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The striped skunk (Mephitis mephitis) is a host of rabies in large areas of Canada and the United States. In each of two experiments, equal numbers of skunks in two groups were inoculated intramuscularly with low doses of a field strain of rabies virus (street rabies virus). In each experiment, skunks in one group surviving to 2 months were killed at this time and selected tissues were used for examination by the polymerase chain reaction (PCR) method or by immunohistochemistry for rabies antigen. Results of detailed examinations using PCR technology (experiment 1) indicated that muscle at the inoculation site contained viral RNA at 2 months postinoculation, when other relevant tissues on the route of viral migration and early entrance into the central nervous system were negative. The cellular location of virus/antigen, as determined immunohistochemically in experiment 2, was striated muscle fibers and fibrocytes. Our results indicate a major role of muscle (tissue) infection at the inoculation site in the long incubation period of rabies in skunks. These and related findings will be useful in rabies control and, if applicable to other species, will be relevant in postexposure treatment.

Twenty-four striped skunks were inoculated intramuscularly (long digital extensor muscle of right pelvic limb) with street rabies virus. Groups of two clinically normal skunks were killed at various times after inoculation; skunks that developed rabies were killed in early stages of the clinical signs. Four clinically normal skunks (numbered 1-4) had slight infection in lumbar spinal ganglia, spinal cord and brain. These four skunks were used for detailed immunohistochemical (rabies antigen) studies that included examination of sections from every segment of the spinal cord, most of the spinal ganglia from the 2nd cervical to the 2nd coccygeal (sections at 25-microns intervals of lumbar, sacral and coccygeal ganglia) and brain (sections at 50-micron intervals). In skunks 1-4, there was increasing distribution of antigen-containing neurons that was not correlated with the time elapsed since inoculation. In three skunks (nos. 1, 2 and 3), antigen-containing neurons were predominantly in caudal regions of the spinal cord, caudal right lumbar and sacral spinal ganglia and certain nuclei/regions of the brain (medial reticular formation, right interpositus and lateral vestibular nuclei, left red nucleus, left motor cortex, and left reticular nucleus of the thalamus). Skunk 4 had more extensive infection than skunks 1-3, but the previous pattern was still evident. The results are consistent with viral entrance into the lumbar spinal cord, initial replication mainly at the L2 and L3 levels, local spread in the cord by propriospinal neurons and early transit to the brain via long ascending and descending fiber tracts (bypassing the grey matter of the rostral spinal cord). These mechanisms could provide for early and rapid dissemination in the brain before a significant immune response develops and could induce behavioral changes before the animal is incapacitated by extensive spinal cord infection. Based on the distribution of antigen-containing neurons, the tracts considered most likely to serve as viral transitways from spinal cord to brain include: rubrospinal, corticospinal, spinothalamic, spino-olivary, vestibulospinal and/or spinovestibular, reticulospinal and/or spinoreticular, cerebellospinal and/or spinocerebellar, and dorsal column pathways.

The proposal that the bizarre behavioral changes which occur during rabies infection are due to selective infection of limbic system neurons was further studied in skunks (a species important in naturally occurring disease). A detailed immunohistochemical study of brains of skunks experimentally infected with either Challenge virus standard (CVS) or street rabies virus revealed only trace amounts of viral antigen in many limbic system neurons and marked differences in viral distribution between street and CVS virus. These data were collected during early stage rabies when behavioral changes occur. Areas which contained heavy accumulations of street rabies virus but low amounts of CVS rabies virus were the neuronal perikarya and processes of the dorsal motor nucleus of the vagus, midbrain raphe, hypoglossal and red nuclei. In contrast, large accumulations of CVS virus were found in the Purkinje cells of the cerebellum, the habenular nuclei and in pyramidal cells throughout the cerebral cortex, while corresponding areas in all street virus-infected skunks contained minimal antigen. These findings were very consistent for animals of the same experimental group and between skunks inoculated both intramuscularly and intranasally with skunk street virus. Skunks inoculated intramuscularly with CVS rabies virus failed to develop rabies. Since, in this model, street virus infection generally produces furious rabies and CVS infection results in dumb rabies, we speculate that the behavioral changes which occur in these two different clinical syndromes are due to the heavy and specific accumulation of virus in different regions of the CNS. These results show that regions other than those of the limbic system may also be involved in the pathogenesis of behavior changes in rabid animals.

In studies to develop an oral rabies vaccine for wildlife, the immune response to and pathogenicity of two types of mutants of rabies viruses were examined. Forty-five small plaque mutants were selected from cultures of ERA rabies virus treated with 8-azaguanine or 5-fluorouracil and tested for pathogenicity in mice. Two of these mutants AZA 1 and AZA 2 (low pathogenicity in mice) were given to skunks by oral (bait), intestinal (endoscope) and intramuscular routes. Additionally, challenge virus standard (CVS) rabies virus and mutants of this and ERA rabies virus (CVS 3766 and 3713, and ERA 3629) that were resistant to neutralization by specific antiglycoprotein monoclonal antibodies (and apathogenic in mice) were tested by various routes in skunks. Skunks given AZA 1 and AZA 2 were challenged at three months postinoculation with street rabies virus. After oral administration, there were very low rates of seroconversion with AZA 1 and AZA 2 and on challenge only 2/7 given AZA 1 and 1/8 given AZA 2 survived. None of the skunks given the other mutants orally seroconverted. AZA 2 produced a high rate of seroconversion (8/8) by the intestinal route and all challenged skunks in this group survived (7/7). All skunks vaccinated intramuscularly with AZA 1 (4/4) or AZA 2 (4/4) developed high levels of rabies neutralizing antibodies and survived challenge. The mutant CVS 3766, while apathogenic when given intracerebrally to adult mice, was consistently pathogenic by this route (and intranasally) in skunks. These results demonstrate that skunks are highly resistant to oral immunization by live rabies virus vaccines and that pathogenicity (by intracerebral route) of the mutant CVS 3766 is markedly different in mice and skunks.

Murine neuroblastoma (NA-C1300) and baby hamster kidney (BHK-21/C13) cell cultures were infected with the Canadian Arctic strain of rabies virus. Subcultures were passed following incubation for 3 to 4 days at 35 degrees C. The supernatant fluids from the BHK cultures demonstrated increasing infectivity in both NA and BHK cells concomitantly with an increase in the number of parent cells staining with an anti-glycoprotein stain. On the other hand, the supernatant fluids from the NA cultures initially showed higher infectivity in NA cells than in BHK cells. This feature was related to a low production of glycoprotein-staining cells in the parent NA cultures. The reduction of infectivity in NA cells of some NA supernatant fluids (and brain suspensions) by anti-nucleoprotein antibodies suggests that nucleocapsid material is, in some manner, capable of infecting NA cells. Infectivity of this virus strain in experimental mice is also related to the production of glycoprotein and may not be correlated with the degree of infection in NA cell cultures.

The authors studied nineteen street virus isolates from different regions of Canada using either anti-nucleocapsid and anti-glycoprotein monoclonal antibodies or cross-protection tests. This study only allowed us to recognize three groups of viruses with different nucleocapsid patterns, and no difference, as far as protection with a Pitmann-Moore vaccine is concerned, between four of these strains.

Striped skunks (Mephitis mephitis) were inoculated into the denervated abductor digiti quinti muscle with street rabies virus. They were killed at various times after inoculation and several tissues were examined by immunofluorescence and light microscopy. Muscle at the inoculation site was examined electron microscopically. Rabies antigen was detected in muscle fibers first on day 7 and persisted until day 28. Light and electron microscopic lesions at the inoculation site included atrophic and degenerating muscle fibers and a few focal and regional endomysial accumulations of macrophages, lymphocytes and plasma cells. Scattered myocytes contained bodies of matrix, virions and anomalous tubular structures on electron microscopic examination. The results indicate that replication of rabies virus may occur in infected muscle fibers at the inoculation site until 28 days after exposure. This could contribute to variations in the incubation period for the first two to three months after exposure. However, the results do not support the contention that virus is contained in striated muscle cells throughout the long incubation periods.

A total of 123 rabies virus isolates from various geographical areas in Canada were characterized by a panel of 43 anti-nucleocapsid monoclonal antibodies. Four major antigenic groups are found in terrestrial mammals: "Canadian Arctic" from Ontario, Quebec and the Northwest Territories; "south-eastern Georgian Bay" from Ontario; "south mid-central skunk" from Alberta, Saskatchewan and Manitoba; and "Brook's, Alberta skunk" from a restricted area in Alberta. Bat isolates can be divided into 4 major antigenic groups: "B-1" in Eptesicus fuscus from Ontario; "B-2" in a variety of bat species from British Columbia eastward into Ontario; "B-3" in Myotis spp. from Ontario and New Brunswick; and "B-4" in E. fuscus from Alberta and Saskatchewan. A single case of bat to horse transmission of rabies virus is recorded. These street isolates are compared with isolates of fixed virus. Epidemiological aspects are discussed.

Several salivary glands and the nasal mucosa of rabid skunks (Mephitis mephitis) contained rabies virus. Generally titers were high in the submandibular, moderate in the parotid and low to moderate in the zygomatic, molar and sublingual salivary glands. The nasal mucosa (glands and epithelium) contained virus at low to moderate titers that occasionally were equal to titers in brain.

Striped skunks (Mephitis mephitis) were inoculated with street rabies virus and immunosuppressed with several doses of cyclophosphamide. Control skunks were inoculated with street virus only. The skunks were killed in terminal stages of the disease and several tissues were collected for examination by immunofluorescence, light microscopy and viral titration. Sera collected at euthanasia from most of the principals did not contain detectable rabies neutralizing antibodies, whereas high titers occurred terminally in controls. Immunofluorescence was much more entensive in submandibular salivary glands of cyclophosphamide-treated than control skunks. Similarly, virus was isolated from this tissue more consistently and at higher titer from principals than from controls. Immunofluorescence was extensive in brains of all skunks (both groups), but virus was isolated consistently only from brains of cyclophosphamide-treated skunks. Most of the cyclophosphamide-treated skunks had very few inflammatory cells in brain and cerebrospinal ganglia. Neuronal degeneration occurred in dorsal root ganglia of both principals and controls. The results suggest that the immune response has no effect on the development of rabies-induced aggressive behavior, that the immune response may inhibit salivary gland infection and that it is not essential for the development of neuronal degeneration in dorsal root ganglia.

Ninety percent of foxes fed commercial ERA vaccine in a specially designed bait developed rabies serum neutralizing antibodies. The vaccine bait did not cause clinical signs of rabies when consumed by foxes, raccoons, skunks, dogs, cats, cattle and monkeys. When presented, in the laboratory, to wild rodents of the species Microtus, Mus musculus and Peromyscus, the vaccine baits caused vaccine-induced rabies only in Mus musculus. Laboratory mice of the CD-1 and CLL strain were susceptible to vaccine-induced rabies; however, studies showed that transmission of virus to other animals did not occur. These studies suggest that the vaccine bait described could be useful in a rabies control program in areas where foxes and wild dogs are the principal vectors.

Striped skunks (Mephitis mephitis) were vaccinated with a vaccinia virus recombinant expressing the rabies virus glycoprotein. Virus neutralizing antibodies to rabies virus were present at 14 days postvaccination by the following routes: scarification (6/6), intramuscular (4/4) and intestinal (5/8). Six out of seven skunks that ate vaccine filled baits had virus neutralizing antibodies at 28 days. When challenged intramuscularly with street virus, the survival rates were 5/7 for the bait-fed group, 4/8 for the intestinal group, 3/4 for the intramuscular group, 5/6 for the animals that were scarified, and 0/8 for controls. This is the first report of a high rate of immunization of skunks with a rabies vaccine administered orally.

Weanling female white Swiss mice were exposed to challenge virus standard rabies virus and street virus isolates from various domestic and wild animals. Virus was given free choice as suspension or as infected mouse brain by stomach tube, by single injection of suspension into the oral cavity of unanesthetized mice, by repeated injection into the oral cavity of anesthetized mice and by single application to the external nares of anesthetized mice. Challenge virus standard virus in mouse brain suspension and a suspension of skunk salivary glands infected with street virus (titers greater than or equal to 10(6)MICLD50/0.03 ml) consistently produced high rates of infection in mice exposed intranasally, low to high rates of infection in mice exposed by forced feeding and other artificial methods of oral exposure and very low rates of infection when given free choice. Street virus isolates passaged intracerebrally in mice had titers less than or equal to 10(4.5) MICLD50/0.03 ml and rarely caused rabies in mice exposed orally or nasally by any method. The results indicate that with the isolates used, virus of high titer (greater than or equal to 10(6)MICLD50/0.03 ml) is required to consistently produce infection in mice by the nasal route and that the mucosa of the nasal cavity probably is the chief route of infection even after oral administration.

Mice experimentally infected with challenge virus standard rabies virus as well as skunks and foxes experimentally infected with street rabies virus were used to demonstrate rabies viral antigen in paraffin-embedded tissue by the peroxidase-antiperoxidase method. Tissues fixed with different fixatives (10% formalin, Bouin's, acetone, ethanol) for various times and fresh frozen tissues were stained by the fluorescent antibody and the peroxidase-antiperoxidase method. Formalin- and Bouin's-fixed tissues were tested with and without use of digestive enzyme (pepsin). The results demonstrated that a procedure using formalin-fixed paraffin-embedded tissue treated with pepsin and stained by peroxidase-antiperoxidase was the best method for both preservation of morphological details and demonstration of antigen.

The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes.

Street rabies virus isolated from 51 specimens from Ontario, Quebec, Manitoba and the Northwest Territories have been typed by a panel of 36 antinucleocapsid monoclonal antibodies. Three main groups were found. The first group comprised those terrestrial mammals originating in Ontario, Quebec and the Northwest Territories. The second group was found in terrestrial mammals from Manitoba. The third heterogenous group was made up of bats from Ontario.

Striped skunks (Mephitis mephitis) were inoculated into the right submandibular salivary gland with street rabies virus. They were killed at various times after inoculation and several tissues were examined by immunofluorescence and light microscopy. Right and left superior cervical, nodose and trigeminal ganglia, medulla oblongata and at least three regions of right and left submandibular salivary glands were examined by the fluorescent antibody technique. Intracerebral titrations of salivary gland suspensions were made in weanling white Swiss mice. Immunofluorescent material (inoculum) was detected in septa and connective tissue surrounding secretory units of the right submandibular gland immediately after inoculation, but otherwise antigen was not detected in either right or left submandibular glands without coincident antigen in the medulla oblongata. This occurred first on day 12 in areas of the gland remote from the inoculation site. Titers of virus were low at this time. Serum neutralizing antibodies occurred by day 7 in a few skunks. The time of development and distribution of antigen strongly suggest that, even after direct inoculation, neural networks are necessary for development of widespread infection of the salivary gland. The early occurrence of serum neutralizing antibodies in some of the skunks suggests that the immune response was activated by virus in the inoculum since immunofluorescence was not detected in any tissue at this time.

Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus.

Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection. These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells. The mortality period in Swiss white mice caused by the various virus suspensions was noted. The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids. On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures. Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude. However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered. Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus.(ABSTRACT TRUNCATED AT 250 WORDS)