J. COMP.

PATH.

1976.

VOL.

TRIGEMINAL CALVES INFECTIOUS

86.

93

GANGLIONITIS AND ENCEPHALITIS INTRANASALLY INOCULATED WITH BOVINE RHINOTRACHEITIS VIRUS

IN

BY

M.

NARITA,

Hokkaido

S.

INUI,

K.

NAMBA

and Y.

SHIMIZV*

Branch Laboratory, National Institute of Animal SaPgoro, Hokkaido, 061-01 Japan

Health,

INTRODUCTION

Many herpesviruses are known to be neurotropic and produce persistent infection in their natural host. The persistence of herpesvirus in the central nervous system (CNS) has been demonstrated by several investigators (Knotts, Cook and Stevens, 1973; Paine, 1964; Plummer, Coleman and Henson, 1972; Plummer, Hollingsworth, Phuangsab and Bowling, 1970). Since herpesviruses were isolated from the trigeminal ganglion of man, monkey and cat (Plummer, 1973), the ganglion has been postulated as a site for persistence of herpesvirus (Paine, 1964; Stevens and Cook, 1971). Infectious bovine rhinotracheitis (IBR) virus has been shown to be the causal agent of meningo-encephalitis in young calves (Bartha, Hajdu, Aldasy and Paczolay, 1969; Hall, Simmons, French, Snowdon and Asdell, 1966). Bagust and Clark (1972) demonstrated that the intranasal inoculation of N569 strain of IBR virus induced infection of the CNS and resulted in nonpurulent meningoencephalitis. The purpose of this report is to describe the lesions in the trigeminal ganglion and CNS in calves intranasally infected with IBR virus. MATERIALS

AND

METHODS

‘Virus.The Los Angeles isolate of IBR virus, supplied by Dr McKercher, University of California, was used. The virus was originally isolated from nasal washing of calves (Madin, York and McKercher, 1956) and serially passagedin bovine kidney (BK) cell culture. Experimental animals. Five Holstein calves 3 to 4 months of age of either sex were used. They were proved

to be free of IBR virus neutralizing

antibody

before

inocula-

tion of virus. Each experimental calf was inoculated intranasafly with 10 ml. of infected culture medium containing 10’ TCID,,. Clinical observation. Clinical observation were made daily on all the calves. Necropsy procedure. Inoculated animals were killed on days 12, 15, 30, 57 and 98 after inoculation. At necropsy, each calf was processed mainly following procedures described by Bagust and Clark (1972). S everal tissues including the brain and trigeminal ganglia were sampled for virus isolation, immunofluorescent and histopathological examination. Virus isolation. Following experimental infection, the nasal secretion of each calf was swabbed every day for the first 14 days and once a week thereafter. Swabs were collected into culture medium and gross debris was removed by centrifugation before inoculation. At necropsy the lung, liver, kidney, spleen, nasal mucosa, trigeminal ganglia, medulla oblongata, and cerebrum were sampled for virological * Present

address:

National

Institute

of Animal

Health,

Kodaira,

Tokyo,

187 Japan.

94

M. NARITA

et al.

examination. Each tissue was homogenized in Earle’s medium, inoculated into primary BK cell cultures and observed microscopically for at least 8 days. Two further passageswere made of each sample. HistopathologyandimmunoJIuorescent study. Blocks of brain and nervous tissueobtained at necropsy were fixed in 10 per cent neutral buffered formalin, sectioned and stained with Mayer’s haematoxylin and eosin (HE.). Immunofluorescent antibody was prepared by the method of Marshall, Eveland and Smith (1958) using gamma globulin obtained from anti-IBR bovine serum. The conjugate was overlaid on the slide in a moist chamber for one hour and was removed with 3 washes of phosphate buffered saline (PBS) at pH 7.2 for 20 min. After that, the sections were examined by fluorescent microscopy. After fluorescent antibody (FA) examination, sections

were washed in PBS, then re-fixed with absolute alcohol for 15 min. These sections were stained with HE. for the identification of specific fluorescent cells. RESULTS

Clinical Observation The clinical response in calves was characterized by a rise in the body temperature between 2 to 5 days after inoculation. During the febrile period, all the calves became depressed. They had a mucoid nasal discharge, were dyspnoeic and coughed; however, the respiratory sound was substantially normal. Diarrhoea was observed in 2 of 5 calves on the day of the highest temperature and persisted 2 to 3 days. Distinct leukopenia was not detected at any stages of infection. The calves recovered from the diseasewithin 8 to 14 days after the febrile period. Viral Examination and Immune-jluorescent Studies The results of viral isolation from the nasal secretion are shown in Fig. 1. The virus was detected 1 to 11 days after inoculation. The virus titre ranged from 10°e5 to 1O7*25TCID,,/ml. The period of virus recovery was broadly coincident with the febrile period. Infection in calf 1 was detected at 12 days by virus isolation and FA tests in the cerebrum, medulla oblongata, trigeminal ganglion and nasal mucosa and in calf 2 at 15 days in the samesites by virus

7.0 6.0 5.0 4.0 3.0 2.0 I-O o-o Days

Fig.

after

inoculation

1. Detection of IBR virus from nasal secretion of intranasally (0) calf 2; (0) calf 3; (m) calf 4; (+) calf 5.

inoculated

calves.

(0)

Calf

1;

RHINOTRACHEITIS

VIRUS

95

IN CALVES

isolation only. In calves 3, 4 and 5 at 30, 57 and 98 days respectively both tests were negative. In the nerve trunks and brain stem of the calf killed 12 days after inoculation, Schwann cells and neuroglia cells exhibited specific immunofluorescence. In the trigeminal ganglion, specific immunofluorescence was observed in satellite cells around the ganglion cell. The stained cells existed apart from the aggregation of neuroglia cells which exhibited no fluorescence at all. No positive fluorescence could be detected in either nerves or ganglia later than 15 days after inoculation. TABLE

Calf

No.

HISTOLOGICAL

LESIONS

Trigeminal ganglion

Mid&a oblongata

Days after infection

12 E

++ +++ ++

z3

:

: P 5

Grade

of lesion : -,

no lesion;

Fig. 2. Infiltration of neuroglia 15 days after inoculation.

IN CALVES

AFTER

Rhinencefihalon

E+ ++ +

f,

I

+

mild;

+ +,

cells, lymphocytes HE. x 520.

moderate;

EXPOSURE

T O IBR VIRUS

Midbrain

Cerebellum

Posterior cerebrum

+ + -

+

-

Anterior cerebrum ++ +

+ + +, severe.

and a mitosis

around

the ganglion

cells from

a calf

Pathological Changes At necropsy, there were no macroscopic lesions in any of the calves. Histologically, inflammatory changes were localized in the bilateral trigeminal ganglia and rhombencephalon. Trigeminal ganglionitis was found in

96

M. NARITA

et ai.

all cases. It was conspicuous in calves killed within one month. Encephalitic changes were most severe in the medulla oblongata and pons in the earlier stage of infection. The other parts of the CNS revealed slight changes (Table 1). An extensive infiltration of inflammatory cells which were observed around the blood vessels of the trigeminal ganglia and nerve fibres were predominantly neuroglial cells (Schwann-like cells), lymphocytes and plasma cells. In the calves examined 12 and 15 days after inoculation, basophilic mononuclear cells were scattered in the area of accumulated neuroglial cells. Mitotic figures of proliferated cells (Fig. 2) and plasma cells were well-defined. Basophilic

Fig. 3. Basophilic and amorphous 15 days after inoculation. Day 12 ( Golf

I )

intranuclear HE. x 780. Doy ! Colf

15 2 )

inclusion

body

Doy (Calf

(arrow)

30 3 )

in a neuroglial

cell from

Doy i Colf

a calf

57 4 1

Fig. 4. Distribution of focal gliosis and perivascular cuffing in the pons and medulla oblongata. 1. Nucl. tractus mesencephalic n. trigemini. 2. Nucl. sensorius superior n. trigemini. 3. Nucl. motorius n. trigemini. 4. Nucl. n. abducentis. 5. Nuclei vestibulares. 6. Nucl. tractus spinalis n. trigemini. 7. Nucl. n. facialis. 8. Nucl. n. hypoglossi. 9. Nucl. tractus solitarii. 10. Nucl. olivaris.

RHINOTRACHEITIS

VIRUS

97

IN CALVES

and amorphous intranuclear inclusion bodies could be recognized in a few neuroglial ce1I.s (Fig. 3). Th e inflammatory changes in the trigeminal ganglia and nerve fibres persisted with gradual diminution of their intensity until 98 days after inoculation. Distribution of the lesions in the rhombencephalon is shown diagrammatically (Fig. 4). The lesions consisted of focal aggregations

Fig, 5. Focal nuclei

aggregation of dense mononuclear of the trigeminal nerve from a calf

Fig. 6. Perivascular the trigeminal

cuffing nerve

cells and a few oligodendroglia 12 days after inoxlation. HE.

x

cells in thelsensory 325.

and diffuse infiltration with microglia cells in the spinal from a calf 30 days after inoculation. HE. x 260.

tract

nuclei[of

of oligodendroglia and microglia cells and lymphocytic perivascular cuffing (Figs 5 and 6). In 4 of 5 calves, well-developed lesions were seen in the pons and medulla oblongata. The most advanced lesions in the rhombencephalon were mostly located in the main sensory and spinal tract nuclei of the trigeminal G

98

M. NARITA

et

d.

nerve and they were confined to the inoculated side. There was no neural degeneration or necrosis at any stage of infection. Intranuclear inclusion bodies were not detected in the CNS. DISCUSSION

It is known that IBR virus produces meningoencephalitis in young calves (Bartha et al., 1969; Hall et al., 1966). Bagust and Clark (1972) demonstrated that, by intranasal inoculation, the IBR virus, originally isolated from a calf with meningoencephalitis, spreads to the CNS by a progressive, ascending infection of the trigeminal nerve and produces a non-purulent meningoencephalitis. In the present experiment, the lesions were consistently observed in the trigeminal ganglia and medulla oblongata of all calves intranasally inoculated with IBR virus. The lesions in the medulla oblongata which were found in the inoculated-side were mostly located in the main sensory and spinal tract nuclei of the trigeminal nerve. From the histopathological changes in the nervous system, trigeminal ganglionitis and focal non-suppurative encephalitis might be considered to be the characteristic lesions in calves intranasally infected with IBR virus: moreover, trigeminal ganglionitis was observed in all calves examined including the calf killed 98 days after inoculation. Goodpasture and Teague ( 1923) and Goodpasture (1925) who reported experimentally induced encephalitis in the rabbit infected with herpes simplex virus, found that cornea1 inoculation produced lesions in the sensory tracts in the brain stem and that inoculation into the masseter muscle produced lesions confined to the trigeminal motor nucleus. They concluded that the distribution of lesions along the neural pathways and the primary effect on the motor ganglion cells after masseter inoculation made it likely that the virus travelled in axis cylinders in a centripetal fashion. Knotts, Cook and Stevens (1974) and Oguchi (1973) demonstrated that herpes simplex virus spread rapidly along the nerve fibres and tracts, and extended more slowly by direct cell to cell extension in mice. After ophthalmic or subcutaneous inoculation with herpes simplex virus in the face, trigeminal ganglionitis was observed, and viral particles and fluorescent antigen were detected in the Schwann cells of the trigeminal nerve. In the case of infections with pseudorabies (Dow and McFerran, 1962) and canine herpesvirus (Percy, Munnel, Olander and Carmichael, 1970), the virus spread from the oro-nasal region to the brain, and produced ganglionitis. These facts indicate that herpesvirus generally reached the CNS through the peripheral nerves and then produced a reaction in the associated ganglion. In the present study, intranasal inoculation of IBR virus consistently resulted in pathological changes with specific immunofluorescence in the trigeminal ganglion. These observations suggest that the virus is capable of travelling in the nerve fibre from the nasal mucosa to the trigeminal ganglion, and there producing a characteristic lesion. Davies and Carmichael (1973), Davies and Duncan (1974) and Sheffy and Davies (1972) demonstrated that the recrudescence of IBR virus infection in

RHINOTRACHEITIS

cattle after tract, IBR CNS study glion

VIRUS

IN CALVES

99

was induced by the administration of synthetic corticosteroids 3 months primary infection. The virus was reisolated from the upper respiratory trigeminal ganglion and medulla oblongata. These facts indicate that virus may produce persistent infection in the trigeminal ganglion and as do other herpes simplex viruses. An important problem requiring is the relationship between pathological changes in the trigeminal ganand persistent infection. SUMMARY

Calves inoculated intranasally with infectious bovine rhinotracheitis (IBR) virus were necropsied 12, 15, 30, 57 and 98 days after inoculation. Tissues were subjected to histopathologicai, fluorescent-antibody tracing and viral examinations. The significant histopathological changes were a non-suppurative inflammation of the bilateral trigeminal ganglia and the central nervous system. Bilateral trigeminal ganglionitis was associated with the proliferation of neuroglial cells. In the central nervous system, non-suppurative encephalitic changes were found almost exclusively in the rhombencephalon. Brain lesions were localized in the inoculated side and mostly in the main sensory and spinal tract nuclei of the trigeminal nerve in the medulla oblongata. These lesions existed until 98 days after inoculation, IBR virus was recovered from the cerebrum, medulla oblongata and trigeminal ganglion of calves necropsied at 12 and 15 days after inoculation. ACKNOWLEDGMENTS

The authors wish to thank Dr. T. Kumagai, of the National Institute of Animal Health and Dr Y. Fujimoto, of the Department of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University for helpful suggestions. REFERENCES

Bartha, A., Ha-jdu, G., AldPsy, P., and Paczolay, Gy. (1969). Occurrence of encephalitis caused by infectious bovine rhinotracheitis virus in calves in Hungary. Acta veterinaria Academiae scientiarum hungaricae, 19, 145-l 51. Bagust, T. J., and Clark, L. (1972). Pathogenesisof meningo-encephalitis produced in calves by infectious bovine rhinotracheitis herpesvirus. Journal of Comparative Pathology, 82, 375-384.

Davies, D. H., and Carmichael, L. E. (1973). R o1e of cell-mediated immunity in the recovery of cattle from primary and recurrent infections with infectious bovine rhinotracheitis virus. Infection and ~mmuni~, 8, 510-518. Davies, D. H., and Duncan, J. R. (1974). The pathogenesisof recurrent infections with infectious bovine rhinotracheitis virus induced in calves by treatment with corticosteroids. Cornell Veterinarian, 64, 340-366. Dow, C., and McFerran, J. B. (1962). The pathology of Aujeszky’s diseasein cattle. Journal

of Comparative Pathology, 72, 337-347.

Goodpasture, E. W. (1925). The axis-cylinders of peripheral nerves as portals of entry to the central nervous system for the virus of herpes simplex in experimentally infected rabbits. American Journal of Pathology, 1, 1l-28. Goodpasture, E. W., and Teague, 0. (1923). T ransmissionof the virus of herpes febrilis along nerves in experimentally infected rabbits. Journal of Medical Research, 44, 139-l 82.

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et al.

Hall,

W. T. K., Simmons, G. C., French, E. L., Snowdon, W. A., and Asdell, M. (1966). The pathogenesis of encephalitis caused by the infectious bovine rhinotracheitis virus. Australian Veterinary Journal, 42, 229-237. Knotts, F. B., Cook, M. L., and Stevens, J. G. (1973). Latent herpes simplex virus in the central nervous system of rabbits and mice. Journal of Experimental Medicine, 138, 740-744. Knotts, F. B., Cook, M. L., and Stevens, J. G. (1974). Pathogenesis of herpetic encephalitis in mice after ophthalmic inoculation. Journal, of Infectious Disease,

130, 16-27. D. G. (1956). Isolation of infectious Madin, S. H., York, C. J., and McKercher, bovine rhinotracheitis virus. Science, 124, 721-722. Marshall, J. D., Eveland, W. C., and Smith, C. W. (1958). Superiority of fluorescein isothiocyanate (Riggs) for fluorescent-antibody technique with a modification of its application. Proceedings of the Society for Experimental Biology and Medicine, 98, 898-900. Oguchi, K. (1973). Neural spread of herpes simplex virus in trigeminal nerve of mouse. Viral and electron microscopic studies. Advances in Neurological Sciences,

18, 362-369. Paine, T. F. (1964). Latent herpes simplex infection in man. Bacteriological Reviews, 28,472479. Percy, D. H., Munnel, J. I;., Olander, H. J., and Carmichael, L. E. (1970). Pathogenesis of canine herpesvirus encephalitis. American Journal of Veterinary Research, 31, 145-156. Plummer, G. (1973). Isolation of herpesvirus from trigeminal ganglia of man, monkey, and cats, Journal of Infectious Disease, 128, 345-348. Plummer, G., Coleman, P. L., and Henson, D. (1972). Chronic infection of the rabbit central nervous system by a slowly growing equine herpesvirus. Infection and Immunity, 5, 172-l 75. Plummer, G., Hollingsworth, D. C., Phuangsab, A., and Bowling, C. P. (1970). Chronic infection by herpes simplex viruses and horse and cat herpesviruses. Infection and Immunity, 1, 351-355. Sheffy, B. E., and Davies, D. H. (1972). Reactivation of a bovine herpesvirus after corticosteroid treatment. Proceedings of the Society for Experimental Biology and Medicine, 140, 974-976. Stevens, J. G., and Cook, M. L. (1971). Latent herpes simplex virus from trigeminal ganglia of mice. Science, 173, 843-845. [Received for publication,

324~ lst, 19751