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.     

Mutants of rabies viruses in skunks: immune response and pathogenicity.

In studies to develop an oral rabies vaccine for wildlife, the immune response to and pathogenicity of two types of mutants of rabies viruses were examined. Forty-five small plaque mutants were selected from cultures of ERA rabies virus treated with 8-azaguanine or 5-fluorouracil and tested for pathogenicity in mice. Two of these mutants AZA 1 and AZA 2 (low pathogenicity in mice) were given to skunks by oral (bait), intestinal (endoscope) and intramuscular routes. Additionally, challenge virus standard (CVS) rabies virus and mutants of this and ERA rabies virus (CVS 3766 and 3713, and ERA 3629) that were resistant to neutralization by specific antiglycoprotein monoclonal antibodies (and apathogenic in mice) were tested by various routes in skunks. Skunks given AZA 1 and AZA 2 were challenged at three months postinoculation with street rabies virus. After oral administration, there were very low rates of seroconversion with AZA 1 and AZA 2 and on challenge only 2/7 given AZA 1 and 1/8 given AZA 2 survived. None of the skunks given the other mutants orally seroconverted. AZA 2 produced a high rate of seroconversion (8/8) by the intestinal route and all challenged skunks in this group survived (7/7). All skunks vaccinated intramuscularly with AZA 1 (4/4) or AZA 2 (4/4) developed high levels of rabies neutralizing antibodies and survived challenge. The mutant CVS 3766, while apathogenic when given intracerebrally to adult mice, was consistently pathogenic by this route (and intranasally) in skunks. These results demonstrate that skunks are highly resistant to oral immunization by live rabies virus vaccines and that pathogenicity (by intracerebral route) of the mutant CVS 3766 is markedly different in mice and skunks.     

Experimental rabies in skunks: Immune response and salivary gland infection

Groups of striped skunks (Mephitis mephitis) were inoculated intramuscularly with graded doses of street rabies virus. At various intervals after inoculation, saliva and sera were tested for rabies virus and neutralizing antibodies, respectively. Skunks that developed rabies were killed in terminal stages of the disease and the following examinations were made: titers of virus and antibody in submandibular salivary glands and brain, extent of immunofluorescence in submandibular salivary glands, and histologic examination of various tissues.

Skunks that received inocula containing 4 × 10⁴ to 4 × 10⁵ mouse intracerebral lethal dose₅₀ (MICLD₅₀) had detectable serum neutralizing antibodies by 7–12 days postinoculation; however, most of the skunks that received lower doses (40 to 4 × 10³ MICLD₅₀) did not have detectable serum neutralizing antibodies until clinical signs began. In the salivary glands, slight and extensive immunofluorescence corresponded to high and low titers of tissue neutralizing antibody. Also low viral titers were associated with high tissue neutralizing antibody titers. There was a close correlation between viral titers in right and left submandibular salivary glands.

The results suggest that the immune response can impede the process of infection of the salivary glands resulting in lack of antigen or low amounts of antigen in this tissue. This could occur through interference with centrifugal neural transport of virus and/or neutralization of virus during transfer from neural elements to epithelial cells. Lack of infectious virus or low viral titers in salivary glands containing antigen and high levels of tissue neutralizing antibodies can be caused partly by postmortem virus neutralization (during viral titration).     

Safety and immunogenicity of a vaccine bait containing ERA strain of attenuated rabies virus.

Ninety percent of foxes fed commercial ERA vaccine in a specially designed bait developed rabies serum neutralizing antibodies. The vaccine bait did not cause clinical signs of rabies when consumed by foxes, raccoons, skunks, dogs, cats, cattle and monkeys. When presented, in the laboratory, to wild rodents of the species Microtus, Mus musculus and Peromyscus, the vaccine baits caused vaccine-induced rabies only in Mus musculus. Laboratory mice of the CD-1 and CLL strain were susceptible to vaccine-induced rabies; however, studies showed that transmission of virus to other animals did not occur. These studies suggest that the vaccine bait described could be useful in a rabies control program in areas where foxes and wild dogs are the principal vectors.     

牛传染性鼻气管炎病毒LNM株致弱疫苗免疫效力的研究

为检测牛传染性鼻气管炎病毒(IBRV)抗体在免疫牛体内的产生及其消长规律,评价IBRV LNM株致弱疫苗的保护效力,并确定免疫持续期,本实验对免疫试验组牛每头颈部肌肉接种制备的IBRV LNM株致弱疫苗104.5 TCID50/mL,监测血清抗体效价,进行免疫期的确定。在疫苗免疫后的6个月、9个月和12个月分别抽取3头免疫组和两头对照组牛采用IBRV-LN01/08强毒株进行攻毒试验,每头牛的攻毒剂量为107.0 TCID50/mL。结果显示疫苗免疫后12个月时,中和抗体效价仍维持在1∶11以上。攻毒结果显示3个时间点强毒攻击后,疫苗对免疫牛均具有良好的保护力。研究表明IBRV弱毒疫苗可有效地保护免疫牛抵抗IBRV强毒株的攻毒,其免疫持续期最少为12个月。     

Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line.

The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes.     

The serological response of young dogs to the Flury LEP strain of rabies virus vaccine

The serological response of puppies from Nigeria to live Flury low egg passage (LEP) rabies vaccine was determined. Two sets of puppies were used: one set from rabies-vaccinated bitches and another set from non-vaccinated bitches. Puppies were vaccinated intramuscularly with Flury LEP strain rabies vaccine and serially bled from the 4th week to the 30th week. Serum rabies virus neutralizing antibodies (VNA) were measured by a modified rapid fluorescent focus inhibition test (RFFIT). Puppies from non-vaccinated bitches responded well to vaccination after the 4th week and through to the 10th week of age, showing a progressive increase in VNA. In contrast, puppies from vaccinated bitches responded well to rabies vaccination only at 10 weeks of age, although detectable maternal rabies VNA and rabies anti-ribonucleoprotein (RNP) antibodies had decreased by 6 weeks post partum.     

Evaluation of an inactivated rabies virus vaccine in domestic ferrets

Efficacy of an SC-administered commercial inactivated vaccine for prevention of rabies was evaluated in domestic ferrets. Ferret immunity was challenged by the IM inoculation of street rabies virus. All ferrets developed titers of rabies virus-neutralizing antibodies within 30 days of vaccination (geometric mean titer [GMT] = 154, n = 41) that were maintained for at least one year (GMT = 106, n = 36), compared with no seroconversion in controls (GMT less than 5, n = 39). Following rabies virus challenge inoculation, 89% (32/36) of vaccinated ferrets survived vs less than 6% (2/38) survival in control ferrets. These results demonstrate the protective efficacy of a commercial, inactivated rabies vaccine of at least one year's duration for domestic ferrets.     

Evidence of canine distemper virus infection in skunks negative for antibody against rabies virus

Between January 1981 and October 1985, brain tissue specimens from 192 skunks that were negative for antibodies against rabies virus were obtained from 2 Illinois Public Health laboratories (A and B). Brain lesions were detected microscopically in specimens from 17 of the 91 (18.7%) skunks from laboratory B and in specimens from 30 of the 101 (29.7%) skunks from laboratory A. Lesions in 3 skunks (1 from laboratory A, 2 from B) were caused by cerebral parasitism. Lesions in the remaining 44 skunks were characterized by perivascular, nonsuppurative, mononuclear cell infiltrates and foci of glial cells of differing severity. The similarity of lesions and the finding of inclusions diagnostic of canine distemper virus (CDV) in some skunks indicated that CDV may be the main cause of neurologic disease in nonrabid skunks. Seventeen of 36 (47.2%) skunks evaluated for antibody against CDV, using an unlabeled antibody-enzyme method, were positive for CDV. Findings in skunks from the 2 laboratories indicated similar annual prevalences of brain lesions in 1982, 1983, and 1984. The highest percentage (40.5%) of nonrabid skunks with encephalitis was found in skunks submitted to laboratory B in 1981, which was concurrent with a rabies epizootic among skunks in Illinois in 1981. The number of skunks from both laboratories with CDV infection peaked during winter-spring. Importance of CDV in skunk population dynamics remains to be elucidated; however, infection with CDV appears to be enzootic and occasionally epizootic in skunks. Because enzootic/epizootic CDV may bias rabies surveillance data, caution in interpretation of surveillance data is necessary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental rabies in skunks: effects of immunosuppression induced by cyclophosphamide.

Striped skunks (Mephitis mephitis) were inoculated with street rabies virus and immunosuppressed with several doses of cyclophosphamide. Control skunks were inoculated with street virus only. The skunks were killed in terminal stages of the disease and several tissues were collected for examination by immunofluorescence, light microscopy and viral titration. Sera collected at euthanasia from most of the principals did not contain detectable rabies neutralizing antibodies, whereas high titers occurred terminally in controls. Immunofluorescence was much more entensive in submandibular salivary glands of cyclophosphamide-treated than control skunks. Similarly, virus was isolated from this tissue more consistently and at higher titer from principals than from controls. Immunofluorescence was extensive in brains of all skunks (both groups), but virus was isolated consistently only from brains of cyclophosphamide-treated skunks. Most of the cyclophosphamide-treated skunks had very few inflammatory cells in brain and cerebrospinal ganglia. Neuronal degeneration occurred in dorsal root ganglia of both principals and controls. The results suggest that the immune response has no effect on the development of rabies-induced aggressive behavior, that the immune response may inhibit salivary gland infection and that it is not essential for the development of neuronal degeneration in dorsal root ganglia.     

Vaccination of Egyptian fruit bats (Rousettus aegyptiacus) with monovalent inactivated rabies vaccine.

Twenty-six captive, adult Egyptian fruit bats (Rousettus aegyptiacus) were tested for the presence of rabies virus neutralizing antibodies (RVNA) using a rapid fluorescent focus inhibition test before and after vaccination. The bats were randomly assigned into three treatment groups: group A (n = 10) bats each received one 0.1-ml dose of monovalent inactivated rabies vaccine, group B (n = 10) bats each received two 0.1-ml doses of vaccine given 30 days apart, and group C (n = 6) bats remained unvaccinated. Plasma was collected from all bats before vaccination and on days 14, 30, 60, and 360. All bats were seronegative before vaccination, and all unvaccinated animals remained negative throughout the study. Rabies virus neutralization titers remained above 0.5 IU/ml from day 30 through day 360 for both vaccinated groups. Group B had significantly higher titers on day 60. This study demonstrated a measurable humoral immune response after vaccination with an inactivated rabies vaccine, with two doses producing a higher level of RVNA. This study confirms the feasibility of a rabies vaccination program for Egyptian fruit bats.     

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.     

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.     

Immunization of foxes Vulpes vulpes by the oral and intramuscular routes with inactivated rabies vaccines.

Inactivated rabies vaccines prepared from common vaccine strains of virus were inoculated into foxes by the intramuscular and intestinal route. There were differences among the vaccines in the duration of antibody produced after intramuscular administration. Inactivated vaccines deposited directly into the lumen of the duodenum by means of a fiberscope caused seroconversion in some foxes, especially following a booster dose, but the antibodies produced were for the most part of short duration. The ERA modified live virus vaccine, in contrast, produced a satisfactory and long lasting antibody after intestinal instillation.     

Effect of maternal immunity on the immune response to oral vaccination against rabies in young foxes.

OBJECTIVE: To determine effect of maternal antibodies on immune response to oral vaccination against rabies in young foxes. ANIMALS: 250 cubs from 48 vixens. PROCEDURE: Sera were obtained from cubs of 36 vaccinated (maternally vaccinated [MV+]) and 12 nonvaccinated (MV-) vixens between 23 and 71 days of age and tested for neutralizing antibodies. Seventy-one MV+ cubs and 33 MV-cubs were vaccinated orally with modified-live virus vaccine SAD B19. Geometric mean titer (GMT) was determined in these cubs approximately 21, 39, and 57 days after vaccination. In a subsequent experiment, 10 vaccinated MV+ cubs, 6 vaccinated MV- cubs, and 6 control cubs were challenge inoculated with virulent rabies virus approximately 100 days after vaccination. RESULTS: Serum GMT of nonvaccinated MV cubs (0.23 U/ml) was significantly greater than that of non-vaccinated MV- cubs (0.15 U/ml). The GMT of vaccinated MV+ cubs 21, 39, and 57 days after vaccination were 2.85, 2.11, and 0.79 U/ml, respectively, and were significantly less than those of vaccinated MV- cubs (12.19, 6.76, and 4.02 U/ml, respectively). All challenge-inoculated cubs with GMT < 0.5 U/ml succumbed to rabies. CONCLUSION AND CLINICAL RELEVANCE: Partially impaired immune response in cubs < 8 weeks old from vaccinated vixens causes insufficient protection against rabies. Inhibition of the immune response persists longer than the period during which maternal antibodies are detectable. Thus, oral vaccination campaigns for young foxes in areas where vaccination has been performed need to be reconsidered.     

A simplified roller bottle platform for the production of a new generation VLPs rabies vaccine for veterinary applications

Rabies is a neglected disease with an estimated annual mortality of 55,000 human deaths, affecting mainly low-income countries. Over 95% of these cases result from virus transmission through the bite of infected dogs and for this reason there is a real need for a cheap and effective rabies veterinary vaccine to be used in mass vaccination campaigns. In this work, we describe the establishment of a simple platform for the production of a virus-like particles based rabies vaccine using mammalian cells and roller bottles as culture system. Adherent cells were cultured during more than 15 days and VLPs were continuously produced and secreted to the culture supernatant. Immunogenicity and protective efficacy of VLPs were tested through rabies virus neutralizing antibody test and NIH potency test. These viral particles induced high titer of long lasting neutralizing antibodies and protected mice against active virus challenge. Therefore, this development represents a promising platform for the production of a new generation and virus-free rabies vaccine candidate for veterinary applications.     

Experimental rabies in skunks: mechanisms of infection of the salivary glands.

Striped skunks (Mephitis mephitis) were inoculated into the right submandibular salivary gland with street rabies virus. They were killed at various times after inoculation and several tissues were examined by immunofluorescence and light microscopy. Right and left superior cervical, nodose and trigeminal ganglia, medulla oblongata and at least three regions of right and left submandibular salivary glands were examined by the fluorescent antibody technique. Intracerebral titrations of salivary gland suspensions were made in weanling white Swiss mice. Immunofluorescent material (inoculum) was detected in septa and connective tissue surrounding secretory units of the right submandibular gland immediately after inoculation, but otherwise antigen was not detected in either right or left submandibular glands without coincident antigen in the medulla oblongata. This occurred first on day 12 in areas of the gland remote from the inoculation site. Titers of virus were low at this time. Serum neutralizing antibodies occurred by day 7 in a few skunks. The time of development and distribution of antigen strongly suggest that, even after direct inoculation, neural networks are necessary for development of widespread infection of the salivary gland. The early occurrence of serum neutralizing antibodies in some of the skunks suggests that the immune response was activated by virus in the inoculum since immunofluorescence was not detected in any tissue at this time.     

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.     

Postexposure prophylaxis for prevention of rabies in dogs

OBJECTIVE: To evaluate postexposure prophylaxis (PEP) in dogs experimentally infected with rabies. ANIMALS: 29 Beagles. PROCEDURE: Dogs were sedated and inoculated in the right masseter muscle with a salivary gland homogenate from a naturally infected rabid dog (day 0). Six hours later, 5 dogs were treated by administration of 2 murine anti-rabies glycoprotein monoclonal antibodies (mAb) and commercial vaccine; 5 received mAb alone; 5 received purified, heat-treated, equine rabies immune globulin (PHT-ERIG) and vaccine; 5 received PHT-ERIG alone; 4 received vaccine alone; and 5 control dogs were not treated. The mAb or PHT-ERIG was administered at the site of rabies virus inoculation. Additional vaccine doses for groups mAb plus vaccine, PHT-ERIG plus vaccine, and vaccine alone were administered IM in the right hind limb on days 3, 7, 14, and 35. RESULTS: All control dogs and dogs that received only vaccine developed rabies. In the PHT-ERIG and vaccine group, 2 of 5 dogs were protected, whereas none were protected with PHT-ERIG alone. Use of mAb alone resulted in protection in 4 of 5 dogs. Administration of mAb in combination with vaccine provided protection in all 5 dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Current national guidelines recommend euthanasia or a 6-month quarantine for unvaccinated animals exposed to rabies. Findings from this study document that vaccine alone following severe exposure was unable to provide protection from rabies. However, vaccine combined with mAb resulted in protection in all treated dogs, revealing the potential use of mAb in PEP against rabies in naive dogs.     

Duration of immunity in foxes vaccinated orally with ERA vaccine in a bait.

Red foxes (Vulpes vulpes) vaccinated orally with the ERA strain of rabies vaccine in a bait were challenged after 83 mo. Ten of 11 foxes that had seroconverted following vaccination resisted challenge with a virulent rabies virus which produced clinical signs of rabies in 6 of 6 unvaccinated foxes. Five of 11 vaccinated animals retained titers of rabies virus neutralizing antibody throughout the period. Although 6 of 11 had no detectable antibody at the time of challenge, 5 of these 6 resisted challenge and had an anamnestic response, as indicated by elevated titers of antibody when measured at day 77 postchallenge. These results show that foxes can be immunized successfully with a single oral dose of ERA vaccine, probably with protection against a lethal rabies challenge, for at least 7 y.