Histopathological, immunohistochemical, and ultrastructural evidence of spontaneous Senecavirus A-induced lesions at the choroid plexus of newborn piglets

Abstract

Epidemic Transient Neonatal Losses (ETNL) is a disease of piglets caused by Senecavirus A (SVA) in which the method of dissemination and associated lesions are not well-defined. This study investigated the possible SVA-induced lesions by examining spontaneous infections in newborn piglets. Histopathology revealed ballooning degeneration of transitional epithelium, nonsuppurative meningoencephalitis, plexus choroiditis, and atrophic enteritis. RT-PCR identified SVA in all tissues evaluated and sequencing confirmed these results. Positive immunoreactivity to SVA was observed in endothelial and epithelial tissues of all organs evaluated. Semithin analysis revealed vacuolization of apical enterocytes of the small intestine, balloon degeneration and necrosis of endothelial cells of the choroid plexus (CP) and nonsuppurative choroid plexitis. Ultrathin evaluation demonstrated hydropic degeneration of apical enterocytes, degeneration and necrosis of endothelium of CP fenestrated capillaries, degeneration of ependymocytes associated with intralesional viral particles. It is proposed that SVA initially infects apical enterocytes of newborn piglets and probably enters the circulatory system with entry to the brain via the CP, by first producing an initial inflammatory reaction, with subsequent encephalitic dissemination. Consequently, SVA probably uses an enteric-neurological method of dissemination.

Figure 1

Gross findings observed in newborn piglets naturally infected by SVA. Coronary band, 4-day-old piglet; observe the erosive lesion (0.7 cm in diameter) at the coronary band of the right forelimb (A). Abdominal cavity, 3-day-old piglet; there is mesocolonic oedema; the intestinal segment is dilated by diarrhoea and the mesocolon is swollen (B). Snout, 4-day-old piglet; there is multifocal ulceration of the skin (arrow) of the snout after rupture of a vesicle (C). Tongue, 6- day-old piglet; there is symmetric ulcerative glossitis (arrow) at the ventral face of the tongue (D). Fore limbs and hooves, 7-day-old piglet; observe ulcerations and crusting lesions (2 cm diameter) at the coronary bands and metacarpus with an erosive lesion at the margin of the coronary band of the forelimbs (E). Palmar footpad, 3-day-old piglet; there is a large ulceration (1.2 cm diameter) at the right footpad (F). Scale in cm.

Figure 2

Graphical distribution of the principal histopathological findings in 43 piglets with SVA. Legend: BDTE, ballooning degeneration of transitional epithelium; ballooning degeneration of renal pelvis (BDRP), tonsilar depletion (TD), fusion of villi (FV), mesenteric lymph node depletion (MLND), necrosis of apical enterocytes (NAE), Peyer’s patch hyperplasia (PPH), vacuolization of enterocytes (VE), necrotizing dermatitis (ND), necrotizing glossitis (NG).

Figure 3

Histopathological findings observed in newborn piglets naturally infected by SVA. Ureter, there is severe hyperplasia and balloon degeneration of the urothelium (A). Urinary bladder, observe ballooning degeneration of the transitional epithelium associated with an intracytoplasmic, eosinophilic inclusion body (arrow) (B). There is severe focally extensive nonsuppurative meningoencephalitis (*, C) and cerebrocortical necrosis of the brain (D); observe several ischemic neurons (arrows). There is severe multifocal nonsuppurative choroid plexitis (*) of the lateral ventricle (E); observe the intraluminal accumulation of fibrin (★) in a fenestrated capillary, degeneration of ependymocytes and (open arrows), ballooning degeneration (arrow head) as compared with normal (closed arrow) ependymocytes (F). There is malacia (★) of the brainstem with gliosis (G), and discrete perivascular cuffing formed by lymphocytes and macrophages at the cortex (H). Haematoxylin & Eosin stain. Bar, A, E 50 µm; B 5 µm; C 100 µm; D,F–H 20 µm.

Figure 4

Immunohistochemical detection of antigens of SVA in epithelial tissues of newborn piglets. Cerebrum; there is positive immunostaining (arrow) at the capillary endothelium (A) and at the ependymal cells (arrow heads) of the choroid plexus (C). Urinary bladder; observe immunoreactivity to SVA at transitional epithelial cells (B). Observe the vacuolization of epithelial cells of the choroid plexus (C). Oral mucosa; observe positive immunolabelling of mucosal epithelium within areas of hydropic degeneration (D,E). Small intestine; there is positive immunolabelling of SVA at apical enterocytes within areas of intracytoplasmic vacuolization (F). Immunoperoxidase. Bar, A 50 µm; B,D-F 20 µm, and C 10 µm.

Figure 5

Semithin sections of the small intestine of piglets naturally infected with SVA. Observe SVA-induced balloon degeneration (open arrow) of the apical enterocyte of the small intestine resulting in displacement of the nucleus to the periphery. Bar, 5 µm.

Figure 6

Semithin evaluation of the choroid plexus of piglets naturally infected with SVA. There is non-suppurative choroid plexitis (★), ballooning degeneration of endothelial cells (open arrows) of fenestrated capillary, degeneration (closed arrow) and swelling of (arrow heads) ependymocytes (A). Observe severe accumulations of macrophages and lymphocytes admixed with fibrin in the lumen (*) of a fenestrated capillary, necrosis (closed arrow) and degeneration (open arrows) of endothelial cells, with swelling and thickening (arrow heads) of ependymocytes (B,C). New methylene blue stain Bar, A-C, 50 µm.

Figure 7

Transmission electron micrograph of the apical enterocytes of the small intestine. Electron microscopy of positive stained demonstrating that normal non-infected apical enterocytes of the small intestine contained numerous enlarged vacuoles (Va), intestinal lumen, L (A). Negative staining revealed that normal enterocytes had vacuoles (Va) of different sizes without viral particles, while infected cells had marked hydropic degeneration (vesicle, V) and viral particle aggregates surround (arrow heads) (B,C). Non-enveloped virus particles (D). Inset: high magnification of virus particles shown in (d) measuring 17 to 20 nm.

Figure 8

Transmission electron micrograph of the choroid plexus of newborn piglets naturally infected with SVA. Positive staining of the CP revealed the fenestrated capillary (arrow heads) containing an influx of lymphocytes (L) and monocytes (M); the tight junction (arrow heads) between adjacent endothelial cells (EC) is shown. N, nucleus (A). Observe the negatively stained balloon degeneration of ependymocytes characterized by intracytoplasmic vesicle (V) that is surrounded by aggregates (arrow heads) of SVA (B). There is an infected endothelial cell of the CP with an apoptotic body (AB), a vesicle (V) and aggregates of SVA (head arrow) (C). Closer demonstration of the non-enveloped virus observed in the cytoplasm of the infected endothelial cell of the CP (D).