Necrotizing Encephalitis in Skunks Caused by Herpes simplex Virus K. M. Chariton, G. C. Dulac, F. C. Thomas and H. K. Mitchell* ABSTRACT
INTRODUCTION
Herpes simplex virus was isolated from the brain of a wild skunk (Mephitis mephitis) which had clinical neurological disease. In the brain of this skunk and in brain of skunks inoculated intracerebrally, there were areas of necrosis in the grey matter of the cerebral cortex and to a lesser extent in the brain stem. Intranuclear inclusions occurred in neurons and glial cells with vascular cuffing by mononuclear cells and a few neutrophils. Skunks inoculated intravenously had extensive focal necrosis in the liver and adrenal glands.
Neurological diseases caused by herpesviruses occur sporadically in several species including man, cattle, horses, dogs, pigs and monkeys (1, 2, 3, 5, 9, 11, 12, 13, 15). Infection of adults by a herpesvirus of their own species usually does not result in severe clinical disease. However, encephalitis or severe disseminated disease may occur in neonatal or young animals through infection with the same virus (4, 11). Herpesvirus encephalitis in adults most frequently is caused by a virus acquired from another species (11). In most species, except equine (3), lesions in the brains of affected animals consist of areas of necrosis of neurons and glial cells, intranuclear inclusions, and perivascular and parenchymal accumulations of inflammatory cells. Spontaneous encephalitis, caused by Herpes simplex, occurs in man, owl monkeys (Aotus trivirgatu.s), gibbons (Hylobates lar), lemurs and skunks (2, 6, 7, 10, 11, 15). A previous report of the disease in skunks indicated that all the isolates were from pet skunks - suggesting that infection occurred through close contact with man (6). This is a report of isolation of Herpes simplex from a wild skunk with encephalitis, and subsequent experimental inoculation of skunks with this agent.
RESUME Les auteurs ont isole le virus herpes simplex du cerveau d'une mouffette sauvage (Mephitis mephitis) qui avait manifeste des signes nerveux. Ils decelerent des foyers de necrose au sein du cortex cerebral et, a un degre moindre, dans la partie posterieure du cerveau, chez cette mouffette et chez celles qu'ils avaient inoculees par la voie intracerebrale. Des neurones et des cellules de la nevroglie contenaient des inclusions intranucleaires; ils noterent aussi la presence de manchons peri-vasculaires, composes de cellules mononucleaires et de quelques neutrophiles. Le foie et les surrenales des mouffettes qu'ils inoculerent par la voie intra-veineuse presentaient des foyers de necrose extensifs.
MATERIALS AND METHODS *Animal Pathology Division, Health of Animab Branch, Agriculture Canada, Animal Diseases Research Institute (B), P.O. Box 11300, Station H, Ottawa, Ontario K2H 8P9 (Charlton, Dulac, Thomas) and Contagious Diseases Division, Health of Animals Branch, P.O. Box 968, Sault Ste. Marie, Ontario P6A 5N5 (Mitchell). Submitted December 16, 1976.
460
A wild skunk, found in a private garage in Sault Ste. Marie, Ontario was described as being lethargic, had tremors, nodding of the head and profuse salivation. The animal was killed and the brain was
Can. J. comp. Med.
submitted to the Animal Diseases Research Institute (Eastern) for rabies diagnosis. Following a negative fluorescent antibody test for rabies, histological and virological studies were undertaken. VIRUS ISOLATION AND IDENTIFICATION
The initial isolation was made in primary pig kidney (PK) cells inoculated with bacteria-free supernatant fluids from a 10% brain suspension in phosphate buffered saline, pH 7.2, centrifuged at 600 g for ten min. In order to increase the titer, the virus was passaged three times in fresh PK cells and a fourth passage having a titer of 104`TCID50/0.1 ml was used in these experiments. Infective cell culture fluids were stained with 2% phosphotungstic acid, pH 7.2, and examined with an Hitachi HU 12 A electron microscope. The skunk agent was positively identified by the serum neutralization (SN) test according to the constant serum (1:10 dilution) - variable virus method, using four tubes per dilution. The neutralization indices were estimated by the method of Reed and Muench (14). SKUNK INOCULATION
Four mature male skunks, purchased from a supplier' were anesthetized with Ketaset2 administered intramusculary at a dosage of 6 mg per pound. Two skunks were inoculated intracerebrally with 10` TCID50 of virus and two were given a similar dose intravenously. They were kept in individual Horsfall cages and observed daily. Skunks which died or were killed were necropsied. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6,u and stained with hematoxylin and eosin. Fresh tissues were used for reisolation of virus as described above.
isolate were obtained from the virus collection of our Institute. The canine herpesvirus was obtained as a seventh dog kidney cell passage from Dr. J. Thorsen.' One additional passage in Madin Darby-canine kidney (MDCK) cell line4 was done in our laboratory. The feline rhinotracheitis virus was a vaccine strain5 passaged once in primary feline kidney cells. Each virus, grown in respective susceptible cells was harvested after two cycles of rapid freezing and thawing. The cell debris was further disintegrated by sonic vibration and filtered through a 0.22 ,u Millipore filter.' The stocks were stored frozen at -70°C in mechanical freezers. IMMUNE SERUMS
Immune serum against the skunk isolate was prepared in two adult male skunks. The animals were immunized by a series of four intramuscular injections given one week apart. Each of the first series of two injections consisted of 1 ml of unconcentrated virulent fluid. One ml of virulent fluid, concentrated 100 times by ultracentrifugation was used for the last two injections. The skunks were bled ten days after the last inoculation and the serum was stored at -20°C until used. Herpes simplex antiserum was obtained from the Canadian Communicable Disease Center in Ottawa; the canine herpes antiserum was supplied by Dr. J. Thorsen and the feline rhinotracheitis antiserum was provided by Dr. D. L. Croghan.7 Other antiserums were from the collection of
this Institute.
RESU LTS VIRUS ISOLATION AND CROSS NEUTRALIZATION TESTS
Initially, the cytopathic effect (CPE) in PK cells was detected at five days post-
VIRUSES
Most viruses used to identify the skunk 'Ruby's Fur Farm, New Sharon, Iowa 50207. 2Ketaset, ketamine hydrochloride, Rogar/STB, London, Ontario.
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3University of Guelph, Guelph, Ontario. 4American type culture collection, 12301 Parklawn Drive, RockviUe, Maryland 20852. 5Pitman-Moore Inc., Washington Crossing, N.J. 08560. 6Millipore Corporation, Bedford, Mass. 01730. 7Veterinary Services Laboratory, Animal and Plant Health Inspection, U.S. Dept. of Agriculture, P.O. Box 70, Ames, Iowa 50070.
461
inoculation. The first lesions appeared as foci of round and refractile cells that remained attached to the glass. Upon subsequent passages, the CPE occurred as early as 24 hours postinoculation and destroyed the monolayers within five days. Inclusion bodies could not be demonstrated in PK cells stained with hematoxylin and eosin or May-Grunwald-Giemsa stains. Electron microscopic examination of infective cell culture fluids revealed numerous herpesvirus particles. The skunk isolate was neutralized by a Herpes simplex antiserum and the Herpes simplex virus was neutralized by the antiserum prepared against the skunk virus. Furthermore, there were no significant cross reactions with any of the other herpesviruses tested (Table I).
against 100 TCID50 of the skunk virus when tested in PK cells. The virus was reisolated from the liver, spleen, kidney and brain of the two skunks inoculated intracerebrally and from the same tissues of the skunk inoculated intravenously and that died on day 7. The virus could not be recovered from the skunk killed on day 21. GROSS LESIONS
The skunk which died following intravenous inoculation, had many small white foci with encircling red zones on the serosal and cut surfaces of the liver (Fig. 1). The adrenal glands were swollen and had
CLINICAL SIGNS AND REISOLATION OF VIRUS IN EXPERIMENTALLY INOCULATED SKUNKS
Both skunks inoculated intracerebrally were lethargic on day 3 and died on day 4. No other clinical signs were observed. One of the skunks inoculated intravenously died on day 7, following a three day illness characterized by anorexia, lethargy and serous nasal exudate. The other intravenously inoculated skunk had oculonasal exudate on day 5 but recovered and subsequently was killed on day 21. This skunk had a serum neutralization titer of 1:320
Fig. 1. Lobe of liver from skunk inoculated intravenously with wild skunk isolate. Many scattered greywhite foci surrounded by narrow red zones.
TABLE I. Results of Cross-neutralization Tests in Cell Culture with the Viral Isolate from a Wild Skunk and Herpesviruses from Other Species Immune Sera Prepared Against
Virus
Bovine RhinoIsolate Canine trafrom Herpes Wild simplex Pseudorabies Herpes cheitis Skunk Virus Virus Virus Virus
Feline Equine Equine Rhino- RhinoVulvotrapneuvagicheitis monitis nitis Virus Virus Virus
Isolate from wild 0.4 0.1 0.1 0.1 0.5 3.7 skunk 3.2a 4.2 4.3 Herpes simplex b 7.2 Pseudorabies 0.3 4.6 Canine Herpes 0.4 Infectious Bovine 3.0 0.2 Rhinotracheitis Feline Rhinotra5. 0 cheitis Equine Rhino3.7 0.2 pneumonitis Equine Vulvo0.2 vaginitis -Log 10 of virus being neutralized by 1:10 dilution of the corresponding antiserum bNot tested
462
0.2
-
3.5
Can. J. comp. Med.
narrow halos (Fig. 3). Most of the inclu'fi ; ::: -vsionsin the skunks inoculated intraceres, X q * \ brally, were grey, homogeneous and filled i;|; >4 0 :the nuclei (Fig. 4). One intracerebrally ::w *s!5eXiF i , X inoculated skunk had foci of necrosis and :^ 9 g. w^ :t!; intranuclear inclusions in the adrenal cor: : :g ;! tex, indicating dissemination via the blood -^5% p#! "zW J: stream from the inoculation site. Both of
the intravenously inoculated skunks had slight perivascular cuffing in the meninges and brain
parenchyma. . 0 Xw iar*%Nd; . sov C a0T(killed on day 21), had an area of spongiosis spinal ~vical em ~ ~
~
k. ~~?
*h41|0 Z0;it*s0t
*
jti
0 ?
One of these skunks
in the lateral funiculus of the cercord. There was accumulation of macrophages and a few gemistocytes this area. ~~~~~~in Many foci of necrosis were scattered in the liver of the intravenously inoculated skunk that died on day 7 (Fig. 5). These foci contained central areas of coagulation necrosis surrounded by congestion and hemorrhage. Hepatocytes in the periphery contained intranuclear inclusions (Fig. 6).
m~~~~~~~~~~~~~~~~~~~~~~~~~~~~.
zrr; k--m
Fig. 2. Herpetic encephalitis in spontaneously infected
skunk, Vascular cuff in the cerebral cortex. H & E. X290.
hemorrhage in the parenchyma and periadrenal tissue. No gross lesions were detected in any of the other inoculated an skunks. Fig 3 Herpetic encephalitis in spontaneously infected
skunkt Eosinophilc intranuclear inclusion
in neuron of
cerebral cortex H & E X275
MICROscopic LESIONS
There was a. necrotizing meningoence-P ie phalitis in the brain of the original sub- c7
mission and in both skunks inoculated in-
tracerebrally. Lesions occurred mainly in the grey matter of the cerebral cortex and to a lesser extent in the brain stem. There * were scattered regions of perivascular cuf-....... fing with lymphocytes, macrophages, and p a few neutrophils (Fig. 2), neuronal degeneration, intranuclear inclusions in neurons and glial cells, and moderatep parenchymal accumulation of lymphocytes, neutrophils and macrophages. Intranuclear inclusions in brain of the original submission wre re and
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'Fig. 4. Herpetic encephalitis in skunk inoculated intracerebrally with wild skunk isolate. Intranuclear Inclusions in neurons and glial cells. Most of the imclusons are grey, homogeneous and fill the nuclei. H. &
463
There was extensive hemorrhage and necrosis in the adrenal cortex of this skunk. The other intravenously inoculated skunk (killed on day 21) had extensive areas of necrosis, mineralization and mononuclear cell accumulation in the adrenal cortex (Fig. 7), and there were regional accumulations of macrophages and lymphocytes in the propria of the turbinates. No inclusions were detected.
j *
Fig. 7. Adrenal gland of skunk inoculated intravenously and killed on day 21. Extensive accumulations of mononuclear cells, mineralization and necrosis in the adrenal cortex. H. & E. X18.
Fig. 5. Liver of skunk inoculated intravenously with wild skunk isolate. Focal necrosis with peripheral zone of congestion and hemorrhage. H. & E. X60.
Fig. 6. Liver of skunk inoculated intravenously with wild skunk isolate. Intranuclear inclusions in hepatocytes near a focus of necrosis. H. & E. X410.
464
DISCUSSION A previous report indicated that Herpes simplex virus was isolated from eight pet skunks in California during the period 1957 to 1966 (6). It was suggested that the eight cases were examples of an anthropozoonosis, the agent apparently being transmitted naturally from human beings to vertebrate hosts of another species. As far as could be determined, the case presented here is the first reported isolation of Herpes simplex from a wild skunk. This skunk was found in an urban area, indicating that human beings may have been the source of infection. We have no
Can. J. comp. Med.
evidence of Herpes simplex infection in of sporadic infection of skunks with this other wild skunks in this, or other areas. agent. The importance of the disease in In tissue culture, the agent described in skunks is due to the fact that the neurothe present report produced CPE similar logical signs may resemble rabies. Since to other herpesviruses, and electron micros- the skunk is an important vector of rabies, copically, the morphology was typical of examination for rabies is justified on the herpesviruses. The results of the cross finding of any skunks with clinical neuroneutralization tests demonstrated that the logical disease. agent was indistinguishable from Herpes simplex, since significant cross reactions occurred with the skunk isolate and a REFERENCES known Herpes simplex, and not with any 1. BAGUST, T. J. and L. CLARK. Pathogenesis of of the other antisera tested. meningoencephalitis produced in calves by infectious Intracerebral inoculation of skunks rebovine rhinotracheitis herpesvirus. J. comp. Path. 82: 376-383. 1972. sulted in severe necrotizing meningoence- 2. BLACKWOOD, W., W. H. McMENEMEY, A. phalitis with occurrence of many type B MEYER, R. M. NORMAN and D. S. RUSSELL. Greenfield's Neuropathology. 2nd Edition. pp. 205inclusions in neurons and glial cells. Simi206. Baltimore: Williams and Wilkins Co. 1967. CHARLTON, K. M., D. MITCHELL, A. GIRARD larly, several other herpesvirus encephali- 3. and A. H. CORNER. Meningoencephalomyelitis in tides frequently are characterized by dehorses associated with equine herpesvirus 1 infection. Vet. Path. 13: 59-68. 1976. struction of neural elements and intranu- 4. CHEVILLE, N. F. Cytopathology in Viral Diseases. Monographs in Virology. Basel: S. Karger. clear inclusion bodies. These include: infec1975. tious bovine rhinotracheitis virus, pseudo- 5. CORNER, A. H. Pathology of experimental Aujeszky's disease in piglets. Res. vet. Sci. 6: 337-343. rabies virus and Herpes simplex in man 1965. 6. EMMONS, R. W. and E. H. LENNETTE. Isolation (1, 2, 5). of herpesvirus hominis from naturally infected pet In herpes simplex encephalitis in man, skunks. Hlth Lab. Sci. 5: 31-37. r968. 7. EMMONS, R. W. and E. H. LENNETTE. Natural there may be large areas of necrosis of the Herpesvirus hominis infection of a gibbon (Hylobates lar). Arch. ges. Virusforsch. 31: 215-218. 1970. cerebral cortex and intranuclear type A 8. GREIG, A. S., G. L. BANNISTER, D. MITCHELL inclusions in neurons and oligodendroglia and A. H. CORNER. Studies on pathogenic porcine enterovisuses II. Isolation of virus in tissue culture (2). Spontaneous encephalitis caused by from the brain and feces of clinical cases. Can. J. Herpes simplex has also been reported in comp. Med. 25: 142-160. 1961. 9. HUNT, R. D. and L. V. MELENDEZ. Herpesvirus the owl monkey and gibbon (10, 16). The infections of non-human primates. A review. Lab. Care 19: 221-234. 1969. naturally infected skunk, described here- 10. Anim. MELENDEZ, L. V., C. ESPANA, R. D. HUNT, M. in, had many type A intranuclear incluD. DANIEL and F. G. GARCIA. Natural herpes simplex infection in the owl monkey (Aotus trivirsions whereas type B inclusions were more gatus). Lab. Anim. Care 19: 38-45. 1969. NAHMIAS, A. J. Herpesviruses from fish to man common in the experimentally inoculated 11. A search for pathobiologic unity. In Pathobiology skunks. This is probably related to the Annual. H. loachim, Editor. pp. lB3-182. New York: Appleton. duration of the lesion as has been reported 12. PERCY, D.1972. H., L. E. CARMICHAEL, D. M. ALBERT, J. M. KING and A. M. JONAS. Lesions previously in Aujeszky's disease in piglets in puppies surviving infection with canine herpes(5) and in Herpes simplex infection in virus. Vet. Path. 8: 37-53. 1971. 13. PERCY, D. H., J. F. MUNNEL, H. J. OLANDER owl monkeys (10). and L. E. CARMICHAEL. Pathogenesis of canine The lesions in the liver and adrenal herpesvirus encephalitis. Am. J. vet. Res. 31: 145156. 1970. glands consisted mainly of focal necrosis 14. REED, L. J. and H. MUENCH. A simple method of estimating fifty per cent end points. Am. J. Hyg. with intranuclear inclusions. Similar le27: 493497. 1938. sions occur in fetal and newborn cattle and 15. SMITH, H. A., T. C. JONES and R. D. HUNT. Veterinary Pathology. 4th Edition. pp. 380-391. horses infected with infectious bovine Philadelphia: Lea and Febiger. 1972. P. C., T. M. YUILL, R. D. BUCHANAN, rhinotracheitis virus and equine rhino- 16. J.SMITH, S. STANTON and V. CHAICUMPA. The Gibbon pneumonitis virus, respectively, in owl (Hylobates lar). A new primate host for Herpesvirus hominis 1. A natural epizootic in a laboratory monkeys with infection due to Herpes simcolony. J. infect. Dis. 120: 292-297. 1969. plex and in piglets with Aujeszky's disease. It has been observed frequently that in most animal species infection with its ADDENDUM own herpesvirus only infrequently results in severe disease, and that natural infecSince this paper was written, the followtion of another species almost always results in severe fatal disease (11). Thus it ing article on isolation of a human herpesis probable that infection of skunks with virus from a pet skunk was published: Herpes simplex is not widespread. In any KANITZ, C. L. and M. E. WOODRUFF. A herpesvirus isolated from a skunk: serologic relationship to other event, the increasing density of humans viruses of the herpes group. Proc. Am. Ass. vet. lab. in some rural areas increases the likelihood Diagn. pp. 1-12. 1976.
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INTRODUCTION
Herpes simplex virus was isolated from the brain of a wild skunk (Mephitis mephitis) which had clinical neurological disease. In the brain of this skunk and in brain of skunks inoculated intracerebrally, there were areas of necrosis in the grey matter of the cerebral cortex and to a lesser extent in the brain stem. Intranuclear inclusions occurred in neurons and glial cells with vascular cuffing by mononuclear cells and a few neutrophils. Skunks inoculated intravenously had extensive focal necrosis in the liver and adrenal glands.
Neurological diseases caused by herpesviruses occur sporadically in several species including man, cattle, horses, dogs, pigs and monkeys (1, 2, 3, 5, 9, 11, 12, 13, 15). Infection of adults by a herpesvirus of their own species usually does not result in severe clinical disease. However, encephalitis or severe disseminated disease may occur in neonatal or young animals through infection with the same virus (4, 11). Herpesvirus encephalitis in adults most frequently is caused by a virus acquired from another species (11). In most species, except equine (3), lesions in the brains of affected animals consist of areas of necrosis of neurons and glial cells, intranuclear inclusions, and perivascular and parenchymal accumulations of inflammatory cells. Spontaneous encephalitis, caused by Herpes simplex, occurs in man, owl monkeys (Aotus trivirgatu.s), gibbons (Hylobates lar), lemurs and skunks (2, 6, 7, 10, 11, 15). A previous report of the disease in skunks indicated that all the isolates were from pet skunks - suggesting that infection occurred through close contact with man (6). This is a report of isolation of Herpes simplex from a wild skunk with encephalitis, and subsequent experimental inoculation of skunks with this agent.
RESUME Les auteurs ont isole le virus herpes simplex du cerveau d'une mouffette sauvage (Mephitis mephitis) qui avait manifeste des signes nerveux. Ils decelerent des foyers de necrose au sein du cortex cerebral et, a un degre moindre, dans la partie posterieure du cerveau, chez cette mouffette et chez celles qu'ils avaient inoculees par la voie intracerebrale. Des neurones et des cellules de la nevroglie contenaient des inclusions intranucleaires; ils noterent aussi la presence de manchons peri-vasculaires, composes de cellules mononucleaires et de quelques neutrophiles. Le foie et les surrenales des mouffettes qu'ils inoculerent par la voie intra-veineuse presentaient des foyers de necrose extensifs.
MATERIALS AND METHODS *Animal Pathology Division, Health of Animab Branch, Agriculture Canada, Animal Diseases Research Institute (B), P.O. Box 11300, Station H, Ottawa, Ontario K2H 8P9 (Charlton, Dulac, Thomas) and Contagious Diseases Division, Health of Animals Branch, P.O. Box 968, Sault Ste. Marie, Ontario P6A 5N5 (Mitchell). Submitted December 16, 1976.
460
A wild skunk, found in a private garage in Sault Ste. Marie, Ontario was described as being lethargic, had tremors, nodding of the head and profuse salivation. The animal was killed and the brain was
Can. J. comp. Med.
submitted to the Animal Diseases Research Institute (Eastern) for rabies diagnosis. Following a negative fluorescent antibody test for rabies, histological and virological studies were undertaken. VIRUS ISOLATION AND IDENTIFICATION
The initial isolation was made in primary pig kidney (PK) cells inoculated with bacteria-free supernatant fluids from a 10% brain suspension in phosphate buffered saline, pH 7.2, centrifuged at 600 g for ten min. In order to increase the titer, the virus was passaged three times in fresh PK cells and a fourth passage having a titer of 104`TCID50/0.1 ml was used in these experiments. Infective cell culture fluids were stained with 2% phosphotungstic acid, pH 7.2, and examined with an Hitachi HU 12 A electron microscope. The skunk agent was positively identified by the serum neutralization (SN) test according to the constant serum (1:10 dilution) - variable virus method, using four tubes per dilution. The neutralization indices were estimated by the method of Reed and Muench (14). SKUNK INOCULATION
Four mature male skunks, purchased from a supplier' were anesthetized with Ketaset2 administered intramusculary at a dosage of 6 mg per pound. Two skunks were inoculated intracerebrally with 10` TCID50 of virus and two were given a similar dose intravenously. They were kept in individual Horsfall cages and observed daily. Skunks which died or were killed were necropsied. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6,u and stained with hematoxylin and eosin. Fresh tissues were used for reisolation of virus as described above.
isolate were obtained from the virus collection of our Institute. The canine herpesvirus was obtained as a seventh dog kidney cell passage from Dr. J. Thorsen.' One additional passage in Madin Darby-canine kidney (MDCK) cell line4 was done in our laboratory. The feline rhinotracheitis virus was a vaccine strain5 passaged once in primary feline kidney cells. Each virus, grown in respective susceptible cells was harvested after two cycles of rapid freezing and thawing. The cell debris was further disintegrated by sonic vibration and filtered through a 0.22 ,u Millipore filter.' The stocks were stored frozen at -70°C in mechanical freezers. IMMUNE SERUMS
Immune serum against the skunk isolate was prepared in two adult male skunks. The animals were immunized by a series of four intramuscular injections given one week apart. Each of the first series of two injections consisted of 1 ml of unconcentrated virulent fluid. One ml of virulent fluid, concentrated 100 times by ultracentrifugation was used for the last two injections. The skunks were bled ten days after the last inoculation and the serum was stored at -20°C until used. Herpes simplex antiserum was obtained from the Canadian Communicable Disease Center in Ottawa; the canine herpes antiserum was supplied by Dr. J. Thorsen and the feline rhinotracheitis antiserum was provided by Dr. D. L. Croghan.7 Other antiserums were from the collection of
this Institute.
RESU LTS VIRUS ISOLATION AND CROSS NEUTRALIZATION TESTS
Initially, the cytopathic effect (CPE) in PK cells was detected at five days post-
VIRUSES
Most viruses used to identify the skunk 'Ruby's Fur Farm, New Sharon, Iowa 50207. 2Ketaset, ketamine hydrochloride, Rogar/STB, London, Ontario.
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3University of Guelph, Guelph, Ontario. 4American type culture collection, 12301 Parklawn Drive, RockviUe, Maryland 20852. 5Pitman-Moore Inc., Washington Crossing, N.J. 08560. 6Millipore Corporation, Bedford, Mass. 01730. 7Veterinary Services Laboratory, Animal and Plant Health Inspection, U.S. Dept. of Agriculture, P.O. Box 70, Ames, Iowa 50070.
461
inoculation. The first lesions appeared as foci of round and refractile cells that remained attached to the glass. Upon subsequent passages, the CPE occurred as early as 24 hours postinoculation and destroyed the monolayers within five days. Inclusion bodies could not be demonstrated in PK cells stained with hematoxylin and eosin or May-Grunwald-Giemsa stains. Electron microscopic examination of infective cell culture fluids revealed numerous herpesvirus particles. The skunk isolate was neutralized by a Herpes simplex antiserum and the Herpes simplex virus was neutralized by the antiserum prepared against the skunk virus. Furthermore, there were no significant cross reactions with any of the other herpesviruses tested (Table I).
against 100 TCID50 of the skunk virus when tested in PK cells. The virus was reisolated from the liver, spleen, kidney and brain of the two skunks inoculated intracerebrally and from the same tissues of the skunk inoculated intravenously and that died on day 7. The virus could not be recovered from the skunk killed on day 21. GROSS LESIONS
The skunk which died following intravenous inoculation, had many small white foci with encircling red zones on the serosal and cut surfaces of the liver (Fig. 1). The adrenal glands were swollen and had
CLINICAL SIGNS AND REISOLATION OF VIRUS IN EXPERIMENTALLY INOCULATED SKUNKS
Both skunks inoculated intracerebrally were lethargic on day 3 and died on day 4. No other clinical signs were observed. One of the skunks inoculated intravenously died on day 7, following a three day illness characterized by anorexia, lethargy and serous nasal exudate. The other intravenously inoculated skunk had oculonasal exudate on day 5 but recovered and subsequently was killed on day 21. This skunk had a serum neutralization titer of 1:320
Fig. 1. Lobe of liver from skunk inoculated intravenously with wild skunk isolate. Many scattered greywhite foci surrounded by narrow red zones.
TABLE I. Results of Cross-neutralization Tests in Cell Culture with the Viral Isolate from a Wild Skunk and Herpesviruses from Other Species Immune Sera Prepared Against
Virus
Bovine RhinoIsolate Canine trafrom Herpes Wild simplex Pseudorabies Herpes cheitis Skunk Virus Virus Virus Virus
Feline Equine Equine Rhino- RhinoVulvotrapneuvagicheitis monitis nitis Virus Virus Virus
Isolate from wild 0.4 0.1 0.1 0.1 0.5 3.7 skunk 3.2a 4.2 4.3 Herpes simplex b 7.2 Pseudorabies 0.3 4.6 Canine Herpes 0.4 Infectious Bovine 3.0 0.2 Rhinotracheitis Feline Rhinotra5. 0 cheitis Equine Rhino3.7 0.2 pneumonitis Equine Vulvo0.2 vaginitis -Log 10 of virus being neutralized by 1:10 dilution of the corresponding antiserum bNot tested
462
0.2
-
3.5
Can. J. comp. Med.
narrow halos (Fig. 3). Most of the inclu'fi ; ::: -vsionsin the skunks inoculated intraceres, X q * \ brally, were grey, homogeneous and filled i;|; >4 0 :the nuclei (Fig. 4). One intracerebrally ::w *s!5eXiF i , X inoculated skunk had foci of necrosis and :^ 9 g. w^ :t!; intranuclear inclusions in the adrenal cor: : :g ;! tex, indicating dissemination via the blood -^5% p#! "zW J: stream from the inoculation site. Both of
the intravenously inoculated skunks had slight perivascular cuffing in the meninges and brain
parenchyma. . 0 Xw iar*%Nd; . sov C a0T(killed on day 21), had an area of spongiosis spinal ~vical em ~ ~
~
k. ~~?
*h41|0 Z0;it*s0t
*
jti
0 ?
One of these skunks
in the lateral funiculus of the cercord. There was accumulation of macrophages and a few gemistocytes this area. ~~~~~~in Many foci of necrosis were scattered in the liver of the intravenously inoculated skunk that died on day 7 (Fig. 5). These foci contained central areas of coagulation necrosis surrounded by congestion and hemorrhage. Hepatocytes in the periphery contained intranuclear inclusions (Fig. 6).
m~~~~~~~~~~~~~~~~~~~~~~~~~~~~.
zrr; k--m
Fig. 2. Herpetic encephalitis in spontaneously infected
skunk, Vascular cuff in the cerebral cortex. H & E. X290.
hemorrhage in the parenchyma and periadrenal tissue. No gross lesions were detected in any of the other inoculated an skunks. Fig 3 Herpetic encephalitis in spontaneously infected
skunkt Eosinophilc intranuclear inclusion
in neuron of
cerebral cortex H & E X275
MICROscopic LESIONS
There was a. necrotizing meningoence-P ie phalitis in the brain of the original sub- c7
mission and in both skunks inoculated in-
tracerebrally. Lesions occurred mainly in the grey matter of the cerebral cortex and to a lesser extent in the brain stem. There * were scattered regions of perivascular cuf-....... fing with lymphocytes, macrophages, and p a few neutrophils (Fig. 2), neuronal degeneration, intranuclear inclusions in neurons and glial cells, and moderatep parenchymal accumulation of lymphocytes, neutrophils and macrophages. Intranuclear inclusions in brain of the original submission wre re and
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'Fig. 4. Herpetic encephalitis in skunk inoculated intracerebrally with wild skunk isolate. Intranuclear Inclusions in neurons and glial cells. Most of the imclusons are grey, homogeneous and fill the nuclei. H. &
463
There was extensive hemorrhage and necrosis in the adrenal cortex of this skunk. The other intravenously inoculated skunk (killed on day 21) had extensive areas of necrosis, mineralization and mononuclear cell accumulation in the adrenal cortex (Fig. 7), and there were regional accumulations of macrophages and lymphocytes in the propria of the turbinates. No inclusions were detected.
j *
Fig. 7. Adrenal gland of skunk inoculated intravenously and killed on day 21. Extensive accumulations of mononuclear cells, mineralization and necrosis in the adrenal cortex. H. & E. X18.
Fig. 5. Liver of skunk inoculated intravenously with wild skunk isolate. Focal necrosis with peripheral zone of congestion and hemorrhage. H. & E. X60.
Fig. 6. Liver of skunk inoculated intravenously with wild skunk isolate. Intranuclear inclusions in hepatocytes near a focus of necrosis. H. & E. X410.
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DISCUSSION A previous report indicated that Herpes simplex virus was isolated from eight pet skunks in California during the period 1957 to 1966 (6). It was suggested that the eight cases were examples of an anthropozoonosis, the agent apparently being transmitted naturally from human beings to vertebrate hosts of another species. As far as could be determined, the case presented here is the first reported isolation of Herpes simplex from a wild skunk. This skunk was found in an urban area, indicating that human beings may have been the source of infection. We have no
Can. J. comp. Med.
evidence of Herpes simplex infection in of sporadic infection of skunks with this other wild skunks in this, or other areas. agent. The importance of the disease in In tissue culture, the agent described in skunks is due to the fact that the neurothe present report produced CPE similar logical signs may resemble rabies. Since to other herpesviruses, and electron micros- the skunk is an important vector of rabies, copically, the morphology was typical of examination for rabies is justified on the herpesviruses. The results of the cross finding of any skunks with clinical neuroneutralization tests demonstrated that the logical disease. agent was indistinguishable from Herpes simplex, since significant cross reactions occurred with the skunk isolate and a REFERENCES known Herpes simplex, and not with any 1. BAGUST, T. J. and L. CLARK. Pathogenesis of of the other antisera tested. meningoencephalitis produced in calves by infectious Intracerebral inoculation of skunks rebovine rhinotracheitis herpesvirus. J. comp. Path. 82: 376-383. 1972. sulted in severe necrotizing meningoence- 2. BLACKWOOD, W., W. H. McMENEMEY, A. phalitis with occurrence of many type B MEYER, R. M. NORMAN and D. S. RUSSELL. Greenfield's Neuropathology. 2nd Edition. pp. 205inclusions in neurons and glial cells. Simi206. Baltimore: Williams and Wilkins Co. 1967. CHARLTON, K. M., D. MITCHELL, A. GIRARD larly, several other herpesvirus encephali- 3. and A. H. CORNER. Meningoencephalomyelitis in tides frequently are characterized by dehorses associated with equine herpesvirus 1 infection. Vet. Path. 13: 59-68. 1976. struction of neural elements and intranu- 4. CHEVILLE, N. F. Cytopathology in Viral Diseases. Monographs in Virology. Basel: S. Karger. clear inclusion bodies. These include: infec1975. tious bovine rhinotracheitis virus, pseudo- 5. CORNER, A. H. Pathology of experimental Aujeszky's disease in piglets. Res. vet. Sci. 6: 337-343. rabies virus and Herpes simplex in man 1965. 6. EMMONS, R. W. and E. H. LENNETTE. Isolation (1, 2, 5). of herpesvirus hominis from naturally infected pet In herpes simplex encephalitis in man, skunks. Hlth Lab. Sci. 5: 31-37. r968. 7. EMMONS, R. W. and E. H. LENNETTE. Natural there may be large areas of necrosis of the Herpesvirus hominis infection of a gibbon (Hylobates lar). Arch. ges. Virusforsch. 31: 215-218. 1970. cerebral cortex and intranuclear type A 8. GREIG, A. S., G. L. BANNISTER, D. MITCHELL inclusions in neurons and oligodendroglia and A. H. CORNER. Studies on pathogenic porcine enterovisuses II. Isolation of virus in tissue culture (2). Spontaneous encephalitis caused by from the brain and feces of clinical cases. Can. J. Herpes simplex has also been reported in comp. Med. 25: 142-160. 1961. 9. HUNT, R. D. and L. V. MELENDEZ. Herpesvirus the owl monkey and gibbon (10, 16). The infections of non-human primates. A review. Lab. Care 19: 221-234. 1969. naturally infected skunk, described here- 10. Anim. MELENDEZ, L. V., C. ESPANA, R. D. HUNT, M. in, had many type A intranuclear incluD. DANIEL and F. G. GARCIA. Natural herpes simplex infection in the owl monkey (Aotus trivirsions whereas type B inclusions were more gatus). Lab. Anim. Care 19: 38-45. 1969. NAHMIAS, A. J. Herpesviruses from fish to man common in the experimentally inoculated 11. A search for pathobiologic unity. In Pathobiology skunks. This is probably related to the Annual. H. loachim, Editor. pp. lB3-182. New York: Appleton. duration of the lesion as has been reported 12. PERCY, D.1972. H., L. E. CARMICHAEL, D. M. ALBERT, J. M. KING and A. M. JONAS. Lesions previously in Aujeszky's disease in piglets in puppies surviving infection with canine herpes(5) and in Herpes simplex infection in virus. Vet. Path. 8: 37-53. 1971. 13. PERCY, D. H., J. F. MUNNEL, H. J. OLANDER owl monkeys (10). and L. E. CARMICHAEL. Pathogenesis of canine The lesions in the liver and adrenal herpesvirus encephalitis. Am. J. vet. Res. 31: 145156. 1970. glands consisted mainly of focal necrosis 14. REED, L. J. and H. MUENCH. A simple method of estimating fifty per cent end points. Am. J. Hyg. with intranuclear inclusions. Similar le27: 493497. 1938. sions occur in fetal and newborn cattle and 15. SMITH, H. A., T. C. JONES and R. D. HUNT. Veterinary Pathology. 4th Edition. pp. 380-391. horses infected with infectious bovine Philadelphia: Lea and Febiger. 1972. P. C., T. M. YUILL, R. D. BUCHANAN, rhinotracheitis virus and equine rhino- 16. J.SMITH, S. STANTON and V. CHAICUMPA. The Gibbon pneumonitis virus, respectively, in owl (Hylobates lar). A new primate host for Herpesvirus hominis 1. A natural epizootic in a laboratory monkeys with infection due to Herpes simcolony. J. infect. Dis. 120: 292-297. 1969. plex and in piglets with Aujeszky's disease. It has been observed frequently that in most animal species infection with its ADDENDUM own herpesvirus only infrequently results in severe disease, and that natural infecSince this paper was written, the followtion of another species almost always results in severe fatal disease (11). Thus it ing article on isolation of a human herpesis probable that infection of skunks with virus from a pet skunk was published: Herpes simplex is not widespread. In any KANITZ, C. L. and M. E. WOODRUFF. A herpesvirus isolated from a skunk: serologic relationship to other event, the increasing density of humans viruses of the herpes group. Proc. Am. Ass. vet. lab. in some rural areas increases the likelihood Diagn. pp. 1-12. 1976.
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