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Brain Lesions and Transmission of Experimental Equine Herpesvirus Type 9 in Pigs
M. Narita, A. Uchimura, K. Kimura, N. Tanimura, T. Yanai, T. Masegi, H. Fukushi and K. Hirai
Vet Pathol 2000 37: 476
DOI: 10.1354/vp.37-5-476
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Vet Pathol 37:476–479 (2000)
Brain Lesions and Transmission of Experimental Equine Herpesvirus Type 9 in Pigs
M. NARITA, A. UCHIMURA, K. KIMURA, N. TANIMURA, T. YANAI, T. MASEGI, H. FUKUSHI, AND
K. HIRAI
Abstract. We demonstrated that pigs are susceptible to acute infection by equine herpesvirus type 9 (EHV-
9). Six 8-week-old SPF pigs were inoculated intranasally and four were inoculated orally with different doses
of EHV-9, and observed for 6 days. Although neurological signs did not develop in any of the infected pigs,
the six intranasally infected pigs and one of the orally infected pigs developed lesions of encephalitis consisting
of neuronal necrosis, neuronophagia, and intranuclear inclusion bodies, distributed mainly in the rhinencephalon.
EHV-9 antigen was localized in the necrotic neuronal cells and was closely associated with the presence of
inclusion bodies. These findings clearly demonstrate that pigs are fully susceptible to EHV-9 infection following
intranasal inoculation (but less so following oral inoculation), and that EHV-9 in pigs has a highly neurotropic
nature.
Key words: Encephalitis; equine herpesvirus type 9; experimental infection; immunohistochemistry;
pathology; pig.
Herpesviruses have been isolated from many different orally with 3 ml of noninfected culture medium as controls.
species. Infection in natural hosts is usually mild and can All pigs were observed clinically for 6 days and then eu-
lead to persistent infection. Some herpesviruses may cross thanatized by an intravenous injection of pentobarbital so-
species barriers nd induce severe to fatal disease in other dium. For virus isolation, the nasal secretion of each pig,
hosts.9 Equine herpesvirus 9 (EHV-9), isolated from Thom- including the two controls, was swabbed before inoculation
son’s gazelles in 1993,2 is the causative agent of fatal me- and at 2, 4, and 6 days after inoculation of the virus. At
ningoencephalitis in the gazelle.10 Previously, it has been necropsy, the olfactory bulb, frontal pole of the brain, tri-
demonstrated that EHV-9 is virulent in suckling mice of the geminal ganglion, and palatine tonsils were collected. Each
ICR strain by intracerebral inoculation and in 4-week-old secretion and minced specimen was cultivated in rabbit kid-
mice by intranasal inoculation, causing both neurological ney cell culture at 37 C for 7 days and was observed for
symptoms and death.2 Presently, the range of natural hosts cytopathic effect. Following gross examination at necropsy,
of EHV-9 is unknown. The infectivity and pathogenicity of tissue specimens from each pig, including the brain, trigem-
EHV-9 was also not clear in domestic animals, including inal ganglia, palatine tonsil, and nasal mucosa, were fixed in
pigs. In this study, 8-week-old pigs were inoculated with a 10% neutral buffered formalin, and representative blocks
virus by different routes and doses to determine the suscep- of the central nervous system (cerebral cortex, frontal pole,
tibility of pigs to EHV-9. motor area, occipital pole, corpus striatum, thalamus, collic-
Twelve 8-week-old, specific-pathogen-free pigs with no ulus caudalis, cerebral peduncles, pons, cerebellum, medulla
serum antibodies against EHV-9, Aujeszky’s disease virus oblongata, spinal cord, and trigeminal ganglia) and other tis-
(ADV), and porcine reproductive and respiratory syndrome sues were embedded in paraffin, sectioned, and stained with
virus (PRRSV) were used. The pigs were divided into three hematoxylin and eosin (HE) for histology. Immunohisto-
groups—orally infected pigs, nasally infected pigs, and un- chemically, viral antigen was demonstrated by the avidin–
infected controls—for the experiments, and the groups were biotin–complex (ABC) immunoperoxidase method with the
housed separately in different blocks to prevent cross infec- Vectastain ABC kit. Anti–EHV-9, and anti-ADV, anti-
tion. EHV-9 isolated from the Thomson’s gazelle2,10 was PRRSV, and anti-hemoagglutinating encephalomyelitis virus
used. The virus was passaged six times in Madin and Darby (HEV) rabbit sera were all used as primary antibodies at
bovine kidney (MDBK) cells, and then twice in a pig kidney dilutions of 1:1,024, 1:2,048, 1:8,192 and 1:2,048, respec-
(PK) cell culture. The infected PK cell culture fluid was used tively. Sections were counterstained with methyl green. Tis-
for the inoculation of the pigs. Before inoculation with EHV- sue sections from pig Nos. 11 and 12 and the serum from a
9, the pigs were anesthetized with ketamine hydrochloride nonimmunized rabbit were used as negative controls.
(30 mg/kg intramuscularly) and xylazine (2.0 mg intramus- Clinically, all pigs (Nos. 1–6) inoculated intranasally and
cularly). Pig Nos. 1 and 2 were inoculated intranasally with one (No. 8) inoculated orally showed a slight febrile reaction
a nebulizer with 3 ml of 107.0 tissue culture infectious dose (over 39.5 C) over 2 to 3 days; the highest temperature of
(TCID)50/ml, pig Nos. 3 and 4 with 3 ml of 105.0 TCID50/ml, pig No. 3 was 40.0 C. No other clinical abnormality was
and pig Nos. 5 and 6 with 3 ml of 103.0 TCID50/ml of EHV- observed in the infected pigs. The two control pigs (Nos. 11
9, respectively. Pigs Nos. 7 and 8 were inoculated orally and 12) remained healthy during the experimental period.
with a syringe with 3 ml of 107.0 TCID50/ml, and pig Nos. 9 Virologically, EHV-9 was recovered from the nasal secre-
and 10 with 3 ml of 105.0 TCID50/ml of EHV-9. Pig No. 11 tions between 2 and 6 days postinoculation in pigs infected
was inoculated intranasally and pig no. 12 was inoculated intranasally, but not orally (Table 1), with titers of 101.0 to
476
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Vet Pathol 37:5, 2000 Brief Communications and Case Reports 477
Table 1. Isolation of EHV-9 from nasal secretions and various tissues of intranasally and orally infected pigs.*
Nasal Secretion (PID)
Challenge Olfactory Trigeminal Palatine
Pig No. Route Dose 0 2 4 6 Lobus Frontal Pole Ganglia Tonsil
1 Nasal 3 107 — 1.75* 2.5 3.0 0.75 — — —
2 Nasal 3 107 — 2.25 1.15 1.5 1.5 3.0 — —
3 Nasal 3 105 — — 1.75 1.0 1.5 3.0 — —
4 Nasal 3 105 — — — 2.0 2.25 3.25 0.75 —
5 Nasal 3 103 — — 1.0 — 2.75 — — —
6 Nasal 3 103 — — 2.0 1.25 2.75 3.0 — —
7 Oral 3 107 — — — — — — — —
8 Oral 3 107 — — — — 1.0 2.5 — —
9 Oral 3 105 — — — — — — — —
10 Oral 3 105 — — — — — — — —
11 Nasal (medium) — — — — — — — —
12 Oral (medium) — — — — — — — —
* Values are expressed as log 10.
103.0 TCID50/ml. The virus was also isolated from the olfac- encephalitis characterized by neuronal necrosis, neuronopha-
tory bulb of the six intranasally infected and one (pig No. gia, focal gliosis, perivascular infiltration of mononuclear
8) of the orally infected pigs, from the frontal pole of four cells, and intranuclear inclusion bodies. The distribution of
(pig Nos. 2, 3, 4, and 6) of the intranasally, and one (pig the encephalitic changes was similar in the six intranasally
No. 8) of the orally infected pigs, and from the trigeminal infected and one (pig No. 8) orally infected pig. In the ce-
ganglion of one (pig No. 4) of the intranasally infected pigs. rebrum, the most prominent lesion was found in the rhin-
The highest titer at the olfactory bulb was 102.75 TCID50/0.1 encephalon and in the cerebral cortex. Neuronal necrosis was
ml in pig Nos. 5 and 6, at the frontal pole of the brain was widespread in the mitral and tufted cell layers of the olfac-
103.25 TCID50/0.1 ml in pig No. 4, and at the trigeminal gan- tory bulbs (Fig. 1) and was accompanied by infiltration of
glion was 100.75 TCID50/0.1 ml in pig No. 4. No virus was macrophages. The affected neurons were characterized by
isolated from the two uninfected control pigs. neuronal pyknosis and basophilia, diffuse to central chro-
No macroscopic lesions were observed in any of the in- matolysis, karyorrhexis, and multifocal cellular debris. Ba-
fected pigs. Microscopic lesions were confined to the central sophilic intranuclear inclusion bodies were present in many
nervous system (Table 2) and consisted of a nonsuppurative neurons and glial cells in this zone. Focal gliosis was found
in the olfactory gyri. Severe neuronal necrosis and many
intranuclear inclusion bodies were detected in the amygda-
Table 2. Distribution of the histologic lesion and viral loid body, in the putamen, and in the caudate nuclei (Fig.
antigen in tissues of pigs infected intranasally or orally with 2). Encephalitic lesions were prominent in the pyramidal and
EHV-9.* large granular cell layers of the lobus frontalis and tempo-
Histopathological Lesion/Detection of EHV-9 Antigen ralis. Slight nonsuppurative meningitis, made up of macro-
phages and lymphocytes, was found in the lobus frontalis.
Olfactory Brain Trigeminal Palatine No lesions were evident in the medulla oblongata, spinal
Pig No. Bulbs Cerebrum Stem Ganglia Tonsil
cord, cerebellum, and trigeminal ganglia, nasal mucosa, pal-
1† 3 /p 3 /p 1 / / / atine tonsils, and other tissues. No macroscopic or micro-
2† 3 /p 3 /p 1 / / / scopic lesions were observed in the two noninfected con-
3† 2 /p 3 /p / / / trols.
4† 2 /p 2 /p / / / Immunohistochemically, EHV-9 antigen was detected in
5† / 1 / / / / the nucleus and cytoplasm of the necrotic neurons. Antigen
6† 2 /p 2 /p / / / localization was closely associated with the presence of in-
7‡ / / / / / tranuclear inclusion bodies and neuronal cell necrosis (Table
8‡ 2 /p 1 /p / / / 2). In pig Nos. 6 and 8, a small amount of the EHV-9 antigen
9‡ / / / / / was also found in neuroglial cells in the mitral and tufted
10‡ / / / / / layer of the olfactory lobus (Fig. 3), as well as in the cortex
11§ / / / / / of the lobus frontalis and temporalis. In four nasally infected
12§ / / / / / pigs (Nos. 1–4), abundant EHV-9 antigen was widely dis-
* Degree of histopathologic lesion: none; 1 slight; 2 tributed in the rhinencephalon (olfactory lobe, amygdaloid
moderate; 3 severe. Detection of HEV-9 antigen: neg- body, putamen, and caudate nuclei), and in the lobus fron-
ative; p positive.
† Nasally infected pigs.
talis, lobus temporalis, lobus parietalis, and lobus occipitalis
‡ Orally infected pigs. (Fig. 4). No EHV-9 antigen was detected in the medulla
§ Uninfected controls. oblongata, cerebellum, trigeminal ganglia, palatine tonsils,
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478 Brief Communications and Case Reports Vet Pathol 37:5, 2000
Fig. 1. Brain, olfactory bulb; intranasal infection, pig No. 4. Neuronal necrosis is extensive in the inner granular and
mitral layer or zone. HE. Bar 25 m.
Fig. 2. Brain, amygdaloid body; intranasal infection, pig No. 3. A basophilic intranuclear inclusion body (arrow) is
present in a dead neuron. HE. Bar 10 m.
Fig. 3. Brain, olfactory bulb; intranasal infection, pig No. 4. Abundant EHV-9 antigen is detected in the inner granular
neurons. Anti–EHV-9 ABC. Bar 25 m.
Fig. 4. Brain, amygdaloid body; intranasal infection, pig No. 3. EHV-9 antigen is present in necrotic neurons. Anti–
EHV-9 ABC. Bar 25 m.
or other tissues in any of the infected pigs. No ADV, PRRSV report10 of gazelles that were naturally infected with EHV-
or HEV antigens were found in any of the tissues of all the 9, and pigs and cattle infected experimentally and naturally
pigs. with the other herpesviruses.3–5
Although neurological signs did not develop in any of the Immunohistolochemically, EHV-9 antigen was demon-
infected pigs, histologically, encephalitis consisting of neu- strated intralesionally in the neuroglial cells and in necrotic
ronal necrosis, neuronophagia, intranuclear inclusion bodies, neurons. The close association of antigen with neuronal ne-
and perivascular cuffing was observed in all six intranasally crosis and intranuclear inclusion bodies suggests that EHV-
infected pigs and in one orally infected pig; lesions were 9 replication led to the neuronal cell death. Moreover, pres-
localized mainly in the rhinencephalon and in the cerebral ence of EHV-9 antigen and the severity of the encephalitis
cortex and were not observed in the cerebellum or spinal corresponded closely with the intranasal route of infection
cord. These findings closely resemble those of a previous and the challenge dose.
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Vet Pathol 37:5, 2000 Brief Communications and Case Reports 479
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