Testing for immunogenic and protective properties of vaccine against tissue-inactivated rabies

The immunogenic and protective potency was tested of vaccine against inactivated tissue rabies developed from the Vnukovo-32 strain, and produced in the Bioveta state corporation at Ivanovice in Haná. In 21 days after an i. m. application of 3 cm3 of vaccine, the average titre of virus-neutralizing antibodies was found to be 1:47. In this period the animals were revaccinated in the same way. The average titre of virus-neutralizing antibodies was 1:99 three months after revaccination, 1:57 in six months and 1:24 in nine months. In this period a challenge test was performed in a dog using the dose of 10(5) MICLD50 of street rabies virus. This dose was implanted i. m. into masticatory muscles. Another dog was infected experimentally 18 months after immunization. The experiment has proved the good immunogenic potency of vaccine against inactivated tissue rabies and its ability to induce the protection of vaccinated dogs from the strees infection with street rabies virus. The control rabies vaccine of foreign make RABISIN was tested in a similar way.     

Rabies Antibody Titres in Vaccinated Reindeer

One dose of inactivated, adjuvanted rabies vaccine of cell culture origin (Rabisin) induced good but short-duration immunity in close to 100% of the 50 semi-domesticated reindeer (Rangifer tarandus tarandus L.) vaccinated. Most of the animals (44) had rabies virus antibody titre ≥1.5 IU/ml at 38 days after vaccination. Five animals had titre 0.5 IU. Antibody titres were not, however, present 1 year after primary vaccination in most animals. About 1 year (360-413 days) after primary vaccination, 22 of the 39 reindeer that could be sampled had rabies virus antibody titre <0.5 IU/ml.     

Gene immunization of mice with plasmid DNA expressing rabies virus glycoprotein

Gene immunization can be an effective vaccine strategy eliciting both humoral and cell-mediated immune responses. We constructed plasmid vectors expressing the full-length Vnukovo-32 rabies virus glycoprotein G under the control of CMV IE promoter and enhancer, adenovirus tripartite leader sequences and poly A signal of SV40. The gene vaccines were evaluated for the ability to elicit neutralizing antibodies and to protect BALB/c mice against lethal rabies virus challenge. First, mice were injected intramuscularly (i.m.) into the left hind leg and by the intradermoplantar (i.d.p.) route with equal amounts of plasmid DNA (0.25-0.1 mg). Two weeks later, immunization was boosted with an additional dose of the DNA. The immunized mice were challenged by intracerebral (i.c.) inoculation of CVS-27 (10-50 LD50) rabies virus. All mice produced anti-rabies virus neutralizing antibodies with a titre of > or = 1:45 after immunization with 0.1-0.4 mg of DNA. In challenge experiments, 83 to 91.6% protection was observed. These results confirm that a DNA vaccine could be a simple and effective solution for preventing the spread of rabies.     

Immune response to Japanese rabies vaccine in domestic dogs

To evaluate the immune response induced by Japanese rabies vaccine for veterinary use as international units (IU), we measured levels of rabies antibody in serum samples from dogs by the rapid fluorescent focus inhibition test (RFFIT). In dogs immunized with a reference vaccine (potency level of 3.1 IU/ml), prepared by the same method as that used to produce commercial vaccine, and its dilutions (1 : 2 or 1 : 4), neutralizing-antibody levels increased to 1.0-2.0 IU/ml over a period of 1 month and then decreased to 0.2-1.5 IU/ml over a period of 1 year after the first vaccination and showed a remarkable increase to 12-47 IU/ml after the second vaccination. Sixty-five (74.7%) of the 87 serum samples from domestic dogs that were tested were seropositive (> or =0.1 IU/ml). However, the seropositive rate in dogs less than 1-year old at the time of vaccination was low (57.1%), and the antibody levels in these dogs were not sufficiently high for the rabies antibody titre in serum to be maintained for 1-year. Levels of rabies antibody in all serum samples were also measured by the virus neutralizing test (VNT), and a strong correlation (r > 0.95) was found between the results of the RFFIT and those of the VNT.     

Immunogenicity and efficacy of the oral rabies vaccine SAD B19 in foxes

Studies on the immunogenicity and efficacy of SAD B19 attenuated rabies virus vaccine in foxes under laboratory conditions were conducted. Twenty-seven foxes (Vulpes vulpes) were offered a vaccine bait containing 10(6.3) FFU/ml SAD B19. Blood samples were collected 60, 110 and 190 days post-vaccination. On day 190 post vaccination the animals and 14 controls were challenged with a canid street rabies virus. Twenty-four of the 26 vaccinated foxes (92.3%) survived the challenge, whereas all the controls died from rabies. The two vaccinated foxes that did not survive the challenge did not show any detectable level of rabies neutralizing antibodies at any time after vaccination. The geometric mean titres (GMT) of foxes that seroconverted after vaccination were 43.5, 33.9 and 43.5 IU/ml 60, 110 and 190 days post-vaccination, respectively. Furthermore, to test the vaccine virus under sub-optimal conditions five naive and nine previously vaccinated vixens received 2 ml SAD B19 (10(6.7) FFU/ml) by direct administration of the vaccine virus into the oral cavity shortly before or during pregnancy. All vixens seroconverted above the threshold of 0.5 IU/ml. No booster effect was observed in the immune response of the previously vaccinated animals.     

Cell-mediated immune response to rabies virus in dogs following vaccination and challenge

Peripheral blood lymphocytes (PBL) from non-vaccinated dogs and from dogs either vaccinated intramuscularly (IM) or subcutaneously (SC) with an inactivated rabies virus vaccine (Rabguard-TC, Norden Laboratories, Lincoln, NE) or intramuscularly with an attenuated rabies virus vaccine (Endurall-R, Norden Laboratories, Lincoln, NE) were exposed in vitro to rabies virus. Blastogenesis of PBL was measured by incorporation of 3H-thymidine into the DNA of proliferating cells in the presence of a suboptimal concentration of phytohemagglutinin (PHA). Following the first vaccination, there was no difference in the blastogenic response of lymphocytes from dogs vaccinated IM with either the inactivated or attenuated rabies virus vaccines. The inactivated rabies vaccine stimulated as great or greater blastogenic response when it was given SC. The PBL from non-vaccinated control dogs were not stimulated by rabies virus. Dogs vaccinated with the inactivated vaccine developed a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus. Nonvaccinated control dogs did not develop a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus.     

Exposure of hooded capuchin monkeys (Cebus apella cay) to a rabid bat at a zoological park.

On 27 May 1999, a big brown bat (Eptesicus fuscus) was discovered on an island exhibit at the Denver Zoo that contained a troop of 15 hooded capuchin monkeys (Cebus apella cay). The monkeys were attacking the bat when it was discovered. The bat was collected and humanely euthanatized without direct handling and submitted to the Colorado Department of Public Health and Environment Virology Laboratory for rabies evaluation. The monkeys had not been vaccinated against rabies virus. The next day, the laboratory confirmed that the bat was positive for rabies. The recommendations from the Colorado Department of Public Health and Environment and the Centers for Disease Control and Prevention were to euthanatize the monkeys or quarantine them and comply with the human nonvaccinated postexposure protocol. A 1-ml dose of a killed rabies vaccine was administered i.m. in the hip on each of days 2, 7, 12, 19, and 33 postexposure, and a single dose of human rabies immune globulin was administered i.m. 5 days postexposure. Blood was collected under anesthesia in order to evaluate the immune response after rabies vaccination from six monkeys 5 days postexposure, six monkeys 19 days postexposure (five of the six monkeys were the same monkeys bled 5 days postexposure), 15 monkeys 67 days postexposure, and 13 monkeys approximately 1 yr postexposure. All of the monkeys developed and maintained levels of rabies virus neutralizing antibody above 0.05 IU/ml by 67 days postexposure. Although a serologic titer of 0.05 IU/ml indicates an adequate human response after rabies vaccination, no similar information is available for nonhuman primates. To date, none of the monkeys has succumbed to rabies.     

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.     

Antibody response to an anti-rabies vaccine in a dog population under field conditions in Bolivia

Rabies remains an important public health issue in Bolivia, South America. Public concern and fears are most focussed on dogs as the source of rabies. The objective of the present study was to assess immunity of an inactivated suckling mouse brain vaccine against canine rabies used for the official vaccination campaigns under field conditions in an endemic area of rabies in Bolivia. A total of 236 vaccinated and 44 unvaccinated dogs in Santa Cruz de la Sierra, selected using stratified random sampling, were investigated in order to obtain owned dog characteristics and antibody titres against rabies in April 2007. The proportion of vaccinated dogs with an antibody titre exceeded the protection threshold value of 0.5 EU/ml was 58% [95% confidence intervals (CI): 52-65], indicating that vaccination is likely to elicit an antibody response (odds ratio 6.3, 95% CI: 1.2-11.5). The range of geometric mean of antibody titre for vaccinated dogs (0.89 EU/ml; 95% CI: 0.75-1.04) was considered to meet the minimal acceptable level indicating an adequate immune response to the vaccine. However, the titre level was not satisfactory in comparison with the results from other field investigations with inactivated tissue culture vaccines. It is recommended for public health authorities to (1) consider modernizing their vaccine manufacturing method because the level of immunity induced by the current vaccine is comparably low, (2) conduct frequent vaccination campaigns to maintain high levels of vaccination coverage, and (3) actively manage the domestic dog population in the study area, which is largely responsible for rabies maintenance.     

Antibody response in mice, rabbits and pigs in response to vaccination with an inactivated oil vaccine containing rotavirus and Escherichia coli strains with K88, K99 and 987P fimbrial antigens]

In trials with mice, rabbits and weanling piglets, four experimental charges of a combined inactivated oil vaccine against diarrhoeas in mammals were tested: the vaccine was to be implanted to sows and it contained porcine rotavirus (PRV); two charges also contained bovine rotavirus and bacterins of enterotoxicogenic strains of E. coli with protective antigens K88, K99 and 987P. At low starting antibody titres the twofold i.m. implantation of 0.2 ml vaccine stimulated in mice the production of antibodies to reach the average titre value of 1:128 against PRV and of 1:256 against BRV; in rabbits the twofold i.m. implantation of 2 ml vaccine stimulated the antibody development to reach the average titres of 1:508 or 1:500, and in weanlings after the twofold i. m. implantation of the vaccine the titres were 1:1028 or 1:469; in mice agglutination antibodies to antigen K88 had the average value of 1:68, to antigen K99 the value of 1:44 and to antigen 987P the value of 1:8192; in rabbits the respective titres were 1:285, 1:136 and 1:6006 and in pigs 1:570, 1:631 and 1:8192. The antibodies to antigen 987P persisted at the same level in pigs for six months. Even though there was a gradual decrease in the antibodies to antigens K88 and K99, at that time the values were 9.8 times, or 15.2 times higher than the starting values, and only the antibodies to PRV dropped to the pre-vaccination level. Repeated administration of vaccine to pigs after six months from revaccination induced, with the exception of antigen 987P, an increase in antibodies in a fortnight to reach such titres that were recorded after revaccination.     

Cerebrospinal fluid analysis of rabid and vaccinia-rabies glycoprotein recombinant, orally vaccinated raccoons (Procyon lotor)

Cerebrospinal fluid obtained from clinically normal free-ranging raccoons was analyzed and compared with CSF obtained from raccoons vaccinated orally with vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine and subsequently challenged peripherally with street rabies virus, and CSF from naive, rabies virus challenge-exposed control raccoons. Significant differences were not found in CSF of free-ranging or V-RG recombinant virus vaccine recipient raccoons, and there was no evidence of CNS invasion by V-RG virus. The CSF of naive, rabies challenge-exposed control raccoons contained high numbers of lymphocytes and monocytes, compatible with rabies virus encephalitis. Although V-RG orally vaccinated challenge-exposed raccoons were protected from lethal rabies virus infection, a mild lymphocytic pleocytosis was evident at 90 days after challenge exposure.     

Use of infrared thermography to detect signs of rabies infection in raccoons (Procyon lotor).

Infrared thermography was evaluated as a technique to determine if raccoons (Procyon lotor) experimentally infected with rabies virus could be differentiated from noninfected raccoons. Following a 10-day adjustment period, raccoons (n = 6) were infected with a virulent rabies street strain raccoon variant by injection into the masseter muscle at a dose of 2 x 10(4) tissue-culture infectious dose (TCID50) in 0.2 ml (n = 4) or 10(5) TCID50 in 1 ml (n = 2). Five of the six raccoons developed prodromal signs of rabies 17 to 22 days postinoculation (PI) and distinctive clinical signs of furious rabies between 19 and 24 days PI. At the time of euthanasia, which occurred 2 days after the onset of clinical signs of rabies, these five raccoons tested positive for rabies virus in brain tissue. Infrared thermal images of each raccoon were recorded twice daily during the preinoculation and PI periods. No apparent differences were identified among thermal temperatures compared among days for the eye, average body surface, and body temperature recorded from subcutaneous implants throughout the experiment for any of the six raccoons. However, increases in infrared surface temperature of the noses and differences in the visual thermal images of the noses were detected when animals began showing clinical signs of rabies. Differences were detected among the mean infrared nose temperatures for the disease progression intervals (F3.12 = 70.03, P < 0.0001). The mean nose temperature in the clinical rabies stage (30.4 +/- 3.5 degrees C) was significantly elevated over the prodromal stage (F1,12 = 151.85, P < 0.0001). This experiment provides data indicating that infrared thermography can be used in an experimental setting to detect raccoons in the infectious stage and capable of exhibiting clinical signs of rabies.     

Protection of dogs against death from experimental rabies by postexposure administration of rabies vaccine and hyperimmune globulin (human).

Two experiments on simulated postexposure treatment were carried out in dogs using human rabies immunoglobulin (RIGH) and human diploid cell vaccine for human use. In one experiment, when animals were challenged by injecting street virus into the masseter muscle and treated with a combination of RIGH and vaccine, 50% of the animals were protected from rabies. In the other trial, in which animals were challenged by injecting the virus into the femoral muscle, treatment with RIGH and vaccine protected all the animals against rabies. To our knowledge this is the highest rate of postexposure survival in animals reported to date. In addition, five out of eight (62.5%) dogs that received RIGH alone after the virus challenge were protected, while none of the animals receiving vaccine alone were protected from rabies. These trials suggest that animals can be protected from rabies by postexposure treatment. The route of exposure and timing of the administration of vaccine and hyperimmune serum would seem to be important.     

Seroconversion in captive African wild dogs (Lycaon pictus) following administration of a chicken head bait/SAG-2 oral rabies vaccine combination

This study determined the proportion of captive juvenile and adult African wild dogs (Lycaon pictus) that developed protective titres of rabies neutralising antibodies following ingestion of a chicken head bait/SAG-2 oral rabies vaccine combination. A single chicken head containing 1.8 ml of SAG-2 vaccine (10(8.0) TCID50/ml) in a plastic blister was fed to each of eight adult and three juvenile wild dogs. Bait ingestion resulted in a significant rise in serum neutralising antibody titres. Overall seroconversion rate was eight out of 11 (72.7%), and all the puppies and five out of eight (62.5%) adults showed potentially protective levels of antibodies on day 31. The mean post-vaccination neutralising antibody titre was within the range reported to be protective against challenge with virulent rabies virus in other species.     

The persistence of rabies virus antibodies in the sera of fox cubs

The persistence of maternal antibodies transfer from rabies-immune vixens to their fox cubs was studied. Eight vixens (Vulpes vulpes) were vaccinated 1 month before pregnancy with Lysvulpen vaccine for oral vaccination of foxes. Twenty-one were foxes born at the first half of April. The geometrical mean titre of rabies neutralizing antibodies of fox cubs sampled in May was 1.31 IU/ml and has dropped successively to 0.54 IU/ml in June samples and to 0.18 IU/ml in July samples. It has been proven that the duration of rabies maternal antibodies in fox cubs was limited to 2 months after birth.     

Serosurveillance of rabies antibodies in dogs in Mumbai region by using indirect ELISA

Rabies is a highly fatal viral infection of the central nervous system affecting all warm-blooded animals including humans. To implement the preventive and control measures, it is important to decide the status of anti-rabies antibodies in dogs. Out of 120 serum samples, 47 (39.2 %) serum samples, showed an antibody titre equal to or above the cut off value of 0.5 IU/ml. The maximum number of dogs showed anti-rabies antibody titres equal to or above the cut-off value of 0.5 IU/ml after <1 month pre-exposure to the rabies vaccine. In 15 serum samples of pet dogs, we observed 13 (86.66 %) dogs with protective anti-rabies antibody titre. Statistical analysis suggests that the age of the animal had no significant effect on anti-rabies antibody titre in vaccinated pet dogs. The overall low seroprevalence of anti-rabies antibody in stray dogs indicates their susceptibility to rabies infection and thus posing a risk of rabies to other animals and humans.     

Survival of rabies virus under external conditions

The survival of the street rabies virus in a 10% suspension, prepared from the salivary gland of a naturally infected fox, was studied under various conditions. A bioassay and titration on mice were used for the identification of the virus in different intervals. The heat inactivation of the virus in a suspension kept in a test tube at the temperatures of 20 degrees C and 37 degrees C was performed in two stages. The rapid reduction of the titre within 24 hours was followed by a slower decrease, reaching total inactivation after 96 hours at both temperatures. When the virus was tested by means of the contamination of various substrates (glass, metal sheet, plant leaf) with 0.1 ml of infection suspension in a thin layer, the longest survival of the virus was recorded at the temperature of 5 degrees C--144 hours. At the temperature of 20 to 21 degrees C the virus kept its activity on the glass and plant leaf for 24 hours and on the metal sheet for 48 hours although the applied drops looked like having dried. The temperature of 30 degrees C combined with intensive sunshine devitalized the virus within 1.5 hours, whereas without sunshine the virus still remained active, at the temperature of 30 degrees C, after 20 hours.     

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.     

Results of vaccination of Asian elephants (Elephas maximus) with monovalent inactivated rabies vaccine

OBJECTIVE: To evaluate the humoral immune response of Asian elephants to a primary IM vaccination with either 1 or 2 doses of a commercially available inactivated rabies virus vaccine and evaluate the anamnestic response to a 1-dose booster vaccination. ANIMALS: 16 captive Asian elephants. PROCEDURES: Elephants with no known prior rabies vaccinations were assigned into 2 treatment groups of 8 elephants; 1 group received 1 dose of vaccine, and the other group received 2 doses of vaccine 9 days apart. All elephants received one or two 4-mL IM injections of a monovalent inactivated rabies virus vaccine. Blood was collected prior to vaccination (day 0) and on days 9, 35, 112, and 344. All elephants received 1 booster dose of vaccine on day 344, and a final blood sample was taken 40 days later (day 384). Serum was tested for rabies virus-neutralizing antibodies by use of the rapid fluorescent focus inhibition test. RESULTS: All elephants were seronegative prior to vaccination. There were significant differences in the rabies geometric mean titers between the 2 elephant groups at days 35, 112, and 202. Both groups had a strong anamnestic response 40 days after the booster given at day 344. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the ability of Asian elephants to develop a humoral immune response after vaccination with a commercially available monovalent inactivated rabies virus vaccine and the feasibility of instituting a rabies virus vaccination program for elephants that are in frequent contact with humans. A 2-dose series of rabies virus vaccine should provide an adequate antibody response in elephants, and annual boosters should maintain the antibody response in this species.     

Rabies antibody titres in vaccinated dogs

In a field study, rabies virus neutralizing antibody titres were determined by the microtest modification of the rapid fluorescent focus inhibition test before and after primary vaccination in 30 puppies, and before and after booster vaccination in 59 previously vaccinated dogs. A commercial modified live virus vaccine was used. Three weeks after primary vaccination the mean antibody titre was 102 ± 90, but only 24 dogs presented for booster vaccination had detectable antibody levels (mean titre 12 ± 16). The antibody responses three weeks after booster vaccination (mean 380 ± 216) were significantly greater than the responses to primary vaccination. It was concluded that previously vaccinated dogs could have an anamnestic response to booster vaccination, even when antibodies were not detected in their sera before revaccination.