Non-suppurative Encephalomyelitis Research Articles

Porcine Astrovirus Infection in Brains of Pigs in Korea.

Recently, neurological diseases associated with astroviruses (AstVs) have been reported in pigs, ruminants, minks, and humans. In 2017, neuro-invasive porcine astrovirus (Ni-PAstV) 3 was detected in the central nervous system (CNS) of pigs with encephalomyelitis in Hungary and the USA. In the process of diagnosing domestic pigs exhibiting neurological signs, histopathologic lesions of non-suppurative encephalomyelitis with meningitis, neuronal vacuolation, and gliosis were detected, and PAstV was identified using reverse transcriptase PCR in CNS samples of four pigs in three farms from August to September in 2020, South Korea. Subsequently, the ORF2 region was successfully acquired from three brain samples, facilitating subsequent analysis. Four genotypes of PAstV (PAstV1, 3, 4, and 5) were detected, and coinfection of PAstV with multiple genotypes was observed in brain samples. This is the first study to report Ni-PAstV infection in pigs in South Korea.

Occurrences of avian encephalomyelitis virus in naturally infected chicks in Saudi Arabia's Eastern Province.

A neurological infectious viral disease, avian encephalomyelitis was initially discovered in 2-week-old commercial chicks in 1930 and classified as a neurotropic viral disease. A neurological outbreak caused by avian encephalomyelitis virus (AEV) in young chicks was first reported in Al-Ahsa in the Kingdom of Saudi Arabia (KSA) in 2010. The aim of this article is to examine the AEV in KSA, Al-Ahsa Province. Gizzard, proventriculus, cerebrum, cerebellum, and medulla oblongata tissue samples were collected from infected chicks for histopathology test and molecular identification. Infected chicks showed neurological signs particularly incoordination, mild head and neck tremors, stretching of legs, and lameness. The average morbidity and mortality rates were 35% and 10%, respectively. At necropsy, no obvious identifiable macroscopic lesions were found in the infected chicks. Nonsuppurative encephalomyelitis was found histopathologically in the central nervous system, mainly in the cerebral molecular layer. Microscopic lesions in the proventriculus showed masses of heavy numbers of small lymphocytes within the muscular layer. RT-PCR followed by sequence analysis revealed that The KSA strain (KJ939252) is intimately related to chicken European strains from Poland (KC912695) and the United Kingdom (AJ225173) with identity 99.6% than Chinese strains (AY225319, AY517471, and AY275539) with identity ranged between 94.6% and 95%. The phylogenetic tree analysis showed that the KSA strain is grouped in a similar clade with chicken European strains. The pattern of disease findings was typical of vertically transmitted AEV. The spread of AEV in Saudi Arabia is most likely due to the trade of birds and bird products with European countries.

Differential role of NSs genes in the neurovirulence of two genogroups of Akabane virus causing postnatal encephalomyelitis.

Akabane virus (AKAV) is a member of the genus Orthobunyavirus, family Peribunyaviridae. In addition to AKAV strains that cause fetal Akabane disease, which is characterized by abortion in ruminants, some AKAV strains cause postnatal infection characterized by nonsuppurative encephalomyelitis in ruminants. Here, we focused on the NSs protein, a virulence factor for most viruses belonging to the genus Orthobunyavirus, and we hypothesized that this protein would act as a neurovirulence factor in AKAV strains causing postnatal encephalomyelitis. We generated AKAV strains that were unable to produce the NSs protein, derived from two different genogroups, genogroups I and II, and then examined the role of their NSs proteins by inoculating mice intracerebrally with these modified viruses. Our results revealed that the neurovirulence of genogroup II strains is dependent on the NSs protein, whereas that of genogroup I strains is independent of this protein. Notably, infection of primary cultured bovine cells with these viruses suggested that the NSs proteins of both genogroups suppress innate immune-related gene expression with equal efficiency. These results indicate differences in the determinants of virulence of orthobunyaviruses.

Congenital malformations caused by Akabane virus in porcine fetuses in southern Japan.

Akabane virus (AKAV) is known as a major teratogenic agent of ruminant fetuses. In this study, we investigated the relationship between porcine abnormal deliveries and AKAV by serology, pathology, and virology investigations using specimens from 16 stillborn fetuses delivered in southern Japan between 2013 and 2015. The major clinical manifestations in stillborn fetuses were hydranencephaly, arthrogryposis, spinal curvature, and both skeletal muscle and subcutaneous edema. Histologic examination of the specimens identified atrophy of skeletal muscle fibers accompanied by adipose replacement. Nonsuppurative encephalomyelitis and decreased neuronal density in the ventral horn of the spinal cord were shown in two separate fetuses, respectively. Neutralizing antibody titers to AKAV were detected in most of the tested fetuses (13/16). The AKAV sequences detected in the affected fetuses in 2013 and 2015 were highly identical and closely related to Japanese AKAV isolates which were isolated in 2013 and sorted into genogroup I of AKAV. Immunohistochemistry visualized AKAV antigens in the neuronal cells of the central nervous system of the fetuses. These findings indicate that AKAV was involved in the birth of abnormal piglets at the affected farm. The clinical manifestations and histopathological features in the stillborn fetuses were very similar to those in ruminant neonates affected by AKAV. To avoid misdiagnosis and to evaluate the precise impact of AKAV on pig reproduction, AKAV should be considered in differential diagnoses of reproductive failures in pigs.

Rustrela Virus-Associated Encephalomyelitis ('Staggering Disease') in Cats from Eastern Austria, 1994-2016.

Clinical cases of 'staggering disease', a nonsuppurative encephalomyelitis associated with gait abnormalities in cats, have been documented for decades in Sweden. In Austria, an increased incidence was observed in the 1990s. Only recently, rustrela virus (RusV) was identified as the causative agent of this clinicopathologic disease entity. In this retrospective study, we analyzed a total of 23 brain and spinal cord samples from Austrian cats with the pathohistological diagnosis of nonsuppurative encephalomyelitis and clinical signs consistent with staggering disease from 1994 to 2016 using reverse transcription real-time polymerase chain reaction (RT-qPCR) and in situ hybridization. We were able to detect RusV nucleic acids in seven of the examined samples. Borna disease virus 1 (BoDV-1) could be excluded in all cases via immunohistochemistry and RT-qPCR. This study confirms that RusV has been a relevant etiological agent of nonsuppurative encephalomyelitis of cats in a geographically and temporally limited disease cluster in Austria, mainly in the 1990s. The geographic distribution of the positive samples in this study is consistent with earlier reports on 'staggering disease' in Austria. Further studies are necessary to confirm the reservoir host of 'staggering disease' in Austria, as well as investigations on the disappearance of this disease and its possible zoonotic potential.

Histopathological, Immunohistochemical and In-Situ Hybridization Findings in Suckling Rats Experimentally Infected With Akabane Genogroups Ⅰ and Ⅱ, Aino and Peaton Viruses

Akabane, Aino and Peaton viruses are closely related arthropod-borne viruses in the genus Orthobunyavirus of the family Peribunyaviridae that can cause congenital abnormalities in cattle, sheep and goats. East Asian Akabane virus strains are subdivided into genogroups Ⅰ and Ⅱ, and the former can also cause non-suppurative encephalomyelitis in post-natal animals. Specific detection of the infecting virus in tissues is essential for accurate diagnosis. Immunohistochemistry (IHC) has been used to identify viral antigen but cannot always detect specific viruses due to potential cross-reactivity of the primary antisera. We compared in-situ hybridization (ISH), based on the use of cocktail probe sets targeted at the RNA of each virus, with IHC for the detection of the specific viruses in tissues of suckling rats inoculated intracerebrally with Akabane (KM-1 or OBE-1 strains), Aino or Peaton viruses at 3 or 7 days of age. Most inoculated rats developed severe neurological signs and histopathological brain lesions including necrosis, spongy degeneration and non-suppurative inflammation. A rabbit polyclonal antiserum immunolabelled antigen of all three viruses within the lesions, whereas ISH specifically detected RNA of each individual virus. The distribution of viral RNA was comparable to that of viral antigens, but tended to be more widespread, especially in immature nervous tissue. Viral antigen and RNA were detected in skeletal muscle and heart of the rats infected with the KM-1 strain of Akabane virus but not with any of the other viruses. This study demonstrates the value of ISH detection of these viruses in a rat model and may prove useful for clarification of the pathogenesis of post-natal arbovirus infection.

Disease surveillance in England and Wales, April 2021

Disease surveillance in England and Wales, April 2021

Diseases of the central nervous system in swine in Southern Brazil

ABSTRACT: Brazil is the fourth largest exporter of pork worldwide, with its production concentrated in the Southern region of the country. To reduce the economic impacts and increase herd health, an efficient diagnosis of diseases is necessary. The frequency, seasonal, and annual distribution of diseases that affecting the swine nervous system in Southern Brazil were determined through a retrospective study conducted from 2008 to 2018. A total of 231 pigs were evaluated, and distributed in the following phases: suckling piglets, nursery, growing, finishing, and adults. Piglets in the nursery phase were the most affected with 58.01%. Infectious diseases accounted for 87.01% (201/231) of lesions in the nervous system. Bacterial meningitis was observed in 58.87% of cases, presenting an endemic distribution, and a propensity to occur in the months of autumn/winter. The highest number of cases of porcine circovirus-2 were observed in 2008 and 2009, with a subsequent decline. Porcine circovirus, salt poisoning, and brain abscesses exhibited no association between the seasonal occurrence and annual distribution. Salt poisoning was observed more frequently in the last years of this study. Both, non-suppurative encephalomyelitis and selenium toxicosis exhibited an association between their occurrence and seasonal distribution in, the months of winter, and summer, respectively.

Complete Genome Sequences of Two Akabane Virus Strains Causing Bovine Postnatal Encephalomyelitis in Japan.

Akabane virus (AKAV) (genus Orthobunyavirus, family Peribunyaviridae) is an arthropod-borne virus that causes congenital abnormalities in ruminants. Here, we report the complete genome sequences of two AKAV strains causing nonsuppurative encephalomyelitis in cattle by postnatal infection in Japan.

Two novel porcine teschovirus strains as the causative agents of encephalomyelitis in the Netherlands

BackgroundPorcine teschovirus (PTV) circulates among wild and domesticated pig populations without causing clinical disease, however neuroinvasive strains have caused high morbidity and mortality in the past. In recent years, several reports appeared with viral agents as a cause for neurologic signs in weanling and growing pigs among which PTV and new strains of PTV were described.Case presentationOn two unrelated pig farms in the Netherlands the weanling pig population showed a staggering gate, which developed progressively to paresis or paralysis of the hind legs with a morbidity up to 5%. After necropsy we diagnosed a non-suppurative encephalomyelitis on both farms, which was most consistent with a viral infection. PTV was detected within the central nervous system by qPCR. From both farms PTV full-length genomes were sequenced, which clustered closely with PTV-3 (98%) or PTV-11 (85%). Other common swine viruses were excluded by qPCR and sequencing of the virus.ConclusionOur results show that new neuroinvasive PTV strains still emerge in pigs in the Netherlands. Further research is needed to investigate the impact of PTV and other viral agents causing encephalomyelitis within wild and domestic pig populations supported by the awareness of veterinarians.

Novel encephalomyelitis-associated astrovirus in a muskox (Ovibos moschatus): a surprise from the archives

BackgroundThe small, single-stranded positive-sense RNA astroviruses are mostly known to be enteric viruses. In recent years, though, different astroviruses were reported in association with neurological disease in various species. In cattle, two distinct neurotropic astrovirus genotype species were described in numerous cases of nonsuppurative encephalomyelitis, with one of these viruses also reported in similar circumstances in several sheep. Here, we retrieved archived formalin-fixed, paraffin-embedded brain tissues of a muskox diagnosed with a comparable disease pattern in 1982 and investigated them for the presence of neurotropic astroviruses with various techniques.ResultsInitially, tissue samples scored positive for both neurotropic astroviruses by immunohistochemistry; however, unexpected results with further immunohistochemical testing, in situ hybridization and qRT-PCR prompted us to submit an RNA extract from the animal’s brain material to next-generation sequencing. We were thus able to obtain the full genome of a novel astrovirus, muskox astrovirus CH18 (MOxAstV-CH18), whose closest relative is an enteric ovine astrovirus. Subsequently, viral RNA could be detected with a specific RT-PCR in the brain of the affected animal, but not in faecal samples from the current muskoxen herd of the animal park where the animal used to be kept.ConclusionsWe identified a novel astrovirus in a historical case of a captive muskox with nonsuppurative encephalomyelitis. Unfortunately, our results and the fact that no material from organs other than of the nervous system was available do not allow any assumption about the epidemiology or pathogenesis of the virus. Still, these findings are yet another piece of evidence that the tropism and species specificity of astroviruses could be more deceptive than generally assumed.

Whole genome analysis of a novel neurotropic bovine astrovirus detected in a Japanese black steer with non-suppurative encephalomyelitis in Japan.

While neurotropic bovine astroviruses (BoAstVs) have been identified in North America and Europe, their presence has never been reported in Asia. In this study, we detected BoAstV in the brain of a steer showing neurological signs. Phylogenetic analysis revealed that the identified virus belongs to the Virginia/Human-Mink-Ovine clade, which contains most of the neurotropic astroviruses including the neurotropic BoAstVs. Similarity plot analysis showed that the virus was closely related to the American BoAstV NeuroS1 strain with respect to the ORF regions and to the European BoAstV CH13 strain in the 3' untranslated region, suggesting the occurrence of intra-genotypic recombination events.

Study of an Outbreak of Highly Pathogenic Avian Influenza H5N8 in Commercial Pekin Ducks (Anas platyrhynchos domesticus) in California

A February 2015 outbreak of highly pathogenic avian influenza (HPAI) H5N8 in a flock of commercial Pekin ducks ( Anas platyrhynchos domesticus) in California was investigated in detail. Approximately 17,349 five-wk-old ducks experienced an increased mortality from a normal of eight birds per day to 24, 18, 24, 33, and 61 birds per day, respectively, in the last 5 days prior to flock depopulation. Clinically, there was decreased water and feed consumption, and approximately 1.0% of the affected flock exhibited neurologic signs. Necropsy of five clinically ill ducks revealed pale, patchy areas on the epicardium in two birds, pale foci of necrosis in the liver of one bird, and airsacculitis in three birds. Histopathology revealed multifocal nonsuppurative encephalomyelitis, myocarditis, myositis, pancreatitis, hepatitis, and glossitis. Immunohistochemistry revealed avian influenza virus (AIV) nucleoprotein in the nucleus and cytoplasm of various cells in the aforementioned organs, as well as in the skin and feathers. Eight of the 10 sera samples tested were positive for avian influenza antibodies by agar gel immunodiffusion serology. Oropharyngeal and cloacal swabs taken from 15 birds, as well as from the lungs, livers, pancreas, and spleen, were positive for AIV by real-time reverse transcriptase (rRT) PCR. AIV was isolated and typed as Eurasian lineage HPAI H5N8, clade 2.3.4.4, by the National Veterinary Services Laboratory, Ames, IA. Extensive surveillance of birds for AIV around the 10-km zone did not reveal any additional cases. Ducks on the affected premises were humanely euthanatized by foam and composted in-house, the houses were heated to 57 C for 4 days, and swabs were taken periodically from the compost to ensure negativity for AIV by rRT-PCR. The compost and litter were then removed, and the house was pressure cleaned, disinfected, and repopulated approximately 120 days after euthanatization of the ducks.

Viruses associated with congenital tremor and high lethality in piglets.

The recently described atypical porcine pestivirus (APPV) has been associated with congenital tremor (CT) type A-II in piglets in different countries. Another important neurological pathogen of pigs is porcine teschovirus (PTV), which has been associated with non-suppurative encephalomyelitis in pigs with severe or mild neurological disorders. There have been no reports of APPV and/or PTV coinfection associated with CT or encephalomyelitis in Brazilian pig herds. The aim of this study was to describe the pathological and molecular findings associated with simultaneous infection of APPV and PTV in piglets with clinical manifestations of CT that were derived from a herd with high rates of CT-associated lethality. In 2017, three piglets from the same litter with CT died spontaneously. The principal pathological alterations in all piglets were secondary demyelination and hypomyelination at the cerebellum, brainstem and spinal cord confirmed by histopathology and luxol fast blue-cresyl violet stain. Additional significant pathological findings included multifocal neuronal necrosis, neuronophagia and gliosis found in the cerebral cortex and spinal cord of all piglets, while atrophic enteritis and mesocolonic oedema were observed in some of them. APPV and PTV RNA were detected in the central nervous system of affected piglets, and PTV was also detected in the intestine and faeces. The pathological alterations and molecular findings together suggest a dual infection due to APPV and PTV at this farm. Moreover, the combined effects of these pathogens can be attributed to the elevated piglet mortality, as coinfections involving PTV have a synergistic effect on the affected animals.

Experimental Infection of Goats with a Newly Isolated Strain of Akabane Virus that Causes Encephalomyelitis

In 2010, there was a large-scale outbreak of bovine encephalomyelitis in Korea, and 15 new strains of Akabane virus (AKAV) were isolated. To identify the pathogenicity of one of these strains, we infected adult goats with AKAV-7 via different routes. Twenty-five female goats were used in this study and were divided into five groups: intracerebral (IC) and intrasubarachnoid (IS) viral inoculation (n=8 each), intravenous (IV) inoculation (n=4), and vaccinated before IV inoculation (n=4), in addition to a negative control animal. All animals inoculated with AKAV-7 had AKAV-neutralizing antibodies at 6-8days post infection (dpi). During the experimental period, infected animals showed no clinical signs. In the IC group, 5/8 goats had non-suppurative encephalomyelitis affecting the cerebrum. Virus S RNA segments were detected in nearly all areas of the brain. In the IS group, 3/8 goats had encephalomyelitis affecting the cerebrum, cerebellum and spinal cord. At 7 and 21dpi, virus S RNA segments were found mostly in the spinal cord, especially around the area of injection (L5-L6). Antibody titres in the serum of the vaccinated group had an early onset and slightly increased titre compared with the IV group. Histopathologically, there were no obvious lesions in the central nervous tissues in the vaccinated group, while one of four goats in the IV group showed encephalomyelitis in the parietal lobe of the cerebrum. The newly isolated AKAV-7 can cause encephalomyelitis in goats after experimental injection. The attenuated AKAV vaccine currently used in Korea may provide partial protective immunity against AKAV-7 infection, but the real effect of the vaccine requires further investigation in goats.

Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations.

Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and little is known about the occurrence and pathogenesis of this parasite in the CNS. The aims of this study were to evaluate the occurrence, viability and load of L. infantum in the CNS, and to identify the neurological histological alterations associated with this protozoan and its co-infections in naturally infected dogs. Forty-eight Leishmania-seropositive dogs from which L. infantum was isolated after necropsy were examined. Cerebrospinal fluid (CSF) samples were analyzed by parasitological culture, quantitative real-time PCR (qPCR) and the rapid immunochromatographic Dual Path Platform test. Brain, spinal cord and spleen samples were submitted to parasitological culture, qPCR, and histological techniques. Additionally, anti-Toxoplasma gondii and anti-Ehrlichia canis antibodies in serum and distemper virus antigens in CSF were investigated. None of the dogs showed neurological signs. All dogs tested positive for L. infantum in the CNS. Viable forms of L. infantum were isolated from CSF, brain and spinal cord in 25% of the dogs. Anti-L. infantum antibodies were detected in CSF in 61% of 36 dogs. Inflammatory histological alterations were observed in the CNS of 31% of the animals; of these, 66% were seropositive for E. canis and/or T. gondii. Amastigote forms were associated with granulomatous non-suppurative encephalomyelitis in a dog without evidence of co-infections. The highest frequency of L. infantum DNA was observed in the brain (98%), followed by the spinal cord (96%), spleen (95%), and CSF (50%). The highest L. infantum load in CNS was found in the spinal cord. These results demonstrate that L. infantum can cross the blood-brain barrier, spread through CSF, and cause active infection in the entire CNS of dogs. Additionally, L. infantum can cause inflammation in the CNS that can lead to neurological signs with progression of the disease.

CASE REPORT: AKABANE DISEASE IN DAIRY CATTLE

A dairy cattle farm in Liuying, Tainan on 29 June 2015. The two-month-old calves in the farm were found with opisthotonus, lameness, paralysis and ataxia. The morbidity is approximately 1.1% and the mortality is 0%. In gross lesions, flat gyri, shallow sulci and meningeal vascular engorgement were seen. Histopathological examinations revealed non-suppurative lymphohistocytic encephalomyelitis and pachymeningitis with hemorrhage, lymphohistocytic perivascular cuffing, microgliosis and demyelination. Nucleic acid was extracted from Spinal cord, then amplified by reverse transcriptase polymerase chain reaction. A 709-base pair amplification product of S segments was detected and showed 99.9% homology to Akabane virus by nucleotide sequencing. Thus, according to the laboratory examination and analysis of the results, this case diagnosed with Akabane disease in dairy cattle.

Experimental infection of cows with newly isolated Akabane virus strain (AKAV-7) causing encephalomyelitis.

Akabane virus (AKAV), an arthropod-transmitted bunyavirus, is a major cause of congenital abnormalities and encephalomyelitis in ruminants. In 2010, there was a major outbreak of encephalomyelitis in Korea and fifteen AKAV strains, including AKAV-7, were isolated from cows. To identify the neuropathogenicity of AKAV-7, we performed experimental infection of cows. Six-month-old female Korean Holstein dairy cattle were inoculated with AKAV-7 by various routes, including intracerebral (IC), intrasubarachnoid space (IS), subcutaneous (SC) and intravenous (IV); a separate group was vaccinated before intravenous infection. Five of the six cows in the IC group and two of the six cows in the IS group showed clinical signs such as locomotor ataxia and paralysis of the hind limbs. Three of six cows died after IC infection 9–12 days post infection (dpi). Histopathologic changes such as nonsuppurative encephalomyelitis were confirmed in various parts of the central nervous system in the IC, IS and SC groups. Early onset of neutralizing antibodies in the serum and lower viral mRNA levels in the peripheral blood mononuclear cells (PBMCs) and various tissues in the vaccinated group was noticeable compared to the unvaccinated group (IV group). We suggest that the AKAV vaccine currently used in Korea may be partially effective for protection against AKAV-7 in cows.

First Report of Congenital Neospora Caninum Encephalomyelitis in Two Newborn Calves in the Argentinean Pampas

Bovine neosporosis is a parasitic disease of major concern in cattle industry worldwide. Although abortion is the more frequent clinical presentation, sporadic congenital clinical bovine neosporosis has been scarcely reported in newborn calves. Neospora caninum encephalomyelitis was diagnosed in two of 314 newborn calves submitted for necropsy and laboratory analysis between 2000 and 2014. The two new born calves had limb dysfunction, loss of conscious proprioception and ataxia. Protozoal multifocal nonsuppurative encephalomyelitis was observed and diagnosis was confirmed using serology, immunohistochemistry and PCR. This is the first report of clinical congenital neosporosis in calves from Argentina.

Japanese encephalitis in a 114-month-old cow: pathological investigation of the affected cow and genetic characterization of Japanese encephalitis virus isolate

BackgroundJapanese encephalitis virus (JEV) is classified into the genus Flavivirus in the family Flaviviridae. JEV can cause febrile illness and encephalitis mainly in humans and horses, and occasionally in cattle.Case presentationIn late September 2010, a 114-month-old cow showed neurological symptoms similar to the symptoms observed in previous bovine cases of Japanese encephalitis (JE); therefore, we conducted virological and pathological tests on the cow. As a result, JEV was isolated from the cerebrum of the affected cow. We determined the complete genome sequence of the JEV isolate, which we named JEV/Bo/Aichi/1/2010, including the envelope (E) gene region and 3’ untranslated region (3’UTR). Our phylogenetic analyses of the E region and complete genome showed that the isolate belongs to JEV genotype 1 (G1). The isolate, JEV/Bo/Aichi/1/2010, was most closely related to several JEV G1 isolates in Toyama Prefecture, Japan in 2007–2009 by the phylogenetic analysis of the E region. In addition, the nucleotide alignment revealed that the deletion in the 3’UTR was the same between JEV/Bo/Aichi/1/2010 and several other JEV G1 isolates identified in Toyama Prefecture in 2008–2009. A hemagglutination inhibition (HI) test was conducted for the detection of anti-JEV antibodies in the affected cow, and the test detected 2-mercaptoethanol (2-ME)-sensitive HI antibodies against JEV in the serum of the affected cow. The histopathological investigation revealed nonsuppurative encephalomyelitis in the affected cow, and the immunohistochemical assay detected JEV antigen in the cerebrum.ConclusionWe diagnosed the case as JE of a cow based on the findings of nonsuppurative encephalomyelitis observed in the central nervous system, JEV antigen detected in the cerebrum, JEV isolated from the cerebrum, and 2-ME-sensitive HI antibodies against JEV detected in the serum. This is the first reported case of JE in a cow over 24 months old.