A 66-day-old, male, mixed, swine (Sus scrofy)Seven of 41 piglets in a herd of 70 sows became affected at approximately 40 to 50 days of age. The affected piglets suddenly developed paralysis of the hind limbs and became recumbent. In spite of the severe flaccid paralysis of the hind limbs, they could move by using their forelimbs. In all cases, their body temperatures were within the normal range, their appetites remained normal, and none died spontaneously.
No gross abnormalities were seen in any of the piglets
Histologically, lesions were limited to the central nervous system (CNS) and peripheral nerve fibers.Â All clinically affected piglets had similar histological changes.Â The changes observed were those of nonsuppurative encephalomyelitis, characterized by perivascular cuffing of the mononuclear cells, focal gliosis, neuronal necrosis and neuronophagia.Â The spinal cord was severely affected and the lesion was seen along the full length of the spinal cord.Â In the ventral horns, nerve cells were degenerated to varying degrees up to, and including, necrosis accompanied by neuronophagia, inflammatory or glial nodules, occasional hemorrhage, and a rather diffuse infiltration of mononuclear cells.Â In the white matter of the spinal cord, perivascular cuffing and infiltration of mononuclear cells and focal gliosis were also observed.Â In addition to the infiltrative changes, severe vacuolar changes and axonal swelling were observed in the white matter of the spinal cord.Â Infiltration of mononuclear cells was observed in the dorsal root ganglia, spinal nerves, and sciatic nerves.Â In some dorsal root ganglia, degenerated ganglion cells and neuronophagia were observed (Fig.Â 2-1).Â Swollen myelin sheaths and axonal spheroids were seen in the spinal roots, and in peripheral nerves, including the brachial plexus and sciatic nerves.Â The cerebellar nuclei and the gray matter of the brainstem were also severely affected.Â In the cerebral hemisphere, only slight perivascular cuffing was present.
Immunohistochemically, PTV antigens were detected in the cytoplasm of large nerve cells and glial cells in the cerebellar nuclei, the gray matter of the brain stem, and the ventral horn of the spinal cord of all examined pigs.Â In the spinal ganglia, PTV antigen was strongly detected in the cytoplasm of ganglion cells.Â In the nervous system, the distribution of PTV antigen was consistent with the lesion distribution.Â In the lesion, no antigens were seen in the central severe area.Â Antigens were mainly seen in the periphery of the severe lesions and, especially, in minimal to mild lesions around areas of perivascular cuffing.
Spinal cord: nonsuppurative, necrotizing, myelitis, with vacuolar changes in the white matter, mixed pig, swine
Dorsal root ganglia: nonsuppurative, ganglionitis
Portions of the cerebrum, brainstem, cerebellum, spinal cord (cervical and lumber enlargement) and tonsils of 4 piglets were taken for virus isolation purposes.Â Each tissue was routinely homogenized in Earle's medium and inoculated onto porcine kidney cell line (CPK) cultures that were then observed microscopically for seven days.Â Three further passages were made of each sample.Â CPK cells were harvested when the cells displayed characteristic cytopathic effects.Â Viral RNA was extracted from the CPK cells with ISOGEN-LS (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions.Â The RT-PCR was performed with a RNA PCR kit (Takara) according to the manufacturer's instructions.Â The amplified product was visualized by standard gel electrophoresis of 10Î¼l of the final reaction mixture on a 2% agarose gel.Â Amplified DNA fragments of specific sizes were located by ultraviolet fluorescence after staining with ethidium bromide.Â Fragment lengths were verified by comparison with a digested lambda standard on the same gel.Â PCR products were purified using a High Pure PCR Product Purification kit (Roche).Â The nucleotide sequences of the purified PCR products were determined by use of BigDye chemistry (Applied Biosystems, Foster City, CA) with the ABI 310 Sequencer (Applied Biosystems).Â The sequences of which were compared with each other and the PTV (Porcine Teschovirus) sequences available in the GenBank/EMBL/DDBJ using GENETYX-WIN version 4.0 (Software Development Co., Ltd, Tokyo, Japan).Â The cytopathogenic agents were recovered from the homogenate of cerebellum and tonsils of 3 piglets including this present piglet, and from the brainstem of this presented piglet.Â All cytopathogenic agents isolated from piglets were amplified in CPK cells, and the resulting extracted RNA was reacted with primers specific for PTV by RT-PCR.Â PCR products isolated from cerebellum and brainstem of this presented piglet were sequenced.Â These sequences were identical and the homology between this sequence and other PTVs were 91.2-95.6%.Â Based on this observation, the pathogenic agent isolated from this pig was identified as PTV.Â Virus was not isolated from the cerebrum and spinal cord.
Enterovirus encephalomyelitis (previously known as Teschen/Talfan disease) caused by at least 9 serotypes of porcine teschovirus (PTV 1, and 2-6, 8, 12, 13 which are responsible for the milder form of the disease) of the picornaviridae family is considered to be of socioeconomic importance.(5) Infection appears to be selective for specific neuronal populations, resulting in a characteristic clinical syndrome of lower motor neuron paralysis.Â A diagnosis of enterovirus encephalomyelitis is made by virus isolation from the central nervous system of pigs showing neural signs(5) The disease was first described as Teschen disease.(5) Less virulent forms of the disease were later recognized in the United Kingdom (Talfan disease) and in Denmark (poliomyelitis suum).(5) These later diseases were described as resulting in lower morbidity and mortality, the clinical syndrome expressed as paresis and ataxia (which seldom progresses to complete paralysis).(1,2) The histological changes were those of nonsuppurative polioencephalomyelitis.(2,3,5) The lesions in the spinal cord in each syndrome were largely confined to the gray matter, particularly the ventral horns, but may selectively involve the dorsal horns in very young pigs.(3) Infections from PTV are most often asymptomatic, and PTV is still frequently isolated from the faces, tonsils and other non-neural organs of apparently unaffected pigs.(4)
In the present cases, the morbidity and mortality were low and the characteristic clinical signs were flaccid paralysis of the hind limbs.Â The nonsuppurative lesions were distributed mainly in the gray matter of the brainstem and the spinal cord.Â These clinical and histological features of the present disease are similar to those of the disease produced by less virulent PTV strains, especially those of Talfan disease.(3,4) In previous reports of experimental Talfan disease, axonal degeneration was seen in the ventral root and sciatic nerves.(1) In the white matter of the spinal cord, slight degenerative changes were seen only in the dorsal funiculus.(1) In contrast, demyelination and axonal degeneration in the present cases, which resulted from a natural outbreak in Japan, appeared in the whole white matter, and in either the ventral or dorsal root.
Immunohistochemically, anti-PTV monoclonal antibody (no.9, IgM) (National Institute of Animal Health, Japan) 6 was used as the primary antibody.Â PTV antigens were detected in cytoplasm of nerve cells, glial cells and endothelial cells in the cerebellar nuclei, the gray matter of the midbrain, pons, and medulla oblongata and the ventral horn of the spinal cord and of ganglion cells in the spinal ganglion corresponding to those lesions characterized as nonsuppurative encephalomyelitis and ganglionitis in the pigs.Â The results suggest that nerve cells of the brainstem and spinal cord, and ganglion cells of the spinal ganglion permit PTV replication and represent the main target cell population of PTV.
The isolation of PTV from CNS is important for diagnosing enterovirus encephalomyelitis.(1) However, it has been reported that isolation of virus from CNS is quite difficult, and virus isolation is not always possible using routine techniques in the cases of enterovirus encephalomyelitis of pigs.(4) The optimum conditions for virus isolation from CNS, including the relationship of clinical signs to the presence of infectious virus and anatomic site where the virus is present in high density, have not been clarified in this disease.Â In the present cases, PTV was isolated from cerebellum and/or brainstem in the pigs slaughtered about three weeks after the onset of neural signs, but not from the cerebrum.Â These results suggest that sampling for virus isolation should be from the cerebellum or brainstem for the successful diagnoses enterovirus encephalomyelitis.
Spinal cord: Poliomyelitis and ganglioneuritis, nonsuppurative, multifocal, marked with neuronal degeneration and necrosis, neuronophagia, gliosis, astrocytosis, satellitosis, and spheroids
The contributor provided an excellent review of porcine teschovirus (PTV) which targets selective neuronal populations in the ventral horn of the spinal cord, brain stem, and ganglion cells of the spinal ganglion resulting in neuronal necrosis, nonsuppurative polioencephalomyelitis and lower motor neuron paralysis.Â In naturally infected cases, the PTV antigens (but no lesions) were also present in bronchial epithelium, tonsillar epithelium, hepatocytes and the myenteric nerve plexus, but not in the cerebral hemispheres.(9) In addition to the typical neurological disease, some strains of the virus have been associated with female reproductive disorders, enteric disease, pneumonia, pericarditis and myocarditis.Â Lesions have been described in the kidney, liver, spleen, adrenal gland and thyroid gland.(9) PTV is frequently isolated from the feces, tonsils and non-neural organs of clinically normal pigs.(9) The proposed pathogenesis includes virus replication within the gut, mucosal lymphoid tissue and local lymph nodes; followed by viremia; and subsequent central nervous system invasion.(9)
Conference participants discussed other viruses affecting the nervous system of pigs.Â Pseudorabies (suid herpesvirus 1) causes nonsuppurative encephalitis primarily affecting the gray matter, neuronal necrosis and ganglioneuritis in the paravertebral ganglia.(7) Eosinophilic intranuclear inclusion bodies are present in the neurons and astroglia.Â Lesions are most severe in the cerebral cortex (differentiating it from porcine teschovirus), brain stem, spinal ganglia and basal ganglia.(7) Very young and aborted pigs typically have small areas of necrosis with eosinophilic intranuclear inclusions in the liver, tonsils, lung, spleen, placenta and adrenal glands.(7) Porcine hemagglutinating encephalitis virus (HEV) is a coronavirus that causes two clinical syndromes in young pigs: neurological signs occur in 4-7-day-old piglets and vomiting and wasting disease occurs in 4-14-day-old piglets.(7) Neurological lesions include nonsuppurative encephalomyelitis affecting the gray matter of the medulla and brain stem, and inflammation within the trigeminal, paravertebral and autonomic ganglia, and the gastric myenteric plexus.(7) Classical swine fever (porcine pestivirus) causes vascular lesions that result in hemorrhage, infarction, necrosis and disseminated intravascular coagulation.(6) Common lesions include hemorrhages in various organs (especially the lymph nodes), renal petechiae and splenic infarction.(6) Neural lesions occur in the gray and white matter, and primarily affect the medulla oblongata, pons, colliculi and thalamus.(10) There is endothelial swelling, proliferation and necrosis; perivascular lymphocytic cuffing; hemorrhage and thrombosis; gliosis; and neuronal degeneration.(10) In utero infections result in cerebellar hypoplasia and spinal cord hypomyelinoogenesis.(7) Two paramyxoviral diseases of pigs include porcine rubulavirus encephalomyelitis (Blue eye disease) and Nipah virus.Â Porcine rubulavirus causes encephalomyelitis, reproductive failure and corneal opacity primarily in Mexico.Â There is nonsuppurative polioencephalomyelitis affecting the thalamus, midbrain and cortex.Â Additional lesions include anterior uveitis, corneal edema, epididymitis, orchitis and interstitial pneumonia.(7) Nipah encephalitis is an emerging disease causing severe and rapidly progressive encephalitis and pneumonia in pigs, other animals and humans.(7) Fruit bats are the natural reservoir.Â There is necrotizing vasculitis and fibrinoid necrosis of arterioles, venules and capillaries with endothelial syncytial cells resulting in large areas of hemorrhage and infarction.Â Eosinophilic intracytoplasmic and intranuclear inclusions are occasionally found in neurons and endothelial syncytia.Â Blood vessels in the lung, brain, glomeruli and lymphoid organs are most commonly affected.(7) Additional lesions include bronchointerstitial pneumonia with necrotizing bronchiolitis, lymphocytic and neutrophilic meningitis, nonsuppurative encephalitis and gliosis.(7)
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