 ° C. The supern a t a n t was filtered t h r o u g h a 450 n m Millipore filter a n d centrifuged a t 100,000 × g for 90 m i n u t e s a t 4°C in a n M S E SS50 ultracentrifuge. T h e resulting pellet was r e s u s p e n d e d in a few drops of distilled water, placed on 100 mesh, f o r m v a r -c o a t e d grids a n d s t a i n e d w i t h 2 per cent p h o s p h o t u n g s t i e acid. Grids were e x a m i n e d u n d e r a Philips 201 CS electron microscope o p e r a t i n g at a n acceleration p o t e n t i a l of 60 kV.
Porcine a n t i -T G E serum was p r e p a r e d in all S P F piglet orally exposed to t h e v i r u l e n t Mitler-3 strMn of T G E virus (2) . Porcine a n t i -h e m a g g l u t i n a t i n g encephalomyelitis virus (HEV) serum was o b t a i n e d from Dr. M. B. PE~SAERT, U n i v e r s i t y of G h e n t , Belgium. T h e sera were h e a t -i n a c t i v a t e d a n d stored frozen u n t i l used. T h e n e u t r a l i z i n g a n t i b o d y titre of T G E s e r u m was 1:2560. I t s o p t i m a l dilution for I E M was 1:50. T h e n e u t r a l i z i n g a n t i b o d y titre of I-IEV a n t i s e r u m was 1:12,288 a n d its o p t i m a l dilution for I E M was 1:400. T h e optimal a n t i b o d y dilution for I E M was considered to be t h a t which produced the largest v i r u s -a n t i b o d y aggregates. Toluidine blue stain. × 370. C SEM of villi from a non-exposed pig. These are long a n d slender w i t h p r o m i n e n t t r a n s v e r s e furrows (arrow). × t 15. D SEM of t h e j e j u n u m from a pig affected with TGE-like disease. The villi are swollen a n d t h e t r a n s v e r s e furrows are missing. × 1 t 5 oration were present in some of them (Fig. 1B) . These alterations were most pronounced in the epithelial cells covering the tips of the villi but they were missing in those lining the crypts of Lieberkiihn.
Examination of small intestines of normal pigs revealed long, finger-like villi of various lengths and configurations. The surfaces of most villi were interrupted by transverse furrows of various depths (Fig. 1 C) . In the small intestines of the affected pigs, villi were swollen and the transverse furrows disappeared. Mierovilli were reduced in number leaving denuded areas on the brush border of the epithelial cells (Fig. 1 D) .
Principal ultrastructural changes were found in the cytoplasm of the villous epithelial cells of the small intestine, The ceil surface and the nucleus were generally intact (Fig. 3) . In some of the affected cells, the cell organelles were missing in rounded areas leaving cleared areas in the cytoplasm (Fig. 4) . Viral particles were present along the border adjacent to the intact areas of cytoplasm (Fig. 4) . Sometimes, viral particles surrounded an electron-dense, coarsely grannlated viral precursor material or viroplasm (Fig. 4 0. The virions consisted of a central electron-opaque core surrounded by an intermediary electron translucent zone and by an electron-opaque outer coat (Fig. 5) . Their average diameter was 70 nm. Viral particles were also present in the dilated apical tubule-vesicular system, free in the cytoplasm (Fig. 6) , among the microvilli (Fig. 5) , or lying free witt~in the dilated cisternae of rough endoplasmic reticulum (Fig. 7) .
Most viral particles were seen in the epithelial cells covering the tips of villi in the jejunum. Their number decreased gradually approaching the inlets of the crypts of Lieberkiihn and no viral particles were found in the epithelial cells lining the crypts. Likewise, viral particles were not found within the nucleus. Sporadically, parallel bundles and fascicles were seen in the cleared areas of cytoplasm (Figs. 3 and 4) , Disruption el microvilli of the brush border and the separation of infected epithelial ce!ls were also demonstrated occasionally.
Coronavirus-like particles were found in negatively stained preparations of the intestinal contents of all piglets. The particles were pleomorphie with short surface projections. The projections formed a single fringe radiating from the electron-opaque central core. The diameter of the particles ranged between 100 and 122 nm. The length of the surface projections was approximately 20 nm (Fig. 8) .
The direct IEM for TGE virus and HEV was negative.
Previous observations and experimental results have assumed the viral etiology of this TGE-like disease. Experimental reproduction of diarrhoea in col ostrum-deprived pigs with bacteria-free fecal samples of sick pigs (1, 11) and the explosive spread of diarrhoea within all age groups have eqr ally led to this assumption.
The results of the present study proved the viral e~iclogy of this TGE-like disease. Virions with an average diameter of 70 nm ~ ere found in the intestinal Fig. 6 . Dilated tubule-vesicular system characteristic for suckling pigs t, h a t --besides maeromoleeules-can also promote the intake of infectious agents. Viral particles (arrow) locating intra-and extraeellular. Mieiovilli of the brush border are disrupted, × 27,900
villous epithelial cells of naturally infected piglets or free in the intestinal lumen, In the infected epithelial cells, viral particles were located mainly within the dilated eisternae of the rough endoplasmie retieulum. In addition, viral part, ides were found free in the cytoplasm or surrounded by a unit membrane. The location of viral particles closely parallels that of TGE virus. The negatively stained particles showed a typical coronavirus morphology. These coronavirus-like particles were morphologically indistinguishable from TGE virus and HEV particles in similar preparations. However, by direct IEM, they were distinct from these two eoronaviruses of swine. The size and morphology of the particles were similar to those of the recently recognized eoronaviruses reported by P~SCSAERT and DE BOUCK (15) and by CHAS~SY and CAaTUmmHT (4) in epidemic diarrhoea of pigs. As mentioned above, TGE virus was excluded as the etiological cause of diarrhoea on the original farms. The histopathology of the intestine and the ultrastruetural changes in villous epithelial cells also differed from those caused by TGE virus. In the case of TGE, the marked shortening of villi of the small intestines that HooPEa and HAELTEaMA~ (10) referred to as villous atrophy proved to be a highly significant lesion. These morphological changes affect the entirety of the villi; they are not restricted to the absorptive cells. Furthermore, simultaneously with the regressive alterations, a marked cell proliferation can be demonstrated in the crypts of Lieberkiihn presumably replacing the destroyed and sloughed epithelial cells.
In the case of TGE-like diarrhoea, the morphological changes are restricted to the cytoplasm of the differentiated villous epithelial cells. In most cases, the cell surface was intact, even if the cytoplasm showed marked pathological changes. In addition, parallel bundles and fascicles were seen in vacuoles in the cytoplasm that were observed neither in normal (Figs. 1 A and 2) nor in TGE virus-infected pigs.
All attempts to isolate and propagate the virus in cell cultures have so fat" been unsuccessful.