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Immunohistochemical demonstration of spread of Aujeszky's disease virus to the porcine central nervous system after intestinal inoculation

Aujeszky's disease virus (ADV) was injected into the duodenal lumen of eight specific pathogen-free pigs aged 5 weeks. The infected pigs did not show any diarrhoea or nervous symptoms, but they developed characteristic necrotizing enteritis and myenteric plexitis, accompanied by follicular necrosis in the Peyer's patches. ADV antigen was detected in the submucosa of the dome area of Peyer's patches, lymphatic follicles, Meissner's and Auerbach's plexuses, solar ganglia and thoracic spinal ganglia. These findings suggest that ADV spreads from the intestinal mucosa to the central nervous system via the autonomic nerves.


Introduction
Aujeszky's disease is a severe, highly fatal disease of young pigs. Aujeszky's disease virus (ADV), a neurotropic herpesvirus, spreads via the nerves from the primary site of infection to the central nervous system (CNS) (Corner, 1965;McFerran and Dow, 1965;Bergmann and Becker, 1967;Rajcani el al., 1969;Sabo et al., 1969;Narita et al., 1991). In piglets, initial replication of ADV in the naso-pharyngeal mucosa and tonsillar epithelium leads to systemic infection with necrotic hepatitis, adrenitis and encephalitis (Corner, 1965;McFerran and Dow, 1965;Olander et al., 1966). Recent studies showed that some strains of ADV produce necrotic lesions in the gastrointestinal tract and myenteric plexuses (Narita et al., 1984(Narita et al., , 1998Ezura et al., 1995;Zhao et al., 1996). However, there has been no demonstration of viral spread to the CNS via the myenteric plexuses in the small intestine and solar ganglia following intestinal infection with ADV. The purpose of the present study was to investigate this point.

Animals
Nine specific pathogen-free pigs aged 5 weeks were used. The animals had no neutralizing antibody against ADV. They were housed separately in a barriermaintained room.

Experimental Procedure
After an intramuscular injection of atropine hydrochloride (0"05g/kg), pigs were anaesthestized with a mixture of halothane and 02, delivered by means of a mask and closed circuit system. Laparotomy was performed by incision along the linea alba.
Eight pigs (nos 1-8) were given 10ml of ADV suspension (10 ~ TCID50/ml) by injection into the lumen of the duodenum; pig 9 (control) was inoculated with sterile culture medium. After closure of the abdomen, the pigs were allowed free access to water. During the experiment, all pigs were observed at least twice daily for clinical signs. The infected pigs were killed in pairs by intravenous overdoses of pentobarbital sodium on post-inoculation day (PID) 3, 5, 7 and 10; the non-infected control (pig 9) was killed on PID 10.

Histopathological and Immunohistochemical Examination
The sites from which specimens were taken are listed in Table 1. Specimens from the gut, spinal cord and ganglia included: two longitudinal specimens from the duodenum; three, four and three specimens from the upper, middle and lower parts of the jejunum, respectively; three and two from the upper and lower parts of the ileum, respectively; two from the caecum; four from the colon; two from the rectum; eight from the cervical spinal cord and ganglia; 12 from the thoracic spinal cord and ganglia; and seven from the lumbar spinal cord and ganglia. The specimens were fixed in buffered 10% formalin, embedded in paraffin wax, sectioned (3gm), and stained with haematoxylin and eosin (HE).
Most of the sections were examined immunohistochemically. Viral antigens were demonstrated by the avidin-biotin complex immunoperoxidase method (ABC-IP), as described previously (Narita et al., 1991), with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Rabbit antisera against ADV, porcine rotavirus, porcine epidemic diarrhoea virus (PEDV) and transmissible gastroenteritis virus (TGEV), kindly provided by Drs T. Imada and T. Tsuda, National Institute of Animal Health, were used as the primary antibody at dilutions of 1 in 2048, 1 in 1024, 1 in 1024 and 1 in 2048, respectively. Sections were counter-stained with methyl green. Tissue sections from pig 9 and serum from a non-immunized rabbit were used for negative control purposes. In addition, sections of small intestine from pigs experimentally infected with rotavirus, PEDV or TGEV were used as positive controls.

Clinical Observations
In pigs 1-8, the initial signs of disease were pyrexia (up to 39"5~ which persisted for 2 or 3 days. No diarrhoea or nervous symptoms were observed. The control pig (no. 9) remained apparently healthy.

Pathology
Macroscopically, no intestinal lesions were observed in any pig. Histologically, characteristic lesions of ADV infection were observed in the small intestine, Site

solar ganglia and thoracic-lumbar spinal cord and associated ganglia, but not in the other tissues (Table 1). On PID 3 and 5, tbur infected pigs (nos 1-4) showed prominent intestinal lesions in the duodenum and jejunum, characterized by karyorrhectic necrotic
foci; these lesions occurred in the epithelium of the villi and crypts, and in associated Pcyer's patches (Fig. 1) and Meissner's ( Fig. 2) and Auerbach's plexuses. The nuclei of the degenerating cells contained a few eosinophilic intranuclear inclusion bodies. In the two pigs killed on PID 7, lesions were found in the myentcric plexuses, but on PID 10, no intestinal lesions were seen. More frequently, necrotic and inflammatory changes were shown in the solar ganglia (Fig. 3) and plexus, and in the thoracic-lumbar spinal cord (Fig.  4) and associated ganglia. The changes were localized mainly to the dorsal and ventral horns and the funiculus dorsalis and funiculus ventralis. Three pigs (nos 4, 5 and 6) showed slight non-suppurative encephalitis in the medulla oblongata, with perivascular cuffing and focal gliosis. Pig 4 had a small necrotic focus with intranuclear inclusion bodies in the adrenal gland. No lesions wcre found in the non-intkcted control pig.

Immunohistochemistry
ADV antigen was observed in the intestinal specimens on PID 3 and 5, and in the solar ganglia on PID 3, 5 and 7. ADV antigen was closely associated with the karyorrhectic necrotic foci (Table 1). ADV antigen was found in the epithelial cells of the villi and crypts. Some was also associated with the dome of Peyer's patches, extending into the deeper regions of the lymphoid follicles (Fig. 5). A moderate amount of ADV antigen was detected in the degenerating neuronal cells of Meissner's ( Fig. 6) and Auerbach's plexuses (Fig. 7), the duodenum and jejunum, solar plexus and ganglia (Fig. 8), and the thoraciclumbar spinal ganglia. Pig 4 showed a small amount of ADV antigen in the adrenal gland. No other viral antigens (porcine rotavirus, PEDV or TGEV) were detected in any pigs.

Discussion
The pathogenesis of experimental ADV is variable, depending on the viral strain, age of the pig, size of the inoculum and route of infection (Kluge et al., 1992). In the present intestinal infections with ADV, the pigs did not develop diarrhoea or nervous symptoms but had characteristic necrotizing enteritis and myenteric plexitis, accompanied by follicular necrosis in the Peyer's patches. The intestinal necrotic lesions closely resembled those described in previous reports of experimentally (Narita et al., 1984;Zhao et al., 1996;Narita et al., 1998) and naturally (Ezura et al., 1995) infected pigs.
Immunohistologically, ADV antigen was demonstrated first on PID 3 in the submucosa of the dome area, the lymphatic follicles of the Peyer's patches, the neuronal cells of Meissner's and Auerbach's plexuses, and the solar plexus and its ganglia. It was subsequently detected in the cells of the thoracic spinal ganglia. The distribution of ADV antigen was closely associated with the necrotic foci, suggesting that ADV replication led to cell death. Moreover, the distribution of ADV antigen in the small intestine resembled that observed in closed intestinal loops inoculated with ADV (Zhao et al., 1996;Narita et al., 1998).
In-vivo experiments suggest that herpes viruses migrate from the inoculation site along peripheral nerves towards the associated peripheral ganglia, and then proceed to the corresponding segments of the CNS (Irie et al., 1989). In ADV infection in piglets, necrotizing enteritis and myenteric plexitis occur (Narita et al., 1984;Ezura et al., 1995) in addition to encephalitis (Olander et al., 1966;Rajcani et al., 1969;Sabo et al., 1969). The neuronal lesions produced in the present study suggested that ADV spread from the myenteric plexuses 7 via the solar plexus and ganglia, and travelled to the spinal ganglia as far as the dorsal and ventral horns of the thoracic-lumbar spinal cord. Thus, it would appear that ADV spreads from the intestinal mucosae to the CNS via autonomic nerves.
Many enteropathogenic viruses, such as PEDV (Pospischil et al., 1981;Ducatelle et al., 1982), porcine rotavirus (Pearson and McNulty, 1977;Narita et al., 1982;Collins et al., 1989) and TGEV (Hooper and Haelterman, 1969;Shepherd el al., 1979), when injected oro-nasally into pigs, produce severe gastroenteritis and villous atrophy; however, infected villous epithelial cells are replaced rapidly as epithelial cells migrate from the crypts of Lieberktihn. In the present study, the pigs inoculated intestinally with ADV showed characteristic necrotizing enteritis in the .jejunum, and necrotic loci in the dome area of Peyer's patches extended into the lymphoid follicles and the myentcric plexuses. Such lesions can therefore be taken to suggest the presence of cnteropathogenic ADV in pigs.