The occurrence of rabies in pre-Columbian Central America: an historical search.

Rabies is considered one of the oldest infectious diseases known to humans. However, the first written reports on rabies cases in the Americas did not appear until the first decade of the 18th century from Mexico. In an attempt to clarify if the disease was already present in pre-Columbian times, we searched for evidence in the Maya and Aztec cultures. Other sources of information were early manuscripts written by the conquistadors and early explorers. We did not identify any unequivocal direct evidence that the disease rabies was known in pre-Columbian Central America but sufficient circumstantial evidence is available suggesting that (bat) rabies was already present in these early times.

Epidemic strain of Venezuelan equine encephalomyelitis virus from a vampire bat captured in Oaxaca, Mexico, 1970.

A vampire bat, Desmodus rotundus, captured in Oaxaca, Mexico, in August 1970, was found to be infected with the epidemic strain of Venezuelan equine encephalomyelitis virus at the same time that an equine epizootic was occurring there.

Experimental rabies in the vampire bat.

Vampire bats were inoculated intramuscularly and subcutaneously with varying doses of rabies virus to simulate bites by rabid animals in nature. Daily saliva samples were then taken from these animals to determine whether they excreted virus and for how long. Vampire bats appear to react to rabies virus as do other animals, with variable incubation periods, some excretion of virus in the saliva, but no prolonged excretion "carrier state."

Intermittent excretion of rabies virus in the saliva of a dog two and six months after it had recovered from experimental rabies.

A dog inoculated with a rabies virus isolate from the saliva of an apparently healthy Ethiopian dog was followed for more than 9 months. Saliva and blood specimens were collected three times weekly and cerebrospinal fluid weekly. Saliva samples collected on days 42 and 169 after the dog's recovery produced fatal rabies infections in mice inoculated intracerebrally.

Early antibody responses to rabies post-exposure vaccine regimens.

The aim of post-exposure rabies vaccine treatment is to induce immunity, measured as neutralizing antibody, as fast as possible. This is especially important in the tropical rabies-endemic areas where simultaneous passive prophylaxis with hyperimmune serum is not practicable in the majority of cases. We compared the rate of production of antibody during the first two weeks, by six vaccine regimens in 118 subjects using two tissue culture vaccines, human diploid cell strain vaccine (HDCSV) and purified Vero cell rabies vaccine (PVRV). No antibody was detected on day 5. On day 7, the highest seroconversion rate was seen in subjects given HDCSV intramuscularly at two sites on days 0 and 3 (7 of 15), but this was not significantly different from the group with the lowest rate: the conventional single-site intramuscular regimen. All subjects had antibody by day 14, at which time the highest geometric mean titer was in the group vaccinated with 0.25 ml doses of diploid cell vaccine given subcutaneously at eight sites. This regimen, together with the standard single-site diploid cell vaccine and an eight-site intradermal regimen of the same product gave significantly higher titers than the two-site intramuscular regimens of either product. No single immunization schedule emerges as best, so the speed of antibody response, economy, and the skill needed for intradermal injection should be considered when deciding on the optimum regimen for use in a particular geographic area.

Rabies serum neutralizing antibody in mongooses from Grenada.

During a four-year study on Grenada, 4,754 mongooses were examined, of which 100 (2.1%) were rabid. Of 1,675 mongooses tested for rabies serum neutralizing (SN) antibody, 498 (30%) were positive. During these four years (1971-74) the antibody prevalence rate increased from 20.8% to 43.2%, whereas the number of rabid mongooses decreased from 3.5% to 0.6%. Naturally acquired antibody was monitored in 20 captive mongooses for up to 35 months, and it was still circulating in 18 when monitoring ceased. The highest titre recorded was 1:1,400, and the rate of fall was greatest in mongooses with high initial titres. High titres of naturally acquired antibody suggest recent rabies activity. 14 mongooses vaccinated parenterally with 1.0 ml of attenuated ERA vaccine showed substantial increases in antibody titres; most had titres greater than 1:1,000 one month later and maintained a high titre for several months. The highest recorded was 1:34,800. The virus titres of brain material from rabid mongooses ranged from 10(1.8) to 10(4.3) and, in some cases, were only slightly lower than those of salivary gland tissue (range, 10(1) to 10(5.6).

Evaluation of an animal rabies vaccine by use of two types of potency tests.

OBJECTIVE: To evaluate the potency of an inactivated animal rabies vaccine for domestic animals by use of 2 types of potency tests after challenge exposure with a laboratory standard virus or 1 of 5 viruses obtained from various wildlife species. ANIMALS: 384 mice vaccinated twice intraperitoneally; 384 mice vaccinated once IM. PROCEDURE: Mice vaccinated with an inactivated, adjuvanted rabies vaccine for domestic animals were challenge exposed with the common fixed challenge virus or 1 of 5 rabies viruses obtained from wild animal species (street viruses) that most commonly transmit the virus in the United States and Canada. Potency tests included 2 types of antigen extinction tests: the National Institutes of Health (NIH) test and the Centers for Disease Control test. RESULTS: Results of both tests indicated that protection was highest against raccoon and bat viruses. Marked differences were detected in the relative potency ratios for the NIH versus the Centers for Disease Control tests, though the relative potencies themselves (against the street viruses) did not differ markedly. CONCLUSIONS: The markedly reduced potency against the street viruses indicated by the NIH test results was suggestive of an inherent bias associated with double intraperitoneal vaccination and intracerebral challenge exposure, whereas the single IM vaccination and IM challenge exposure reduced that bias.

Recovery from clinical rabies of 2 dogs inoculated with a rabies virus strain from Ethiopia.

Two dogs, inoculated with a strain of rabies virus from Ethiopia, showed typical signs of rabies 8 days after inoculation. After 3 or 4 days with a deterioration in the physical condition, both animals began to recover, as shown by increased muscular movements, reaction to stimuli, awareness of surroundings, and attempts to rise. Both animals recovered completely, although 1 then died of Pseudomonas bacterial pneumonia. An increase in serum-neutralizing antibody and in CSF or brain-neutralizing antibody was noted in both animals. Such concentrations have been noted only in animals or persons that recovered from rabies.

Oral vaccination of dogs fed canine adenovirus in baits.

Six groups of 5 dogs each were fed dilutions of canine adenovirus-2, either as raw liquid or after insertion into cornmeal baits. By the fourth week after vaccination, 29 of the 30 dogs developed high titers of serum-neutralizing antibodies to the virus.

Minimal interferon induction in dogs, using a modified polyriboinosinic-polyribocytidylic acid complex.

Adult Beagles failed to respond to high concentrations of interferon (IF) when they were injected with a nuclease-resistant complex poly I:C with poly-L-lysine and carboxymethylcellulose (PICLC), by the IV or intrathecal route. An IV dose of 1 mg of PICLC/kg of body weight was lethal to 1 of 3 adult dogs, but induced IF in only 2 dogs. Smaller doses were less toxic, but also were less effective. The injection of a high dose of a known IF inducer (3 X 10(8) egg LD50 of Newcastle disease virus) also failed to induce IF in Beagles. Interferon could not be induced in vitro when primary cultures of neonatal dog lung or kidney were treated with cultures of neonatal dog lung or kidney were treated with PICLC. When these primary cell cultures were compared with the cell line Madin-Darby canine kidney in an IF assay, no difference in sensitivity to IF-induced protection from infection with vesicular stomatitis virus could be shown. This indicated that the sensitivity of the Madin-Darby cell line was not the only factor in determining the lack of IF response in dogs and indicates that the dogs are poor responders to IF induction.

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.

Immunization of arctic foxes (Alopex lagopus) with oral rabies vaccine.

Arctic foxes (Alopex lagopus) were successfully immunized against rabies using an orally-administered, liquid SAD-BHK21 live virus vaccine in a sausage bait. Immunization was determined by serologic response and by resistance to challenge with an arctic rabies virus strain. Virus was not shed in saliva following oral vaccination, indicating that arctic foxes would not infect other foxes after ingesting this vaccine. High antibody levels were present in all experimental foxes 2 wk following initial vaccination. A booster vaccination at 56 wk induced a significant serologic response within 1 wk, suggesting an anamnestic response but titers began to decline within 8 wk in most foxes. Foxes were observed for 16 mo following the challenge and exhibited no symptoms of rabies. The SAD-BHK21 rabies vaccine in a sausage bait system has a strong potential for vaccinating wild populations of arctic fox.

Plasma marking of arctic foxes with iophenoxic acid.

Six arctic foxes (Alopex lagopus) were marked with iophenoxic acid (IA), a substance which elevates concentrations of protein-bound iodine in blood plasma. Buccal absorption of IA was determined by placing 20 mg IA dissolved in 100% ethyl alcohol on the tongue. Blood samples collected from 1 to 36 wk following exposure showed that all foxes were marked already at 1 wk and continued until 13 wk; two foxes were still marked at 36 wk. Clearance rates for iodine varied with initial dose response, and those foxes with high 1-wk iodine concentrations excreted iodine more rapidly than those with lower initial concentrations; by 13-wk excretion rates were similar.

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.

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.

Acceptance of simulated oral rabies vaccine baits by urban raccoons.

In summer 1986, a study was conducted to evaluate raccoon (Procyon lotor) acceptance of oral baits that could be used for rabies vaccination. One thousand wax-coated sponge bait cubes were filled with 5 mg of a seromarker (iophenoxic acid), placed in polyethylene bags, and hand-distributed in an 80 ha area within an urban National Park in Washington, D. C. (USA). After 3 wk, target and nontarget animals were trapped and blood samples collected to evaluate bait uptake. Thirty-three of 52 (63%) raccoons had elevated blood iodine levels indicating they had eaten at least one bait, 13 (25%) were negative, and six (12%) had marginal values. These results indicate that sponge baits hand-placed at a density of 12.4/ha can reach a significant proportion of an urban raccoon population. Implications for oral rabies vaccination of raccoons are discussed.

Acceptance of candidate baits by domestic dogs for delivery of oral rabies vaccines.

Protocols for evaluating oral rabies vaccine baits for domestic dogs were field tested in central Mexico, after which dog-food manufacturers and suppliers to the pet-food industry were advised as to potential ingredients for use in prototype dog baits. Bait-preference trials in which confined dogs were used were then undertaken, followed by field tests of free-ranging farmer-owned dogs in three towns in the Nile River Delta region of Egypt. Both confined and free-ranging dogs showed strong preferences for certain baits or bait coatings (poultry, beef tallow, cheese, egg and a proprietary product). Fish-meal polymer baits, widely used for wildlife species, were less preferred. In Egypt, a commercial dog-food-meal bait coated with beef tallow and dry cheese, was consumed at a rate approaching that of a chicken-head bait. The percentage baits that were actually eaten after they had been offered to dogs, ranged from 71-96% for household dogs tested in Mexico, 65-91% for confined dogs (beagles and mixed breeds) tested in the United States, and 32-88% for farmer-owned dogs tested in Egypt.

Effect of inosiplex on the humoral and cell-mediated immune responses to intradermal human diploid cell rabies vaccine.

Antigen-stimulated lymphocyte transformation was studied in recipients of intradermal human diploid cell rabies vaccine (HDCV). HDCV was administered intradermally at 8 different anatomical sites, 0.1 ml each, on day 0; followed by another 4-site injection on day 7. Rabies antigen-stimulated in vitro proliferative response was evident as early as 7 days after starting immunization. It reached a peak on day 14 and had declined by day 28. The cellular proliferative response preceded and roughly correlated with the antirabies antibody response. Simultaneous administration of inosiplex, an antiviral and immunopotentiating drug, during the first 10 days of intradermal HDCV immunization did not result in heightened antibody titres or cell-mediated immune response to the vaccine. The number of T cells and the lymphocyte proliferative response to phytohaemagglutinin in inosiplex-treated vaccinees were similarly not significantly different from untreated controls. Our results confirm other previous findings that a specific cell-mediated immune response can be consistently and rapidly induced by an intradermal regimen of HDCV immunization. The addition of inosiplex to this regimen did not enhance the humoral or cell-mediated immune responses to the vaccine. The apparent lack of immunostimulating effect of inosiplex in this setting may be the result of several factors such as the immunization schedule and the immunologic parameters examined.