Studies of ERA/BHK-21 rabies vaccine in skunks and mice.

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.     

Experimental rabies in skunks: mechanisms of infection of the salivary glands.

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.     

Experimental rabies in skunks: Immune response and salivary gland infection

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 × 10⁴ to 4 × 10⁵ 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 × 10³ 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).     

Mutants of rabies viruses in skunks: immune response and pathogenicity.

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.     

Infection of Bergmann glia in the cerebellum of a skunk experimentally infected with street rabies virus.

Rabies virus is a highly neuronotropic virus and glial cell infection is not prominent in the central nervous system (CNS). Paraffin-embedded tissues from the cerebella of skunks experimentally infected with either a skunk salivary gland isolate of street rabies virus or the challenge virus standard (CVS) strain of fixed rabies virus were examined with immunoperoxidase staining for rabies virus antigen by using an anti-rabies virus nucleocapsid protein monoclonal antibody. A skunk infected with street rabies virus showed prominent infection of Bergmann glia. Although infected Purkinje cells were observed, they usually demonstrated a relatively small amount of antigen in their perikarya. A CVS-infected skunk showed many intensely labeled Purkinje cells and a relatively small number of infected Bergmann glia. These findings indicate that although rabies virus is a highly neuronotropic virus, street rabies virus strains do not always demonstrate strict neuronotropism in the central nervous system.     

Immune response in skunks to a vaccinia virus recombinant expressing the rabies virus glycoprotein.

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.     

Experimental rabies in skunks: oral, nasal, tracheal and intestinal exposure.

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.     

Experimental rabies in skunks: persistence of virus in denervated muscle at the inoculation site.

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.     

Rabies virus in the salivary glands and nasal mucosa of naturally infected skunks.

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.     

The demonstration of rabies antigen in paraffin-embedded tissues using the peroxidase-antiperoxidase method: a comparative study.

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.     

Terminal Dissemination of Rabies Virus in Selected Rat Tissues

Fifty rats were divided into 3 groups and challenged via the foot pad route with a fixed and 2 different strains of street rabies virus in order to study the dissemination of the virus and the affinity for certain tissues of the rat.

The incubation period for rats inoculated with fixed rabies is shorter than with street virus, being 5 to 7 days compared with 10 to 12 days.

Rats inoculated with the fixed strain were less aggressive and irritable than rats inoculated with street virus.

The fixed strain demonstrated a greater affinity for the tissues studied as compared to the street strains of virus.

Both the fixed and street strains revealed a low affinity for the parotid gland since no virus could be demonstrated in 14 of 20 in the fixed virus group and 27 of 30 in the street virus group.

     

Virus isolation from saliva and salivary glands of cattle naturally infected with paralytic rabies

The infectivity of saliva, salivary and mammary glands, muscle, lung, kidney and liver of 87 cattle infected with paralytic rabies (positive viral isolation from brains) was studied. Fifty percent dilutions of saliva and tissue samples were inoculated intracerebrally into 10- to 15-day-old mice. Viral isolation in mice was confirmed by direct rabies fluorescent-antibody test and the antigenic variant of the isolates characterized by monoclonal antibodies. Rabies virus was isolated from 4.6% of salivary glands and from 1.6% of saliva samples. The rest of the peripheral tissues were negative. Cerebral and peripheral isolates belonged to vampire-bat antigenic variants. These results indicate that cattle infected by vampire bats may be a source of infection for man. The infection risk would depend on the type of contact between rabid cattle and man.     

Evidence of canine distemper virus infection in skunks negative for antibody against rabies virus

Between January 1981 and October 1985, brain tissue specimens from 192 skunks that were negative for antibodies against rabies virus were obtained from 2 Illinois Public Health laboratories (A and B). Brain lesions were detected microscopically in specimens from 17 of the 91 (18.7%) skunks from laboratory B and in specimens from 30 of the 101 (29.7%) skunks from laboratory A. Lesions in 3 skunks (1 from laboratory A, 2 from B) were caused by cerebral parasitism. Lesions in the remaining 44 skunks were characterized by perivascular, nonsuppurative, mononuclear cell infiltrates and foci of glial cells of differing severity. The similarity of lesions and the finding of inclusions diagnostic of canine distemper virus (CDV) in some skunks indicated that CDV may be the main cause of neurologic disease in nonrabid skunks. Seventeen of 36 (47.2%) skunks evaluated for antibody against CDV, using an unlabeled antibody-enzyme method, were positive for CDV. Findings in skunks from the 2 laboratories indicated similar annual prevalences of brain lesions in 1982, 1983, and 1984. The highest percentage (40.5%) of nonrabid skunks with encephalitis was found in skunks submitted to laboratory B in 1981, which was concurrent with a rabies epizootic among skunks in Illinois in 1981. The number of skunks from both laboratories with CDV infection peaked during winter-spring. Importance of CDV in skunk population dynamics remains to be elucidated; however, infection with CDV appears to be enzootic and occasionally epizootic in skunks. Because enzootic/epizootic CDV may bias rabies surveillance data, caution in interpretation of surveillance data is necessary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental oral and nasal transmission of rabies virus in mice.

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.     

Rabies surveillance in the United States during 2007

SUMMARY: During 2007, 49 states and Puerto Rico reported 7,258 cases of rabies in animals and 1 case in a human to the CDC, representing a 4.6% increase from the 6,940 cases in animals and 3 cases in humans reported in 2006. Approximately 93% of the cases were in wildlife, and 7% were in domestic animals. Relative contributions by the major animal groups were as follows: 2,659 raccoons (36.6%), 1,973 bats (27.2%), 1,478 skunks (20.4%), 489 foxes (6.7%), 274 cats (3.8%), 93 dogs (1.3%), and 57 cattle (0.8%). Compared with numbers of reported cases in 2006, cases in 2007 increased among dogs, bats, foxes, and skunks while decreases were reported among cattle, cats, and skunks. Increases in numbers of rabid raccoons during 2007 were reported by 11 of the 20 eastern states where raccoon rabies was enzootic, and reported cases increased by 1.7% overall, compared with 2006. On a national level, the number of rabies cases in skunks during 2007 decreased by 1.1% from the number reported in 2006. Texas reported the greatest number (n = 362) of rabid skunks and the greatest overall state total of animal rabies cases (969). No cases of rabies associated with the dog/coyote rabies virus variant were reported. The United States remains free of dog-to-dog transmission of canine rabies virus variants. The total number of cases of rabies reported nationally in foxes increased 14.5%, compared with 2006. Increases in the number of reported rabid foxes were attributable to greater numbers of foxes reported with the Arctic fox rabies virus variant in Alaska, the Texas gray fox rabies virus variant in Texas, and the raccoon rabies virus variant in Virginia. The 1,973 cases of rabies reported in bats represented a 16.6% increase over numbers reported in 2006. Cases of rabies in dogs and in sheep and goats increased 17.7% and 18.2%, respectively, whereas cases reported in cattle, cats, and horses and mules decreased 30.5%, 13.8%, and 20.8%, respectively. In Puerto Rico, reported cases of rabies in mongooses decreased 51.5%, and rabies in domestic animals, presumably attributable to spillover infection from mongooses, increased 25%. One human rabies case was reported from Minnesota during 2007. Although typing of the rabies virus variant in this case was not possible, an investigation of this case indicated a bat as the most likely source of exposure.     

Rabies surveillance in the United States during 2001

During 2001, 49 states and Puerto Rico reported 7,437 cases of rabies in nonhuman animals and 1 case in a human being to the Centers for Disease Control and Prevention, an increase of < 1% from 7,364 cases in nonhuman animals and 5 human cases reported in 2000. More than 93% (6,939 cases) were in wild animals, whereas 6.7% (497 cases) were in domestic species (compared with 93.0% in wild animals and 6.9% in domestic species in 2000). The number of cases reported in 2001 increased among bats, cats, skunks, rodents/lagomorphs, and swine and decreased among dogs, cattle, foxes, horses/mules, raccoons, and sheep/goats. The relative contributions of the major groups of animals were as follows: raccoons (37.2%; 2,767 cases), skunks (30.7%; 2,282), bats (17.2%; 1,281), foxes (5.9%; 437), cats (3.6%; 270), dogs (1.2%; 89), and cattle (1.1%; 82). Nine of the 19 states where the raccoon-associated variant of the rabies virus has been enzootic reported decreases in the numbers of rabid raccoons during 2001. Among states with extensive wildlife rabies control programs, Ohio reported (other than rabies in bats) 1 case of rabies in a raccoon that was associated with the epizootic of rabies in raccoons and 1 case in a bovid that was infected with a bat variant of the rabies virus, compared with no cases reported in any terrestrial animals during 2000. Texas reported 1 case associated with the dog/coyote variant of the rabies virus (compared with no cases in 2000) and 20 cases associated with the gray fox variant of the virus (a decrease of 50% from reported cases in 2000). Reports of rabid skunks in Massachusetts and Rhode Island, states with enzootic raccoon rabies, exceeded reports of rabid raccoons for the fifth consecutive year. A similar situation may soon exist in the state of Maine (32 rabid skunks and 34 rabid raccoons during 2001). Nationally, the number of rabies cases in skunks during 2001 increased by 2.7% over those reported in 2000. Texas reported the greatest number of rabid skunks ever documented during a single year by any state, as well as the greatest numerical increase in rabid skunks (778 cases in 2001, compared with 550 in 2000; an increase of 228 cases, or 41.5%) and the largest overall state total of rabies cases (1,043) during 2001. Arizona reported the greatest percentage increase in rabid skunks (247.1%), representing an increase from 17 rabid skunks in 2000 to 59 in 2001. Nineteen of these cases were infected with a bat variant of the rabies virus, documenting a spillover event followed by unprecedented detection of temporal enzootic transmission of a bat variant in a terrestrial species. The number of cases of rabies reported in bats during 2001 (1,281 cases) increased 3.3% and surpassed the previous year's record (1,240 cases) as the largest number of reported cases ever recorded for this group of mammals. Cases of rabies reported in dogs (89) and cattle (82) decreased by 21.9 and 1.2%, respectively; these are the lowest numbers reported for rabid cattle and dogs since the dawn of national rabies record keeping (ca 1938). Cases in cats (270) increased by 8.4% over those reported in 2000, whereas rabies among sheep and goats declined 70%, from 10 cases in 2000 to 3 cases (goats only) in 2001. Rabies among horses and mules declined 1.9% (52 cases in 2000 to 51 cases in 2001). Reported cases of rabies in mongooses in Puerto Rico increased 18.6%, compared with the previous year (70 cases in 2001 from 59 cases in 2000), whereas cases of rabies in dogs declined 15.3% (15 to 13). One case of rabies in a human being reported by California during 2001 was the result of infection with a canine variant of the rabies virus acquired outside the United States.     

Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line.

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.     

Challenges to controlling rabies in skunk populations using oral rabies vaccination: A review

Controlling rabies in skunk populations is an important public health concern in many parts of the United States due to the potential for skunk rabies outbreaks in urban centres and the possible role for skunks in raccoon rabies variant circulation. Oral rabies vaccination (ORV) programmes have supported wildlife rabies control efforts globally but using ORV to control rabies in skunk populations has proven more challenging than with other target species, like foxes, coyotes and raccoons. A review of published studies found that some ORV constructs are immunogenic in skunks and protect against virulent rabies virus challenges, especially when delivered by direct installation into the oral cavity. However, in field ORV programmes using currently available vaccine‐bait formats and distribution methods targeting other rabies reservoir species, skunks often fail to seroconvert. Field effectiveness of ORV in skunks appears to be limited by poor bait uptake or inadequate ingestion of vaccine rather than from poor vaccine efficacy. Observations of captive skunks revealed vaccine spillage when handling and biting into baits such that modification of bait formats might improve field effectiveness. In addition, a dose–response relationship between bait distribution density and post‐baiting seroconversion among skunks was observed across the limited number of field studies. Additional research is needed to identify opportunities to modify ORV baits and distribution strategies to improve the viability of ORV as a rabies control strategy in skunks.     

Experimentally induced rabies in four cats inoculated with a rabies virus isolated from a bat

Four cats were inoculated IM with rabies virus isolated from the salivary gland of a naturally infected big brown bat (Eptesicus fuscus). The 4 cats developed clinical signs of rabies after a median incubation period of 42 days. The median duration of clinical illness was 5 days. Results of fluorescent antibody evaluation, mouse inoculation, and tissue culture isolation indicated large differences in virus concentrations in various areas of the CNS of individual cats. These differences also were observed between cats. Rabies virus was isolated from the salivary glands and saliva of 2 cats; urinary bladder was the only other nonneural tissue found infected. Our observations indicated that cat rabies can be caused by bat rabies virus; that cats thus infected have infectious saliva during aggressive behavior and can therefore transmit the disease; and that adequate specimens of hippocampus, cerebellum, and brain stem are essential for reliable determination of rabies infection. The findings support recommendations for regular rabies vaccination of cats, even in areas of rabies-free terrestrial mammals.