Rapid clearance of SAG-2 rabies virus from dogs after oral vaccination.

This study investigated the safety, efficacy, and clearance of SAG-2, an attentuated rabies virus, after oral vaccination in dogs. Nineteen dogs consumed baits containing lyophilized vaccine, but residual SAG-2 virus was recovered in only one of 57 oral swabs, collected one hour post-vaccination. Seven vaccinates were euthanized between 24 and 96 h after consuming a bait. Rabies virus RNA was detected in tonsils from all seven dogs by nested RT-PCR, with primers to the viral glycoprotein. Genomic, sense-transcripts, and m-RNAs were detected in five of seven tonsil samples using primers to the rabies virus nucleoprotein gene, as well as in four of seven samples from the buccal mucosa and one of seven from the tongue. Rabies virus antigen was detected in all tonsils by an immunohistochemistry test, confirming the RT-PCR results. In addition, virus was isolated from one tonsil sample collected at 96 h, providing supportive evidence of viral replication. Ten of 12 (83%) of the vaccinated dogs demonstrated an anamnestic response, with viral neutralizing antibody titers (> or =0.5 IU/ml), after rabies virus challenge. These ten dogs survived, whereas all control dogs succumbed to rabies. Attenuated rabies viruses, such as SAG-2, replicate in local tissues of the oral cavity and can be cleared relatively quickly, without viral excretion, leading to protective immunity against the disease.

A comparative study of the fluorescent antibody test for rabies diagnosis in fresh and formalin-fixed brain tissue specimens.

Many diagnostic methods have been used to detect rabies virus antigen. The preferred method for routine diagnosis of rabies in fresh or frozen brain tissues is the fluorescent antibody test (FAT). In this study, the FAT was used to evaluate the rabies status of fresh/frozen brain specimens from more than 800 rabies-suspected cases, in more than 14 different species of animals. A comparable brain specimen from each case was fixed in 10% buffered formalin and examined by the FAT. The evaluation of rabies status between fresh and formalin-fixed tissues was in agreement in more than 99.8% of the cases. When fresh tissue is not available for testing, these results validate the use of this procedure for routine diagnosis of rabies in formalin-fixed brain tissues.

Laboratory investigation of human deaths from vampire bat rabies in Peru.

In the spring of 1996, multiple cases of an acute febrile illness resulting in several deaths in remote locations in Peru were reported to the Centers for Disease Control and Prevention (CDC). The clinical syndromes for these cases included dysphagia and encephalitis. Because bat bites were a common occurrence in the affected areas, the initial clinical diagnosis was rabies. However, rabies was discounted primarily because of reported patient recovery. Samples of brain tissue from two of the fatal cases were received at CDC for laboratory confirmation of the rabies diagnosis. An extensive array of tests on the formalin-fixed tissues confirmed the presence of both rabies viral antigen and nucleic acid. The virus was shown to be most closely related to a vampire bat rabies isolate. These results indicate the importance of maintaining rabies in the differential diagnosis of acute febrile encephalitis, particularly in areas where exposure to vampire bats may occur.

DNA immunization protects nonhuman primates against rabies virus.

More than 40,000 people die annually from rabies worldwide. Most of these fatalities occur in developing countries, where rabies is endemic, public health resources are inadequate and there is limited access to preventive treatment. Because of the high cost of vaccines derived from cell culture, many countries still use vaccines produced in sheep, goat or suckling mouse brain. The stability and low cost for mass production of DNA vaccines would make them ideal for use in developing countries. To investigate the potential of DNA vaccines for rabies immunization in humans, we vaccinated Macaca fascicularis (Cynomolgus) monkeys with DNA encoding the glycoprotein of the challenge virus standard rabies virus, or with a human diploid cell vaccine (HDCV). The monkeys then were challenged with a non-passaged rabies virus. DNA or HDCV vaccination elicited comparable primary and anamnestic neutralizing antibody responses. All ten vaccinated monkeys (DNA or HDCV) survived a rabies virus challenge, whereas monkeys vaccinated with only the DNA vector developed rabies. Furthermore, serum samples from DNA- or HDCV-vaccinated monkeys neutralized a global spectrum of rabies virus variants in vitro. This study shows that DNA immunization elicits protective immunity in nonhuman primates against lethal challenge with a human viral pathogen of the central nervous system. Our findings indicate that DNA vaccines may have a promising future in human rabies immunization.

Immunogenicity, efficacy and safety of an oral rabies vaccine (SAG-2) in dogs.

A study of immunogenicity and efficacy of Street Alabama Gif (SAG-2) attenuated rabies virus vaccine in laboratory beagles was conducted. Four groups of ten dogs each received either 1.0 ml of SAG-2 orally on the tongue or 1.5 ml in baits. On day 180 postvaccination, all dogs were challenged with a street rabies virus. The antibody response in groups that received the vaccine directly on the tongue was higher than in those vaccinated with baits, but the difference between groups was not statistically significant. All vaccinated dogs survived, whereas 80% of controls died of rabies. Our findings demonstrate that the SAG-2 is a safe and effective vaccine for oral immunization of canines.

A field evaluation of baits for delivering oral rabies vaccines to raccoons (Procyon lotor).

Eight field trials were conducted in 1989 and 1990 in Georgia (USA) and Maryland (USA) to evaluate baits and baiting strategies for delivering oral rabies vaccines to raccoons (Procyon lotor). Bait packets consisting of corn meal and egg batter-based baits enclosed in plastic bags were placed at 1.0-m diameter, raked tracking stations and checked daily. Packets were well accepted by raccoons; they visited 31 to 44% of the tracking stations where they removed 69 to 90% of the packets within 4 to 5 days. All or nearly all baits were removed from plastic bags and less than 1% of the baits were found only partially eaten. No rejection of water-filled paraffin ampules in baits was observed. The use of an odor attractant on bait packets did not appear to enhance bait discovery when packets were placed on raccoon travel routes. An attractant did enhance discovery when baits were placed off-road in a simulated aerial baiting test. Nontarget species comprised 31 to 53% of all visits to the stations; they took 28 to 55% of the baits but did not appear to adversely affect bait availability for raccoons. A total of 2,300 baits, each containing a wax ampule holding 10 mg of a physiological marker (iophenoxic acid), were distributed at a rate of 82 baits/km2 on 2,800 ha of Sapelo Island, Georgia. Thirty-five (65%) of 54 raccoons collected following bait placement had eaten one or more baits as indicated by elevated levels of iodine in the blood serum.

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.

An immune stimulating complex (ISCOM) subunit rabies vaccine protects dogs and mice against street rabies challenge.

Dogs and mice were immunized with either a rabies glycoprotein subunit vaccine incorporated into an immune stimulating complex (ISCOM) or a commercial human diploid cell vaccine (HDCV) prepared from a Pitman Moore (PM) rabies vaccine strain. Pre-exposure vaccination of mice with two intraperitoneal (i.p.) doses of 360 ng ISCOM or 0.5 ml HDCV protected 95% (38/40) and 90% (36/40) of mice, respectively, against a lethal intracerebral (i.c.) dose with challenge virus strain (CVS). One 360 ng i.p. dose of ISCOM protected 87.5% (35/40) of mice against i.c. challenge with CVS. Three groups of five dogs were vaccinated intramuscularly (i.m.) with 730 ng of rabies ISCOM prepared from either the PM or the CVS rabies strains, and they resisted lethal street rabies challenge. Postexposure treatment of mice with three or four 120 ng i.m. doses of ISCOM protected 90% (27/30) and 94% (45/48), respectively, of mice inoculated in the footpad with street rabies virus, but three doses of HDCV conferred no protection. When four doses of HDCV were administered postexposure, 78% (32/41) of the mice died of anaphylactic shock; 21% (11/52) of mice had already died of rabies 4 days after the third vaccine dose was administered.

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.

The protective role of humoral neutralizing antibody in the NIH potency test for rabies vaccines.

Intraperitoneal vaccination of mice with rabies vaccine results in both dosage-dependent rabies virus neutralizing antibody titres and protection from lethal intracerebral (i.c.) challenge with fixed strain CVS rabies virus. Pre-exposure adoptive intravenous transfer of naive or immune cells did not significantly protect naive Balb/c mice from lethal i.c. CVS challenge, but immune serum and anti-rabies glycoprotein monoclonal antibodies (individually and in combination) did confer significant protection when administered before or up to 24 h after lethal i.c. rabies virus challenge.

Protection of mice with vaccinia virus recombinants that express the rabies nucleoprotein.

The role of rabies virus nucleoprotein (N) in protection against rabies was examined with recombinant vaccinia viruses expressing the N of the Challenge Virus Standard strain. Two chimeric plasmids were constructed with the open reading frame of the N gene placed downstream of the vaccinia P7.5 promoter (early/late class) or the vaccinia P11 promoter (late class), with each expression cassette flanked by vaccinia thymidine kinase (TK) sequences to enable marker rescue by TK insertional inactivation. Two recombinants were isolated that expressed the rabies N in infected cells as determined by radioimmunoprecipitation and immunofluoresence microscopy with an anti-N monoclonal antibody. Two groups of 25 ICR mice inoculated intradermally with the recombinants and challenged with 75 MFPLD50 of street rabies virus showed high survival ratios (22/25 and 21/25). Intramuscular inoculation, however, was not protective against 25 MFPLD50. The intradermally vaccinated mice developed non-neutralizing antibodies against rabies N.

Formulation and evaluation of baits for oral rabies vaccination of raccoons (Procyon lotor).

Captive raccoons were offered a variety of vaccine containers and bait components in a series of three-choice tests. Paraffin wax ampules were the most readily accepted vaccine container. Preferred bait components included corn and shellfish oils, deep fried corn meal batter, and egg, apple and buttermilk flavorings. These results, together with factors including ease of bait formulation, cost, and suitability for field use, were used to develop an experimental delivery system for an oral rabies vaccine. The developed system was composed of a polyurethane sleeve (1.5 x 5.5 cm) dipped in a commercial food batter mix together with corn meal, milk and egg. The sleeve was deep fried in corn oil and a 2.0 ml ampule containing a recombinant rabies vaccine was then inserted into the sleeve bait. These baits were presented to 10 captive raccoons. Nine of the 10 animals developed high levels of rabies virus neutralizing antibodies. Field tests are needed to determine if the delivery system developed also is effective for wild raccoons.

Rabies diagnostic reagents prepared from a rabies N gene recombinant expressed in baculovirus.

A gene encoding the nucleoprotein (N) of rabies virus was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus. Recombinant gene expression was controlled by the strong polyhedrin gene promoter. Insect cells (Spodoptera frugiperda) infected by a baculovirus recombinant containing the rabies virus N gene produced abundant amounts of a novel 55-kilodalton protein of a size comparable to that of the rabies virus N protein, as demonstrated by polyacrylamide gel electrophoresis. This new gene product possessed the antigenic and immunogenic properties of native viral N protein, as shown by the ability of the new protein to react in immunoprecipitation and immunofluorescence assays with antirabies antibodies, to serve as a substitute for infectious rabies virus in adsorbing suspensions for diagnostic tests, and to induce high-titered antiserum. The baculovirus expression system provides a safe, convenient, and inexpensive source of rabies virus N protein for the production of both antiserum and adsorbing suspensions for use in rabies diagnoses.

Efficacy of rabies vaccines against Duvenhage virus isolated from European house bats (Eptesicus serotinus), classic rabies and rabies-related viruses.

Isolates of rabies from separate enzootics can be distinguished by their reactions with panels of monoclonal antibodies (mAbs) directed to different sites on the nucleocapsid and glycoproteins of the virus. Estimates of antigenic relatedness can be made by comparing similarities among groups. In this manner it can be shown that while classic strains of rabies react with most of the mAbs, the rabies related Lyssaviruses (Mokola, Lagos and Duvenhage) react with only a few of the mAbs and isolates of rabies from Eptesicus serotinus bats in Europe are intermediate between the two groups. Mice immunized intraperitoneally with human diploid vaccine (HDCV) or animal vaccines (Rabisin and Rabiffa) were protected against a challenge with DBV, DUV-1 and most classic rabies strains. HDCV gave only partial protection against human virus isolates from Finland and Saudi Arabia. The HDCV did not protect mice against challenges with Lagos bat or Mokola virus (rabies-like viruses). The animal vaccines, however, did protect mice against Lagos bat virus, but not against Mokola. Dogs immunized with Rabisin were protected against an intracerebral challenge with DBV. Dogs developed rabies-neutralizing antibody titres after intramuscular or intravenous inoculation with live DBV or DUV-1 virus; these dogs were protected against an intramuscular canine street rabies virus challenge. We conclude that the rabies vaccines tested protect against DBV/DUV-1 and classic street rabies strains, but not Mokola.

Successful oral rabies vaccination of raccoons with raccoon poxvirus recombinants expressing rabies virus glycoprotein.

Two infectious raccoon poxvirus (RCN) recombinants for expressing rabies virus surface spike glycoprotein (G) were produced by homologous recombination between raccoon poxvirus DNA and chimeric plasmids previously used for production of vaccinia virus recombinants. Expression of G protein was controlled by vaccinia virus promoter P7.5 (early/late class) or by P11 (late class). Immunoprecipitation of infected cell extracts indicated that both of the RCN recombinants directed faithful expression of G protein. Raccoons that were fed polyurethane baits loaded with either recombinant quickly developed high levels of rabies virus neutralizing antibodies and were protected when challenged with lethal raccoon rabies street virus.

Oral administration of an attenuated strain of canine adenovirus (type 2) to raccoons, foxes, skunk, and mongoose.

An attenuated strain of canine adenovirus type-2 (CAV-2) was administered orally to 2 foxes (Vulpes fulva), 6 raccoons (Procyon lotor), a skunk (Mephitis mephitis), and a mongoose (Herpestus auropunctatus). Blood was collected weekly from the animals to monitor CAV-2 virus-neutralizing antibody titers. All animals had increases in titers. Sera from 8 foxes, 30 mongooses, 52 raccoons, and 22 skunks trapped in the field had naturally occurring antibody to CAV-2.

Pathogenesis of rabies virus from a Danish bat (Eptesicus serotinus): neuronal changes suggestive of spongiosis.

Rabies virus strains isolated from a European bat (Eptesicus serotinus) in Denmark (DBV), a North American big brown bat (Eptesicus fuscus) in New York State (NY-bat), and a human in South Africa (Duvenhage strain (DUV-1) were studied by using a panel of monoclonal antibodies and by inoculating mice, cats, and dogs. The ten Danish virus isolates from the same bat species reacted identically with a panel of monoclonal antibodies. Immunofluorescence, monoclonal antibody, and histopathologic studies showed that the Danish bat isolates were similar to Duvenhage, and to some degree, to classical rabies virus. All isolates produced fatal infections in mice when inoculated by the intracerebral, footpad, and oral routes. Dogs and cats inoculated intracerebrally with the DBV and DUV-1 virus strains died of rabies-like illnesses within 10 days. Although no dogs that were inoculated intramuscularly or intravenously showed signs of disease, all developed neutralizing antibodies and resisted challenge with lethal dose of street rabies virus. All dogs inoculated with the NY-bat virus, with the exception of those inoculated intravenously, showed classical signs of rabies and one of the intramuscularly inoculated dogs recovered. Cats inoculated intramuscularly also died of rabies-like illness within 15 days. At necropsy, rabies antigen was detected by immunofluorescence in frozen sections of several organs, including brain and salivary glands. Histopathologic and electron microscopic studies of the central nervous system of mice, dogs and cats that died of DBV infection showed neuronal cytoplasmic changes considered to be a form of spongiosis.

Peripheral distribution of virus in dogs inoculated with two strains of rabies virus.

Forty-seven Beagles were inoculated IM with an Ethiopian strain or a Mexican strain of rabies virus to study the pathogenesis of street rabies virus in dogs. Thirty-nine dogs died of rabies, with incubation periods lasting 9 to 69 days. Of the dogs that died, 82% had shown typical signs of rabies, but 18% died without any noticeable signs of illness. Eight dogs that remained healthy during an observation period lasting more than 2 years did not produce detectable amounts of rabies virus-neutralizing antibodies; however, when challenge exposed with a large dose of the homologous rabies virus inoculum, these 8 dogs responded with high antibody titers, but challenge-exposed control dogs died of rabies. Infective virus was isolated from the saliva and cerebrospinal fluid of dogs before any signs of rabies were noticed; rabies virus-neutralizing antibodies were not detected in the serum and cerebrospinal fluid before illness. In this study, viral antigen was not detected in the skin biopsy specimens taken before signs of rabies were noticed. At necropsy of the 39 dogs, rabies virus was detected in most tissues examined. Viral antigen was detected in the skin tissues of 14 (36.8%) of the 38 dogs examined. The presence of viral antigen in the skin seemed to correlate with the presence of virus in the salivary glands, but virus in the salivary glands did not indicate the presence of virus in the skin. Eleven (44%) of the 25 dogs which had virus in the salivary glands did not have any detectable amount of viral antigen in the skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabies virus in the tonsils of a carrier dog.

A female dog, inoculated with a rabies isolate from the saliva of an apparently healthy Ethiopian dog, developed rabies but later recovered without supportive treatment. Rabies virus was isolated from the saliva collected 42, 169 and 305 days after recovery. Sixteen months after it recovered, the dog suddenly died after giving birth to two stillborn puppies. At necropsy, viral antigen could be detected in the tonsils and the brain tissue, but viable virus was isolated from the Palatine tonsils only.