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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.

Non-neural tissues, from three male and four female stripped skunks (Mephitis mephitis), 5 to 7 months old, and one male and two female red foxes (Vulpes vulpes), 12 to 16 months old, experimentally infected with street rabies virus, were examined by light microscopic immunohistochemical and electron microscopic methods. This is the first report of ultrastructural lesions in rabies-infected adrenal medulla, cornea, and nasal glands. Using the streptavidin biotin peroxidase technique, antigen was detected in mucous cells and interstitial neurons and their processes in the submandibular salivary gland, in chromaffin cells of the adrenal medulla, in epidermal cells of the skin, in external root sheath cells of hair follicles, and in corneal epithelial cells. Electron microscopically, matrix (viral nucleocapsid), virions, and anomalous viral products were common in most tissues examined, but their relative proportions varied. The results suggested that replication with minimal accumulation of matrix and anomalous viral growth products was characteristic of growth in tissues (submandibular salivary gland) that frequently produce high titers of virus, whereas replication with large amounts of matrix and anomalous structures occurred in tissues (adrenal gland and nasal gland) that generally contained low or moderate titers of virus. Novel findings included viral budding into secretory granules, increase in microfilaments in infected mucogenic cells, and continuity of viral convoluted membranous profiles with rough endoplasmic reticulum of chromaffin cells and nasal glandular cells. The presence of viral antigen and developing virus in extra-neural tissues constitutes a potential risk of non-bite exposure to people in certain groups/occupations.

An experimental beta-propiolactone (BPL)-inactivated rabies virus vaccine was evaluated for the oral immunization of captive raccoons (Procyon lotor) and red foxes (Vulpes vulpes). None of 10 red foxes administered a single 1.0 ml dose of BPL-inactivated rabies virus vaccine (PM strain; 100 or 500 micrograms protein) per os developed detectable anti-rabies virus-neutralizing antibodies (VNA) at any time over 8 wk of observation. Foxes were excluded from further study. In two different groups of five to six raccoons, each administered a single 1.0 ml dose of BPL-inactivated rabies virus vaccine (ERA strain) per os, at concentrations of 100 or 400 micrograms protein, only a single animal in each group demonstrated evidence of seroconversion within 4 wk. In contrast, instillation of a single dose (500 micrograms protein) of BPL-inactivated rabies virus vaccine (ERA strain), directly into the small intestine via fiberoptic endoscope, or ERA vaccine (800 micrograms protein) instillation to the buccal cavity by needle-less syringe, resulted in the production of rabies-specific VNA and protection against lethal rabies infection in three of six, and in four of six raccoons, respectively; all seven control raccoons succumbed to street virus challenge. These preliminary challenge studies, while somewhat encouraging, demonstrate that considerable quantities of purified viral antigen are required for even minimal oral efficacy against lethal rabies infection. At the present time, therefore, potent, self-replicating, attenuated, or recombinant viruses offer the most versatile, economic, efficacious, and safe solutions to terrestrial rabies control of free-ranging carnivores.

A new recombinant rabies vaccine (human adenovirus 5 containing the rabies glycoprotein gene) was given to striped skunks (Mephitis mephitis) and red foxes (Vulpes vulpes). Groups of skunks received the vaccine in baits, by direct instillation into the mouth, or intramuscularly. Foxes were given vaccine by direct instillation into the oral cavity (DIOC). Selected groups of vaccinated skunks and foxes were challenged with street rabies virus. There were high rates of seroconversion (generally with high antibody titers) in both foxes and skunks, with survival of all challenged vaccinated animals (all challenge controls developed rabies). In skunks, vaccine given DIOC was effective over a broad range of doses (10(8.7), 10(7.6) and 10(6.4) median tissue culture infective doses). There was no evidence of pathogenic effects. The results indicate that this adenovirus recombinant has considerable potential as a wildlife oral rabies vaccine.

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.

Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies.

Three attenuated rabies virus vaccines (SAD-B19, ERA/BHK-21, AZA 2) were compared for efficacy and safety in the striped skunk (Mephitis mephitis) by the oral and intranasal routes. The SAD-B19 and ERA/BHK-21 vaccines were given orally; all three vaccines were given intranasally. Oral administration of SAD-B19 and ERA/BHK-21 vaccines induced neither seroconversion nor significant protection against rabies challenge. One skunk which consumed a SAD-B19 vaccine-laden bait succumbed to vaccine-induced rabies. Intranasal instillation of the three vaccines resulted in the deaths of two of six (AZA 2), three of six (ERA/BHK-21) and six of six (SAD-B19) skunks.

In North America, the number of cases of rabies diagnosed in skunks generally exceeds that in either raccoons or foxes. Enzootic skunk rabies occurs mainly in four geographic regions: (1) southern Ontario and Quebec and upper New York State; (2) the north central United States and the Canadian provinces of Manitoba, Saskatchewan, and Alberta; (3) California; and (4) south central United States (Texas and several adjacent states). Rabies in these areas (in skunks and, to a large extent, in other terrestrial mammals) is caused mainly by three street virus variants, as determined by monoclonal antibody testing (one variant for areas 2 and 3 and separate variants for each of areas 1 and 4). Experimental studies suggest that the species specificity (e.g., raccoon vs. skunk) of enzootic rabies is due, at least partly, to differences in the pathogenicity of variants of rabies virus.

Striped skunks (Mephitis mephitis) experimentally infected with street rabies virus developed spongiform lesions that light- and electron-microscopically were indistinguishable from those found in the transmissible spongiform encephalopathies of man and animals. These previously unreported lesions were also detected in naturally occurring cases of rabies. The spongiform lesions consisted of round or oval vacuoles in the neuropil, rarely in neuronal perikarya. The most severely affected areas were the thalamus and cerebral cortex. The implications of this finding include similarities in the pathogenetic mechanisms of rabies and the traditional spongiform encephalopathies and the possibility of lesion variation due to differences in rabies viral strains. The spongiform lesions of rabies will require consideration in differential diagnosis.

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.

The pathogenesis of rabies spongiform lesions in striped skunks (Mephitis mephitis) and red foxes (Vulpes vulpes) was studied by light and electron microscopy and peroxidase-antiperoxidase immunocytochemistry. Studies in skunks included use of several street virus variants (different antigenic profiles as tested by monoclonal antibodies) different routes of inoculation (intranasal, intracerebral and intramuscular), immunosuppression of infected skunks, different preparations of virus (brain and salivary gland suspensions and infective tissue culture fluids), and sequential development of the lesions. Foxes (Vulpes vulpes) were infected intramuscularly with a street virus isolate. Except for the group of immunosuppressed skunks, all animals that developed clinical signs of rabies had encephalitis characterized by varying degrees of mononuclear perivascular cuffing, focal gliosis, and Negri bodies. Spongiform change occurred in the neuropil of the grey matter (especially thalamus and cerebral cortex) in rabid animals from all groups, but not in controls or exposed animals that did not develop rabies. Rabies antigen (detected by peroxidase-antiperoxidase immunocytochemistry) occurred only in small amounts in many thalami; some vacuolated areas were devoid of antigen. Ultrastructurally, there was a gradation in lesions from small to large membrane-bound vacuoles in cellular processes (mainly dendrites, less frequently axons) and to large tissue spaces containing granular and/or membranous material. These studies indicate that rabies spongiform change occurs in skunks given street virus of several different antigenic profiles and challenge virus standard rabies virus and that the distribution of the lesions has remarkable similarities to those of the traditional spongiform encephalopathies. The occurrence of the lesion is not affected by the immune response, the route of inoculation of virus, the preparation (suspension of salivary gland or brain, or tissue culture fluid), or the incubation period. The paucity of antigen in many thalami suggests that incorporation of viral components into vacuolar membranes is not necessary for development of the spongiform change. The development of the lesions includes formation of small membrane-bound vacuoles in cellular processes, rapid enlargement (less than 3 days) with compression of adjacent neural tissue, and rupture resulting in the large tissue spaces readily visible by light microscopy.

ERA rabies vaccine virus grown in BHK-21 13S cells (ERA/BHK-21) and street rabies virus were titrated in mice by intracerebral, intranasal and intramuscular inoculation. Mice were also given undiluted ERA/BHK-21 in baits. Skunks were given undiluted ERA/BHK-21 in baits and by intramuscular, intranasal and intestinal inoculation. Virus neutralizing antibody titers against rabies virus were measured over a three month observation period. The surviving skunks were challenged by intramuscular inoculation with rabies street virus from a skunk salivary gland suspension. When titrated in mice, ERA/BHK-21 had titers of 10(7.0), 10(5.2) and 10(3.9) median lethal doses per mL by the intracerebral, intranasal and intramuscular routes, respectively. All skunks (8/8) inoculated intranasally developed paralytic rabies by 12 days after exposure to ERA/BHK-21 virus. None of the skunks that developed vaccine-induced rabies had infectious virus in the submandibular salivary glands. Vaccine-induced rabies also occurred in 1/8 skunks in the intramuscularly inoculated group and in 1/8 in the intestinally inoculated group. The survival rates of challenged skunks in the various groups were as follows: intramuscular, 7/7; intestinal, 2/7; bait, 0/8; and control, 0/8. These results indicate that ERA/BHK-21 virus has a significant residual pathogenicity in mice and in skunks by some routes of inoculation. Skunks given vaccine intramuscularly were protected against challenge, while those skunks given the vaccine in baits were not.

Brain tissue from 64 rabies suspect specimens were inoculated intracerebrally into twenty 9-12 gm adult Swiss white mice. Two mice from each specimen were killed on specific days postinoculation and examined for the presence of rabies virus by the fluorescent antibody staining technique. In this way a positive diagnosis was made in the majority of cases between postinoculation days 4 and 12 when the incubation period of these same specimens ranged between eight and 20 days.

Striped skunks were inoculated intracerebrally with the scrapie agent (suspension of brain from a naturally infected Suffolk sheep) or intramuscularly with street rabies virus (suspension of salivary glands from naturally infected skunks). Those given the scrapie agent developed clinical signs of weakness, posterior ataxia, and emaciation after incubated periods of 8 to 23 months. Those inoculated with rabies virus developed clinical signs of rabies (aggressive behavior, hyperexcitability, ataxia and paralysis) after incubation periods of 20 to 62 days. The gross lesions in the brains of the skunks given the scrapie agent consisted of marked atrophy of the thalamus and moderate atrophy of the cerebrum. No gross lesions occurred in the rabid skunks. Histologically, the type of spongiform lesion in rabies was the same as that in scrapie. However, spongiform change of rabies infected brains was less extensive (only rarely affected the basal ganglia, hippocampus or hypothalamus) than that of brains infected with the scrapie agent and was characterized by fewer numbers of small vacuoles (as a proportion of total number of vacuoles) than occurred in scrapie spongiform change.

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.

Striped skunks (Mephitis mephitis) were inoculated into the abductor digiti quinti muscle with street rabies virus isolated from salivary glands of rabid skunks. Using the immunofluorescence technique, antigen was detected in muscle cells at the inoculation site before it was detected in the central nervous system. Neurons and their processes in nearly all regions of the brain, spinal cord, cerebrospinal ganglia, and peripheral nerves contained antigen in terminal stages of the disease. Electron microscopically, matrix (viral nucleocapsid), virions, and anomalous viral products were mainly in neuronal perikarya and dendrites, and less often in myelinated axons. Matrices, virions, and crystalloid structures were in muscle fibers at the inoculation site. Viral budding occurred on endoplasmic reticulum, neurotubules, and neuronal plasma membrane. In the brain and dorsal horn of the spinal cord, virus budded from the postsynaptic and adjacent dendritic or perikaryal plasma membrane. There was simultaneous esotropic uptake of these particles by adjacent axon terminals. The results strongly suggest that direct transneuronal transfer of virus from perikarya and dendrites to adjacent axon terminals is a mechanism in dissemination of rabies in the central nervous system of striped skunks. Variation in the length of the incubation period may be due partly to replication or virus in myocytes at the inoculation site and subsequent transfer to peripheral nerves.

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.

A horse showing clinical signs of a neurological disorder was killed and various diagnostic tests for rabies were carried out. Histopathlogy revealed a nonsuppurative encephalitis. Fluorescent antibody test and mouse inoculation test were negative. A positive diagnosis of rabies was based on a high antibody titer (1:10,000) to rabies virus in brain tissue.

Groups of striped skunks (Mephitis mephitis) were inoculated intramuscularly with graded doses of street rabies virus. At various intervals after inoculation, saliva and sera were tested for rabies virus and neutralizing antibodies, respectively. Skunks that developed rabies were killed in terminal stages of the disease and the following examinations were made: titers of virus and antibody in submandibular salivary glands and brain, extent of immunofluorescence in submandibular salivary glands, and histologic examination of various tissues. Skunks that received inocula containing 4 x 10(4) to 4 x 10(5) mouse intracerebral lethal dose50 (MICLD50) had detectable serum neutralizing antibodies by 7-12 days postinoculation; however, most of the skunks that received lower doses (40 to 4 x 10(3) MICLD50) did not have detectable serum neutralizing antibodies until clinical signs began. In the salivary glands, slight and extensive immunofluorescence corresponded to high and low titers of tissue neutralizing antibody. Also low viral titers were associated with high tissue neutralizing antibody titers. There was a close correlation between viral titers in right and left submandibular salivary glands. The results suggest that the immune response can impede the process of infection of the salivary glands resulting in lack of antigen or low amounts of antigen in this tissue. This could occur through interference with centrifugal neural transport of virus and/or neutralization of virus during transfer from neural elements to epithelial cells. Lack of infectious virus or low viral titers in salivary glands containing antigen and high levels of tissue neutralizing antibodies can be caused partly by postmortem virus neutralization (during viral titration).

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.

A young man developed virologically proved hydrophobic rabies three months after being scratched on the cheek by a bat in Northern Alberta. He became comatose after 8 days and died 5 weeks after vigorous therapy with immune globulin, interferon and Vidarabine, and excellent maintenance of oxygenation. Electroencephalographic deterioration was gradual. At postmortem examination, the brain showed widespread loss of nerve cells, mild diffuse inflammatory changes and no Negri bodies. The lesions of massive laminar necrosis of the cerebral cortex, total loss of Purkinje cells with preservation of the granule cell layer, and severe softening of the amygdala are attributed to direct viral action rather than to anoxia or brain swelling with respirator brain.

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.

Striped skunks (Mephitis mephitis) were exposed to challenge virus standard rabies virus by feeding infected mouse brain in suspension or as intact brain free choice, by forced feeding of suspension, and by intranasal, intratracheal and intraintestinal instillation of suspension. All of five skunks exposed intranasally, two of five exposed intratracheally and two of ten exposed by forced feeding developed rabies. None of the skunks exposed to challenge virus standard virus, by other methods, became rabid. Most of the survivors, when challenged intramuscularly with street rabies virus at six months, developed rabies. The results indicate that the skunk is much more susceptible to challenge virus standard rabies virus given intranasally than by the other methods used. When disease occurs following oral administration, infection may be associated with prolonged contact with buccal mucosa or accidental contact with nasal mucosa. Survivors had little or no protection when challenged intramuscularly with street rabies virus.

Foxes were vaccinated orally (by bait), gastrically (by stomach tube) and by scarification with a vaccinia recombinant virus expressing the rabies glycoprotein. Neutralizing antibodies against rabies virus were detected at two weeks postvaccination in 8/8 foxes in the bait-fed group, in 3/6 foxes inoculated by stomach tube and in 2/2 of the scarified foxes. After challenge at three months postvaccination with street rabies virus, all foxes that had developed antibodies were protected. The high rate of seroconversion, high levels of antibodies, and resistance to challenge suggest that this recombinant virus might be a suitable vaccine for oral immunization of foxes against rabies.