Protection of non-human primates against rabies with an adenovirus recombinant vaccine.

Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials.

High prevalence of antibodies against canine adenovirus (CAV) type 2 in domestic dog populations in South Africa precludes the use of CAV-based recombinant rabies vaccines.

Rabies in dogs can be controlled through mass vaccination. Oral vaccination of domestic dogs would be useful in the developing world, where greater vaccination coverage is needed especially in inaccessible areas or places with large numbers of free-roaming dogs. From this perspective, recent research has focused on development of new recombinant vaccines that can be administered orally in a bait to be used as adjunct for parenteral vaccination. One such candidate, a recombinant canine adenovirus type 2 vaccine expressing the rabies virus glycoprotein (CAV2-RG), is considered a promising option for dogs, given host specificity and safety. To assess the potential use of this vaccine in domestic dog populations, we investigated the prevalence of antibodies against canine adenovirus type 2 in South African dogs. Blood was collected from 241 dogs from the Gauteng and KwaZulu-Natal provinces. Sampled dogs had not previously been vaccinated against canine adenovirus type 1 (CAV1) or canine adenovirus type 2 (CAV2). Animals from both provinces had a high percentage of seropositivity (45% and 62%), suggesting that CAV2 circulates extensively among domestic dog populations in South Africa. Given this finding, we evaluated the effect of pre-existing CAV-specific antibodies on the efficacy of the CAV2-RG vaccine delivered via the oral route in dogs. Purpose-bred Beagle dogs, which received prior vaccination against canine parvovirus, canine distemper virus and CAV, were immunized by oral administration of CAV2-RG. After rabies virus (RABV) infection all animals, except one vaccinated dog, developed rabies. This study demonstrated that pre-existing antibodies against CAV, such as naturally occurs in South African dogs, inhibits the development of neutralizing antibodies against RABV when immunized with a CAV-based rabies recombinant vaccine.

Enhancing comparative rabies DNA vaccine effectiveness through glycoprotein gene modifications.

Enhancing DNA vaccine effectiveness remains a challenge, especially if the desired goal is immunization efficacy after a single dose. The glycoprotein gene from the rabies virus Evelyn-Rokitnicki-Abelseth (ERA) strain was modified by mutation at amino acid residue 333 from arginine to glutamine. The modified and original unmodified glycoprotein genes were cloned separately and developed as DNA vaccines for immunization in mice. The intramuscular (IM) route using a single dose (100 microg) of a modified DNA vaccine showed virus neutralizing antibody induction by d30, and 80% of the mice survived a challenge in which 100% of unvaccinated controls succumbed. Similar results were obtained using a single dose (10 microg) by the intradermal (ID) route with one-tenth amount of the DNA administered. Administration of single dose of DNA vaccine with unmodified G did not result in the production of detectable levels of virus neutralizing antibody by d30. The results of the IM and the ID routes of administration were statistically significant (P<0.01). Based on these preliminary results, a modified glycoprotein gene from the ERA rabies virus strain may be an ideal candidate for DNA vaccine efficacy enhancement.

Mongoose rabies in southern Africa: a re-evaluation based on molecular epidemiology.

Relative to the developed world, rabies has been poorly studied in the vast African continent. The southern African countries of Zimbabwe and South Africa, however, are known to sustain a great diversity of lyssaviruses, with large biological variations amongst genotype 1 (rabies viruses) at present more apparent here than elsewhere on the continent. One recognized biotype of rabies virus in the subcontinent appears to be specifically adapted to a variety of mongooses, belonging to the Viverrinae subfamily (family Herpestidae) and are commonly referred to as viverrid viruses, although the term mongoose rabies would be more correct, considering the taxonomic status of the host species involved. It was our objective to study the genetic relationships of 77 rabies virus isolates of this mongoose biotype, isolated in South Africa and Zimbabwe, towards elucidation of the molecular epidemiology of this interesting group of African viruses. In our study of a 592 nucleotide sequence encompassing the cytoplasmic domain of the glycoprotein and the G-L intergenic region of the viral genomes, we provide the first comprehensive data on the molecular epidemiology of these viruses and indicate a history of extended evolutionary adaptation in this geographical domain. The molecular epidemiological observations reported here are highly unlikely to be limited to the small geographical areas of South Africa and Zimbabwe and illustrate the need for lyssavirus surveillance in the rest of sub-Saharan Africa and throughout the entire continent.

A comparison of DNA vaccines for the rabies-related virus, Mokola.

Mokola virus, a rabies-related virus, has been reported to date from the African continent only. Like rabies virus, it is highly pathogenic, causes acute encephalitis, and zoonotic events have been documented. Although believed to be rare, there has been an unexplained increase in the number of isolations of the virus in South Africa in recent years. We have cloned and sequenced the glycoprotein (G) and nucleoprotein (N) genes from a South African Mokola virus, and used these in the construction of different DNA vaccines for immunization against Mokola virus. Four vaccines, utilizing different promoters and DNA backbone compositions, were generated and compared for efficacy in protection against Mokola virus. In one of these, both the Mokola virus G and N genes were co-expressed. Two of the single G-expressing DNA vaccines (based on pSG5 and pCI-neo, respectively) protected laboratory mice against lethal challenge, despite major differences in their promoters. However, neither vaccine was fully protective in a single immunization only. Serological assays confirmed titers of virus-neutralizing antibodies after immunization, which increased upon booster vaccine administration. A third construct (based on pBudCE4) was less effective in inducing a protective immune response, despite employing a strong CMV enhancer/promoter also used in the pCI-neo plasmid. Dual expression of Mokola virus G and N genes in pBudCE4 did not enhance its efficacy, under the conditions described. In addition, no significant utility could be demonstrated for a combined prime-boost approach, as no cross-protective immunity was observed against rabies or Mokola viruses from the use of pSG5-mokG or vaccinia-rabies glycoprotein recombinant virus vaccines, respectively, even though both vaccines provided 60-100% protection against homologous virus challenge.

Prevalence of psammoma bodies in meninges and choroid plexuses of raccoons (Procyon lotor) from Parramore Island, Virginia.

Microscopic evidence of multifocal mineralizations (psammoma bodies) were seen in brains of 33/53 (62%) raccoons (Procyon lotor) necropsied on Parramore Island, Virginia. Most mineralized foci had concentric laminations and were present in small capillaries of meninges of the brain (15/33), in choroid plexus (3/33), or at both these sites (13/33). In 2 raccoons, the lesions were confined to the meninges of the proximal cervical spinal cord. In most cases, the affected vessels appeared to have been completely occluded. However, no evidence of ischemic changes in the brain parenchyma was seen, and none of the raccoons had abnormal neurologic signs prior to euthanasia. The condition appears to be a common incidental histopathologic finding in raccoons from the eastern United States. Although the exact cause of this condition is not known, a primary vascular insult with resultant dystrophic mineralization of the affected vessels is suspected.

The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure.

To provide a more defined and safer replacement for the human rabies immune globulin (HRIG) from pooled serum which is currently used for treatment of exposure to rabies virus we have developed a series of human rabies virus-specific monoclonal antibodies. Mouse-human heterohybrid myeloma cells producing rabies virus-specific human monoclonal antibodies were prepared using B cells obtained from volunteers recently-immunized with a commercial rabies virus vaccine (HDCV). Cell lines producing antibody which neutralized the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus strain in vitro were cloned and the resulting monoclonal antibodies characterized for isotype, specificity against a variety of rabies virus isolates, and neutralization capacity. The ability of the monoclonal antibodies to neutralize a variety of rabies virus strains in vitro correlated with their binding specificity for these viruses in an enzyme-linked immunoadsorbant assay (ELISA). A number of these antibodies have proven suitable for the formulation of a prophylactic human monoclonal antibody-based reagent which would provide significant advantages to the HRIG in having defined, reproducible specificity, lessened possibility of contamination with viral pathogens, and consistent availability.

Rabies in an American bison from North Dakota.

In North Dakota (USA) during April 1998, a ranched female bison (Bison bison) was found dead. At gross necropsy, there was profound hair loss and consolidated lung lobes. Intracytoplasmic neuronal inclusions suggestive of Negri bodies were observed in the brain stem and hippocampus, and a diagnosis of rabies was confirmed by the fluorescent antibody test. Antigenic typing demonstrated the occurrence of a rabies virus variant associated with skunks from the upper midwestern USA. This case of a rabid bison was one of only four such instances recorded from the USA over the past 40 yr, and is the first case report of rabies in a bison that reports clinical, pathologic, and antigenic findings. Although rabies in bison is rare, veterinarians and wildlife managers that work closely with such non-traditional species are reminded of the dangers that zoonoses such as rabies present.

Viral excretion in domestic ferrets (Mustela putorius furo) inoculated with a raccoon rabies isolate.

OBJECTIVE: To determine susceptibility, incubation and morbidity periods, clinical signs of infection, serologic response, and excretion of virus in domestic ferrets inoculated with rabies virus of raccoon origin. ANIMALS: 54 domestic ferrets. PROCEDURE: 5 groups of ferrets were inoculated IM with the rabies virus. Oral cavity swab specimens and saliva were obtained for virus isolation. Blood was obtained for virus-neutralizing antibody determination. If clinical signs were severe, ferrets were euthanatized immediately. Salivary gland and brain tissue was collected for virus isolation and rabies diagnosis, respectively. RESULTS: Of 51 inoculated ferrets, 19 (37%) were euthanatized with clinical signs of rabies. Mean incubation period was 28 days (range, 17 to 63 days). Clinical signs included ataxia, cachexia, inactivity, paresis, paraparesis, bladder atony, tremors, hypothermia, lethargy, constipation, paralysis, and anorexia. Two rabid ferrets manifested aggressive behavior. Mean morbidity period was 4 to 5 days (range, 1 to 8 days). Virus antigen was detected in brain tissue from all rabid ferrets (n = 19). Two rabid ferrets had detectable virus-neutralizing antibody. Of 32 ferrets that survived, only 1 seroconverted; survivors remained clinically normal throughout the observation period. Rabies virus was isolated from salivary glands of 12 of 19 (63%) rabid ferrets, and 9 (47%) shed virus in saliva. Initiation of virus excretion ranged from 2 days before onset of illness to 6 days after onset. CONCLUSIONS AND CLINICAL RELEVANCE: Rabies should be considered in the differential diagnosis for ferrets that have acute onset of paralysis or behavioral changes and a condition that rapidly deteriorates despite intense medical intervention.

Pathogenesis of experimentally induced rabies in domestic ferrets.

OBJECTIVE: To determine susceptibility, incubation and morbidity periods, clinical signs, serologic response, and excretion of virus in domestic ferrets inoculated with rabies virus. ANIMALS: 55 domestic ferrets. PROCEDURE: 5 groups of 10 ferrets were inoculated with rabies virus, IM, at doses of 10(5.5) to 10(1.5) median mouse intracerebral lethal dose. Ferrets were observed and behavior was recorded. Rectal temperature, body weight, and samples from the oral cavity and samples of saliva and blood were obtained. Virus isolation was attempted, using intracranial mouse inoculation and cell culture. Virus neutralizing antibodies were determined by rapid fluorescent focus inhibition test. Ferrets were euthanatized immediately if clinical signs were severe. Rabies was confirmed by direct immunofluorescent antibody test. RESULTS: Mean incubation period was 33 days (range, 16 to 96 days). Clinical signs included ascending paralysis, ataxia, cachexia, bladder atony, fever, hyperactivity, tremors, and paresthesia. Mean morbidity period was 4 to 5 days (range, 2 to 10 days). Virus antigen was detected in brain tissue from all clinically rabid ferrets. Ferrets given the highest viral dose were euthanatized and had VNA; ferrets receiving the next dilution also were euthanatized, but only 4 had seroconverted. Of 17 ferrets that survived, 5 seroconverted. Survivors remained clinically normal except for 1 that recovered with severe paralytic sequelae. Rabies virus was isolated from the salivary gland of 1 ferret that was euthanatized. CONCLUSIONS AND CLINICAL RELEVANCE: Rabies should be considered as a differential diagnosis in any ferret that has acute onset of paralysis or behavioral changes and a condition that rapidly deteriorates despite intense medical intervention.

Lymphosarcoma in a raccoon (Procyon lotor).

A case of lymphosarcoma in a captive adult female raccoon (Procyon lotor) from northeastern Pennsylvania (USA) was observed in 1991. Prior to its death the raccoon had lost weight. At necropsy the carcass was in poor body condition and had pale mucous membranes. The thoracic and abdominal lymph nodes were enlarged, soft, and pale tan. Microscopically, there was effacement of normal lymph node architecture by sheets of mononuclear cells. These were well-differentiated small lymphocytes with distinct cell borders. Nuclei of these cells were darkly stained and mitotic figures were frequently seen. Similar but lesser numbers of neoplastic cells were seen in the parenchyma of liver, spleen, and the pancreas. Since the neoplasm involved several organs, we propose that the condition was of multicentric origin. Gross lesions, histopathologic findings and the organs involved differed from a previously described case of lymphosarcoma in a raccoon.

Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America.

The silver-haired bat variant of rabies virus (SHBRV) has been identified as the etiological agent of a number of recent human rabies cases in the United States that are unusual in not having been associated with any known history of conventional exposure. Comparison of the different biological and biochemical properties of isolates of this virus with those of a coyote street rabies virus (COSRV) revealed that there are unique features associated with SHBRV. In vitro studies showed that, while the susceptibility of neuroblastoma cells to infection by both viruses was similar, the infectivity of SHBRV was much higher than that of COSRV in fibroblasts (BHK-21) and epithelial cells (MA-104), particularly when these cells were kept at 34 degrees C. At this temperature, low pH-dependent fusion and cell-to-cell spread of virus is seen in BHK-21 cells infected with SHBRV but not with COSRV. It appears that SHBRV may possess an unique cellular tropism and the ability to replicate at lower temperature, allowing a more effective local replication in the dermis. This hypothesis is supported by in vivo results which showed that while SHBRV is less neurovirulent than COSRV when administered via the intramuscular or intranasal routes, both viruses are equally neuroinvasive if injected intracranially or intradermally. Consistent with the above findings, the amino acid sequences of the glycoproteins of SHBRV and COSRV were found to have substantial differences, particularly in the region that contains the putative toxic loop, which are reflected in marked differences in their antigenic composition. Nevertheless, an experimental rabies vaccine based on the Pittman Moore vaccine strain protected mice equally well from lethal doses of SHBRV and COSRV, suggesting that currently used vaccines should be effective in the postexposure prophylaxis of rabies due to SHBRV.

Pyogranulomatous peritonitis associated with Nocardia sp.-like organisms in a raccoon (Procyon lotor).

During 1992 raccoons (Procyon lotor) were live-trapped in northeastern Pennsylvania (USA). In one of these animals a localized pyogranulomatous peritonitis was seen. Grossly a large mass with a central area of liquifactive necrosis was present in the anterior abdomen. Microscopically the lesion contained multiple colonies of filamentous organisms with histomorphologic and histochemical charcteristics resembling Nocardia sp. This appears to be the first report of Nocardia infection in the raccoon.

A Sarcocystis neurona-like organism associated with encephalitis in a striped skunk (Mephitis mephitis).

A Sarcocystis neurona-like organism was associated with granulomatous encephalitis in an ataxic male juvenile striped skunk (Mephitis mephitis) from Cape Cod, Massachusetts. Various stages of schizonts and merozoites of S. neurona were seen within some of the granulomata.

Experimental Toxoplasma gondii infection in raccoons (Procyon lotor).

Six raccoons (Procyon lotor) without detectable Toxoplasma gondii antibodies were used. Four raccoons were inoculated orally (2 with oocysts and 2 with tissue cysts) with ME49 strain of T. gondii and 2 raccoons were not inoculated with T. gondii. All raccoons remained clinically normal. Raccoons were killed between 59 and 61 days after inoculation and portions of their heart, skeletal muscle, and brain were digested in pepsin solution, and homogenates were bioassayed in mice. Toxoplasma gondii was isolated from all 4 inoculated raccoons; from the heart of 3, skeletal muscles of 2 and the brain of none. All 4 inoculated raccoons developed antibody titers > or = 1:1,600 in the modified direct agglutination test (MAT) using whole formalinized tachyzoites. Toxoplasma gondii antibody titers of the raccoons not inoculated with T. gondii remained < 1:25, and T. gondii was not isolated from their tissues. It was concluded that muscle tissue from multiple sites including the heart was the tissue of choice for conducting parasitologic surveys for T. gondii in raccoons. Evaluation of the sera of the experimentally infected raccoons in the Sabin-Feldman dye test, latex agglutination test, and the indirect hemagglutination tests indicated that the MAT detected antibodies faster and in higher titers than did the other serological tests.