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.

Conjugative metabolism of 1,2-dibromoethane in mitochondria: disruption of oxidative phosphorylation and alkylation of mitochondrial DNA.

1,2-Dibromoethane (DBE) is an environmental contaminant that is metabolized by glutathione S-transferases to a haloethane-glutathione conjugate. Since haloethane-glutathione conjugates are known to alkylate nuclear DNA and cytoplasmic proteins, these effects were investigated in isolated rat liver mitochondria exposed to DBE by measuring guanine adducts and several aspects of oxidative phosphorylation including respiratory control ratios, respiratory enzyme activity, and ATP levels. Mitochondrial large-amplitude swelling and glutathione status were assessed to evaluate mitochondrial membrane integrity and function. When exposed to DBE, mitochondria became uncoupled rapidly, yet no large-amplitude swelling or extramitochondrial glutathione was observed. Mitochondrial GSH was depleted to 2-53% of controls after a 60-min exposure to micromolar quantities of DBE; however, no extramitochondrial GSH or GSSG was detected. The depletion of mitochondrial glutathione corresponded to an increase of an intramitochondrial GSH-conjugate which, based on HPLC elution profiles and retention times, appeared to be S,S'-(1,2-ethanediyl)bis(glutathione). Activities of the NADH oxidase and succinate oxidase respiratory enzyme systems were inhibited 10-74% at micromolar levels of DBE, with succinate oxidase inactivation occurring at lower doses. ATP concentrations in DBE-exposed mitochondria in the presence of succinate were 5-90% lower than in the controls. The DNA adduct S-[2-(N(7)-guanyl)ethyl]glutathione was detected by HPLC in mtDNA isolated from DBE-exposed mitochondria. The results suggest that respiratory enzyme inhibition, glutathione depletion, decreased ATP levels, and DNA alkylation in DBE-exposed mitochondria occur via the formation of an S-(2-bromoethyl)glutathione conjugate, the precursor of the episulfonium ion alkylating species of DBE.

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.

Epidemiology of human rabies in the United States, 1980 to 1996.

PURPOSE: To summarize the epidemiologic, diagnostic, and clinical features of the 32 laboratory-confirmed cases of human rabies diagnosed in the United States from 1980 to 1996. DATA SOURCES: Data were obtained from case reports of human rabies submitted to the Centers for Disease Control and Prevention by state or local health authorities. STUDY SELECTION: All cases of human rabies reported in the United States from 1980 to 1996 in which infection with rabies virus was confirmed by laboratory studies. DATA EXTRACTION: Patients were reviewed for demographic characteristics, exposure history, rabies prophylaxis, clinical presentation, treatment, clinical course, diagnostic laboratory tests, identification of rabies virus variants, and the number of medical personnel or family members who required postexposure prophylaxis after coming in contact with an exposed person. DATA SYNTHESIS: 32 cases of human rabies were reported from 20 states. Patients ranged in age from 4 to 82 years and were predominantly male (63%). Most patients (25 of 32) had no definite history of an animal bite or other event associated with rabies virus transmission. Of the 32 cases, 17 (53%) were associated with rabies virus variants found in insectivorous bats, 12 (38%) with variants found in domestic dogs outside the United States, 2 (6%) with variants found in indigenous domestic dogs, and 1 (3%) with a variant found in indigenous skunks. Among the 7 patients with a definite exposure history, 6 cases were attributable to dog bites received in foreign countries and 1 was attributable to a bat bite received in the United States. In 12 of the 32 patients (38%), rabies was not clinically suspected and was diagnosed after death. In the remaining 20 cases (63%), the diagnosis of rabies was considered before death and samples were obtained specifically for laboratory confirmation a median of 7 days (range, 3 to 17 days) after the onset of clinical signs. Of the clinical differences between patients in whom rabies was diagnosed before death and those in whom it was diagnosed after death, the presence of hydrophobia or aerophobia was significantly associated with antemortem diagnosis (odds ratio, 11.0 [95% CI, 1.05 to 273.34]). The median number of medical personnel or familial contacts of the patients who received postexposure prophylaxis was 54 per patient (range, 4 to 179). None of the 32 patients with rabies received postexposure prophylaxis before the onset of clinical disease. CONCLUSIONS: In the United States, human rabies is rare but probably underdiagnosed. Rabies should be included in the differential diagnosis of any case of acute, rapidly progressing encephalitis, even if the patient does not recall being bitten by an animal. In addition to situations involving an animal bite, a scratch from an animal, or contact of mucous membranes with infectious saliva, postexposure prophylaxis should be considered if the history indicates that a bat was physically present, even if the person is unable to reliably report contact that could have resulted in a bite. Such a situation may arise when a bat bite causes an insignificant wound or the circumstances do not allow recognition of contact, such as when a bat is found in the room of a sleeping person or near a previously unattended child.

Serosurvey for selected viral diseases and demography of African wild dogs in Tanzania.

African wild dogs (Lycaon pictus) are endangered, with only 3,000-5,000 remaining in the wild. It is believed that wild dogs are unusually vulnerable to viral diseases, particularly rabies and canine distemper (CDV). However, canine distemper has been confirmed by laboratory diagnosis in only one free-living wild dog. The 43,000 km2 Selous Game Reserve (SGR; Tanzania) holds approximately 900 adult wild dogs. In a study area of 2,600 km2, the population maintained high density (> or = 1 dog/20.5 km2) from 1991 to 1996. The population was stable, varying 18% below and 9% above the mean density over the 6-yr period. Serum samples (n = 22) collected over 3 yr showed that most individuals were exposed to CDV (59%:95% confidence interval = 43-76% seropositive) and canine parvovirus (68%:95% CI = 54-81% seropositive), although none were seropositive for rabies (0%:95% CI = 0-17%). CDV titers were positively related to age, with no seropositive dogs younger than 1.9 yr. At least five of 13 dogs positive for CDV seroconverted during the study. Dogs with high CDV titers did not survive better in the years after sampling (mean survival +/- SE for those that died = 638 +/- 92 days,). Variation in mean litter size was inversely related to CPV exposure in the SGR and elsewhere. Annual mortality rates were low in comparison to other populations for all age classes (pups: 31 +/- 8%, n = 127, yearlings: 22 +/- 10%, n = 93, adults: 20 +/- 6%, n = 235). Annual mortality rates fluctuated little between 1992 and 1996. These data show that wild dog populations, like those of other canids, can remain stable and demographically healthy despite exposure to CDV and CPV.

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.

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.

Human diploid cell rabies vaccine purified by zonal centrifugation: a controlled study of antibody response and side effects following primary and booster pre-exposure immunizations.

Systemic allergic reactions following booster immunizations have complicated rabies pre-exposure prophylaxis with the human diploid cell rabies vaccine licensed in the US (conventional HDCV). We conducted two studies comparing an HDCV purified by zonal centrifugation to conventional HDCV. In a study of primary pre-exposure immunization, volunteers received one of four regimens: three 1.0-ml intramuscular (i.m.) or 0.1-ml intradermal (i.d.) doses of conventional or purified HDCV over 28 days. Although volunteers vaccinated i.m. had significantly greater rabies neutralizing antibody titres (VNA) 49 days, 91 days and 26 months after immunization began than volunteers vaccinated i.d. (p less than 0.005-p less than 0.05), there were no significant differences between vaccines. In a study of booster immunizations, 77 volunteers immunized with conventional HDCV 2 years earlier received a 0.1-ml i.d. booster with either conventional or purified HDCV. VNA was significantly greater with the conventional HDCV on days 7 and 28 after booster, but not on day 365. A moderate or severe reaction was reported by 5 (13%) of the 40 persons who received boosters with conventional HDCV, versus none of 37 who received the purified HDCV (p = 0.03). Purified HDCV appears to be preferable to conventional HDCV for booster vaccination.

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.

Use of the avidin-biotin peroxidase system to detect rabies antigen in formalin-fixed paraffin-embedded tissues.

We stained rabies-infected nervous and salivary-gland tissues fixed in formalin or acetone and embedded in paraffin with the avidin-biotin peroxidase system. With this system, rabies-virus antigen was detected in neurons, glandular acinar cells, and vascular endothelial cells more effectively than by immunofluorescence, especially when tissues were enzyme-digested with pronase before immunoperoxidase staining. The avidin-biotin peroxidase system should be useful for routine diagnosis, retrospective studies of rabies, and identification of specific cells involved in the spread of virus in rabies-infected hosts.

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.

Effect of adjuvant vaccines on dengue virus type 2 immune ascitic fluid production.

In a comparison of dengue type 2 immune mouse ascitic fluid immunization schedules, a schedule in which adjuvant vaccines were not used produced neutralizing antibody titers that were specific and a mouse mortality rate that was lower, resulting in a greater yield of ascitic fluids. In the schedules in which emulsified adjuvant vaccines were used, the quality of the emulsion had little effect on antibody titer produced.