key: cord-1005985-qsdxuykl authors: Sueyoshi, M.; Tsuda, T.; Yamazaki, K.; Yoshida, K.; Nakazawa, M.; Sato, K.; Minami, T.; Iwashita, K.; Watanabe, M.; Suzuki, Y.; Mori, M. title: An immunohistochemical investigation of porcine epidemic diarrhoea date: 1995-07-31 journal: Journal of Comparative Pathology DOI: 10.1016/s0021-9975(05)80069-6 sha: 79a53f2ac148a3ee70d11b5d2700b7a42301ada5 doc_id: 1005985 cord_uid: qsdxuykl Summary A sudden outbreak of epidemic diarrhoea of piglets occurred in Japan, the principal features being watery diarrhoea, dehydration and high mortality in newborn animals. The microscopical lesions were villous atrophy in the small intestine, the villous enterocytes being vacuolated and cuboidal in shape. The villus-crypt ratio was severely reduced, varying from 1:1 to 3:1. Transmission electron microscopy showed numerous coronaviruses within the cytoplasm of enterocytes and among microvilli. Specific antigens of porcine epidemic diarrhoea (PED) virus were detected in the cytoplasm of enterocytes by the streptavidin-biotin (SAB) technique. Infected cells, which were most abundant in the villous epithelia of the jejunum and ileum, were present in small numbers in the large intestine, the crypt epithelia, the lamina propria and Peyer's patches. The study suggests that the SAB technique is useful for the diagnosis of PED. Porcine epidemic diarrhoea (PED) virus is distinguishable antigenically from transmissible gastroenteritis virus (TGEV) and haemagglutinating encephalomyelitis virus (HEV) (Horvath and Mocsari, 1981; Pensaert et al., 1981) . PED has been reported in England (Wood, 1977; Chasey and Cartwright, 1978) , Belgium (Pensaert and Debouck, 1978) , Canada (Turgeon et al., 1980) , Hungary (Horvath and Moesari, 1981) and Germany (Pospischil et al., 1981) . In Japan, PED or PED-like disease occurred from late 1982 to early 1983 (Takahashi et al., 1983; Kuwahara et al., 1988) but was not reported again until outbreaks occurred on a number of farms between September 1993 and June 1994. Newborn piglets developed signs of diarrhoea, dehydration and vomiting, death occurring within a few days. Mortality ranged from 30% to 80% and c. 14000 deaths occurred. Adult pigs showed only transient inappetence and the milk secretion of sows was sometimes depressed. PED virus antigens were demonstrated by the direct immunofluorescence technique in the intestine of experimentally infected pigs (Debouck and Pensaert, 1980; Debouck et al., 1981; Pospischil et al., 1981) . This paper 0021-9975/95/050059 + 09 $12.00/0 9 1995 Academic Press Limited describes the demonstration of PED virus antigens in the intestine of naturally infected piglets by the hitherto untried streptavidin-biotin technique. Twenty-one piglets aged 2 to 7 days were collected from five large Japanese swine breeding farms, with 100-2600 sows, during outbreaks of diarrhoeal disease (Table 1 ). The piglet mortality varied from 30% to 60% and c. 10 000 newborn piglets died. On these farms, vaccines against transmissible gastroenteritis (TGE),Japanese porcine encephalitis, porcine parvovirus infection, swine fever, Aujeszky's disease and swine erysipelas had been in regular use. Piglets were treated for diarrhoea with colistin or oxolinic acid, but without success. TGE was excluded by failure to isolate the virus from the small and large intestine of all 21 piglets on a porcine kidney cell line (CPK cell) and in Vero cell cultures, and by failure to demonstrate elevated neutralizing antibody titres against TGEV in the serum of nine convalescent piglets (19-day-old) on farms 1 and 2 (Table 1) . Rotavirus infection was also excluded by the absence of specific antigen in the intestinal content of all 21 piglets collected, as shown by the Rotaclone Kit (Cambridge Biotech, MA, USA). Bacteriological examination of samples from 13 diarrhoeic piglets (intestinal contents, liver, lung and kidney) failed to reveal pathogenic bacteria. Control material for the PED virus antigen tests (see below) was obtained from a 25-day-old specific pathogen-free (SPF) piglet, a 25-day-old SPF piglet infected orally with the virulent TGEV strain Shizuoka-20, and four newborn piglets in which TGE had been diagnosed by direct immunofluorescence. Specimens were taken after slaughter from the stomach, duodenum, jejunum, ileum, caecum, colon, liver, spleen, lung, heart, kidney, adrenal, tonsil, mesenteric lymph node, pancreas, cerebrum, cerebellum, mesencephalon, medulla oblongata, hypophysis, spinal cord, trigeminal ganglion, and coeliac ganglion. The tissues were fixed in 10% phosphate-buffered formalin, embedded in paraffin wax, sectioned, stained with haematoxylin and eosin (HE), and examined by light microscopy. The samples obtained from the jejunum were used. They were fixed in 2"5% glutaraldehyde-phosphate buffer (pH 7.4) at 4~ for 2 h, washed in 0" 1 M phosphate buffer and post-fixed in 1% osmium tetroxide-phosphate buffer at 4~ for 1 h. After having been dehydrated, they were embedded in epoxy resin. Ultrathin sections were then cut, stained with uranyl acetate and lead citrate, and examined with a Jeol transmission electron microscope (JEM-1010). The distribution of PEDV antigens was investigated by the streptavidin-biotin (SAB) technique, with light microscopy. The SAB kit (Scytek Laboratories, Logan, Utah, USA) was used to examine formalin-fixed, paraffin wax-embedded sections of the gastrointestinal tract and lung. After de-waxing, endogenous peroxidase activity was blocked by treatment with 0-3% H202, and the sections were then washed in 0"05 M Tris buffer (pH 7"6). Reagents in the SAB kit were prepared according to the manufacturer's instructions. Sections were counterstained with methyl green. Rabbit antiserum against PED virus (strain 83P-5) was used as primary antibody. The specificity of the antiserum has been reported previously by Kusanagi et al. (1992) , Macroscopically, the intestine ofdiarrhoeic piglets was distended with yellowish watery contents and Sometimes contained flecks of curdled undigested milk. The intestinal wall was thin and transparent (Fig. lb) . The stomach was also distended and in 14 piglets the wall was thin. The mesenteric lymph nodes were occasionally enlarged. Microscopically, the villi were atrophied in the small intestine of 20 piglets ( Table 1 ). The villus-crypt ratio was reduced, varying from c. 1:1 to 3:1 (Fig. 2a, b) . Atrophied villi often consisted of strikingly vacuolated emerocytes. Numerous enterocytes on the villi were cuboidal to low columnar and the brush border was indistinct (Fig. 2c) . Some cells on the crests of the villi appeared distorted and the nuclei were pyknotic. The mucosal lamina propria was infiltrated slightly with lymphocytes, eosinophils and neutrophils, and was occasionally congested and oedematous. However, inflammatory exudate was not a striking feature. The lesions were most common and severe in the jejunum and ileum, and were sometimes observed in the duodenum. The caecum and colon showed no obvious changes except for vacuolation of the superficial enterocytes. No characteristic lesions were observed in other organs except slight pneumonia and slight renal nephrosis in some piglets. Microvilli of infected superficial enterocytes were disoriented. Many viral particles were seen in vacuoles or cytoplasmic organelles situated mainly in the supranuclear cytoplasm. Some viruses were seen between the microvilli. The viral particles were pleomorphic, measuring between 70 nm and 140 nm. They were mainly spherical or oval. Mature viruses (100-140 nm diameter) with a number of projections (c. 20 nm) were observed within the membranebounded vacuoles in the enterocytes (Fig. 3) . PEDV antigens were detected in high concentration in the enterocytes in 18 of 21 piglets by the SAB technique. The antigen-containing enterocytes were profuse in the small intestinal mucosa but sparse in the large intestine (Table 2 ; Fig. 4 ). The infected cells in the duodenum were fewer than in the jejunum and ileum but greater than in the caecum and colon. In infected piglets, antigen-containing cells invariably occurred in the jejunum. These infected enterocytes tended to be less numerous in severe (Fig. 2d ) than in moderate (Fig. 4b) villous atrophy. Infected cells were not seen in two piglets that showed severely atrophied villi. Antigen was confined to the cytoplasm, and could not be detected in the nuclei. Most infected cells were not arranged continuously in the epithelial layer (Figs 2d and 4a-d) and the border between infected and uninfected cells was distinct. Infected cells were observed occasionally in the crypts (Fig. 4a ) and in the lamina propria and Peyer's patches in the ileum (Fig. 4c) . PEDV antigen could not be detected in Brunner's glands of the duodenum, stomach or lung, or in the intestine of control animals (a healthy SPF piglet, a piglet experimentally infected with TGEV, and four piglets in which natural TGE had been diagnosed). In an outbreak of epidemic diarrhoea in piglets in Japan, TGEV, rotavirus and bacteria were excluded as possible aetiological agents by appropriate tests. However, the SAB technique, with PEDV antiserum, gave positive results in the small intestine, caecum and colon. This closely resembled the findings reported by Debouck et al. (1981) in pigs experimentally infected with PEDV and examined by the immunofluorescence technique. The site of viral replication of PEDV was clearly different from that of TGEV, which is the small intestine (Hooper and Haelterman, 1966) . In addition, PEDV was absent from the lung, thus differing from porcine respiratory coronavirus (Cox et al., 1990) . From the results, the diarrhoeal disease was diagnosed as PED. This infection seems to lead to diffuse and severe villous atrophy in the small intestine. Debouck et al. (1981) reported that the mean villus-crypt ratio varied from 2"3:1 to 1 "5:1 in experimental PED. In the present study, however, the villus-crypt ratio in the most severely affected piglets was 1:1 and the lesions were as severe as those of TGE. Furthermore, villous atrophy was observed in the duodenum, in contrast to the findings of Ducatelle et al. (1981) in experimental pigs. The differences between the results obtained in experimental infection and those in the present study may have been due to differences in the virulence of the strains or in the infecting dose. The number of enterocytes infected with PEDV tended to decrease with increasing severity of villous atrophy. Possibly in piglets with severe villous atrophy, the PEDV may already have left the enterocytes, or the infected cells may already have been eroded. Thus the distribution of PEDV antigens may vary according to the stage of the disease. In the SAB technique, fixed material may be used, no cryostat is needed, and the sections show adequate detail and are relatively permanent. The method would therefore seem useful as a diagnostic method for PED and for studying the distribution of PEDV antigens. Virus-like particles associated with porcine epidemic diarrhoea Sites of replication of a porcine respiratory coronavirus related to transmissible gastroenteritis virus. Research in Veterinary Science Experimental infection of pigs with a new porcine enteric coronavirus CV 777 The pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, CV 777 Three-dimensional sequential study of the intestinal surface in experimental porcine CV 777 coronavirus enteritis Growth of transmissible gastroenteritis virus in young pigs Ultrastructural changes in the small intestinal epithelium of suckling pigs affected with a transmissible gastroenteritis (TGE)-like disease Isolation and serial propagation of porcine epidemic diarrhoea virus in cell cultures and partial characterization of the isolate Passage in piglets of a coronavirus associated with porcine epidemic diarrhoea A new coronavirus-like particle associated with diarrhoea in swine An immunoelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, CV 777, and several coronaviruses Light microscopy and ultrahistology of intestinal changes in pigs infected with epizootic diarrhoea virus (EVD): comparison with transmissible gastroenteritis (TGE) virus and porcine rotavirus infections An outbreak of swine diarrhoea of a new type associated with coronavirus-like particles in Japan C oronavirus-like particles associated with diarrhea in baby pigs in We thank Dr H. Kuwahara, Nippon Institute for Biological Science, Ome, Tokyo 198, Japan, for providing the PEDV antiserum. We also thank Messrs K. Kawasaki, Y. Ando and T. Fujisawa for assistance.