Thermo-stability of the oral rabies virus vaccines SAD B19 and SAD P5/88

The thermo-stability of two widely used oral rabies vaccine viruses, SAD B19 and SAD P5/88, was examined under various laboratory and field conditions. In the laboratory, both vaccine viruses were kept at 35 degrees C and titrated after 3 days. The titer of both vaccine viruses was also determined after 4 and 7 days when stored at 20 degrees C. Furthermore, vaccine baits were placed in three different micro-environments during two successive 21-day observation periods (11.9.01-2.10.01 and 2.10.01-23.10.01); (i) wooded area--location A, (ii) grassy meadow--location B, and (iii) barren soil--location C. Baits were re-collected 0, 3, 6, 9, 12, 15 and 21 days after distribution and the vaccine virus was subsequently titrated. The temperature stress for the vaccine baits was highest at location C, followed by location B and the lowest at location A. The vaccine baits were exposed to higher temperatures and higher temperature fluctuations at location B and C during trial I than during trial II. However, for both vaccines the loss of titer was more pronounced during trial II than during trial I. It is therefore suggested that under the given climatic conditions, the stability of the virus was hardly influenced by the temperatures and temperature fluctuations.     

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.     

The safety and efficacy of immunizing foxes (Vulpes vulpes) using bait containing attenuated rabies virus vaccine.

Foxes given ERA rabies vaccine baits were challenged at one, six, 12 and 24 months later and showed a resistance to challenge in 80%, 78%, 60% and 44% of individuals respectively. All animals showing seroconversion following vaccination, resisted challenge at 24 months, suggesting that successful vaccination by the oral route could confer a relatively long term duration of immunity. The trials showed that fox pups did not immunize as easily as adult foxes using ERA rabies vaccine baits. Back-passage studies and the consumption of ERA injected mice by foxes failed to show any reversion of the vaccine virus to a virulent state. The fox and mouse are shown to be highly susceptible to rabies street virus, while the domestic species tested are consisderably more resistant. Monkeys were found to be intermediate in susceptibility to the virus. Safety tests carried out on various species of wildlife showed only the mouse to be susceptible to infection from ingesting the vaccine in the form of a bait. ERA rabies vaccine was shown to be safe in monkeys even when high titred virus was administered by the oral route.     

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.     

The prevalence of Sarcocystis spp in dogs and red foxes.

Protozoan parasites of the genus Sarcocystis have been recognised for many years as intramuscular cysts of numerous vertebrates. It is only comparatively recently that the two-host nature of the life cycle has been recognised and that the intramuscular cysts are a stage in the developmental cycle of coccidian parasites of flesh eating mammals (Fayer 1974, Fayer and Johnson 1973, 1974, Rommel and others 1972, Dubey 1976). Carnivores ingest the intramuscular cysts from herbivores and presumably from other animals too and eventually shed sporulated tetrazoic sporocysts in their faeces. The cystic stages which occur in the flesh of herbivores are probably non-pathogenic but the earlier stages in which schizonts develop in vascular endothelium may be severely pathogenic. Sarcocystis cruzi, S ovicanis and S porcifelis are known to be severely pathogenic in cattle, sheep and pigs respectively (Dubey 1976). Observations on the prevalence of Sarcocystis spp in the faeces of working farm dogs, greyhounds and foxes (Vulpes vulpes) are recorded.     

Measles vaccine in dogs: efficacy against aerosol challenge with virulent canine distemper virus.

Fifty young Beagle pups were used in studies on the efficacy of measles virus vaccine in providing protection against virulent canine distemper (CD) virus given intranasally. Among 29 dogs vaccinated with measles virus vaccine and subsequently exposed to virulent CD virus, 1 died, 7 developed relatively severe signs of CD, 15 had mild signs of distemper, and 6 remained clinically normal. Of 15 unvaccinated dogs similarly exposed to virulent CD virus, 11 succumbed to distemper. Six pups vaccinated with modified live-virus (MLV) CD virus vaccine remained clinically normal following immunity challenge.     

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.     

Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from breeding foxes,raccoon dogs and minks in China

Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. Genetic/antigenic heterogeneity has been observed among the various CDV strains, notably in the haemagglutinin (H) gene, that appears as a good target to gather epidemiological information. Based on sequence analysis of the H gene, wild-type CDV strains cluster into distinct geographic lineages (genotypes), irrespective of the species of isolation. The sequence of the H gene of 28 CDV strains detected from both vaccinated and non-vaccinated breeding foxes, raccoon dogs and minks from different geographical areas of China during the years 2004–2008 was determined. All the CDV strains but two (strains HL and HLJ2) were characterized as Asia-1 genotype and were highly similar to each other (96.2–99.7% at the amino acid [aa] level) and to other Asia-1 strains (96.1–99.5% aa) previously detected in China. The CDV strains HL and HLJ2 were both collected from foxes in Heilongjiang province in 2005. Strain HL resembled CDVs of the Arctic genotype (GR88-like) and displayed high aa identity (98.0%) to the Chinese canine strain Liu. By converse, strain HLJ2 was barely related to CDVs of the Asia-2 genotype (88.7–90.3% aa identity), and could represent a novel CDV genotype, tentatively proposed as Asia-3. These results suggest that at least three different CDV genotypes, distantly related (81.8–91.6% aa identity) to the vaccine strains, Onderstepoort-like (America-1 genotype), are currently circulating in breeding foxes, raccoon dogs and minks in China, and that the genotype Asia-1 is predominant. Whether the diversity between wild-type CDVs and the vaccine strains may affect, to some extent, the efficacy of the vaccines deserves further investigations.     

First molecular detection of Pneumocystis spp. in red foxes (Vulpes vulpes linnaeus, 1758) and raccoon dogs (Nyctereutes procyonoides gray, 1834)

Fungal organisms of the genus Pneumocystis may cause Pneumocystis pneumonia (PCP) in humans, but also domestic and wild mammals. Almost every animal species hosts its own genetically distinct Pneumocystis species, however information is sparse. In this study, 62 red foxes (Vulpes vulpes) and 37 raccoon dogs (Nyctereutes procyonoides) were collected in North-East Germany. The lung tissues of the animals were analysed by a new designed specific pan-Pneumocystis mtLSU rRNA gene PCR and sequencing. With this PCR, detection and discrimination of all known Pneumocystis spp. in a single step should be possible. This first detection of Pneumocystis spp. in 29/62 (46.8%) red foxes and 29/37 (78.4%) raccoon dogs indicated, that they harbour two dissimilar strains, as seen by specific single nucleotide position changes (SNPs). Nevertheless, five samples with contrary SNPs showed a probable inter-species transmission.     

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.     

Comparative efficacy of an injectable vaccine and an intranasal vaccine in stimulating Bordetella bronchiseptica-reactive antibody responses in seropositive dogs

OBJECTIVE: To compare antibody responses to intranasal and SC Bordetella bronchiseptica vaccines in seropositive dogs. DESIGN: Randomized controlled study. ANIMALS: 40 young adult Beagles vaccinated against B bronchiseptica. PROCEDURE: Dogs were randomly assigned to 1 of 4 groups (intranasal vaccine, SC vaccine, intranasal and SC vaccines, no vaccine) and vaccinated on day 0. Serum and salivary B bronchiseptica-reactive antibody responses were measured on days 0 through 7, 10, 14, 21, and 28. RESULTS: Dogs that were vaccinated with the SC vaccine, alone or in combination with the intranasal vaccine, had a significant increase in serum concentration of B bronchiseptica-reactive IgG beginning on day 5 and persisting through day 28. Dogs that were vaccinated with the intranasal vaccine alone had a significant increase in serum concentration of B bronchiseptica-reactive IgG beginning on day 10 and persisting through day 28, but serum IgG concentration in these dogs was significantly less than concentration in dogs that received the SC vaccine. Neither vaccine had a demonstrable effect on salivary concentrations of B bronchiseptica-reactive IgA or IgG. On day 10, all vaccinated groups had significantly higher serum IgA concentrations than did unvaccinated control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the SC B bronchiseptica vaccine may be used to stimulate antibody responses in seropositive dogs. There was no apparent benefit to administering these vaccines simultaneously. Intranasal vaccines may not be effective for booster vaccination of dogs previously exposed to or immunized against B bronchiseptica. Dogs should be vaccinated at least 5 days prior to exposure to B bronchiseptica.     

Three-year rabies duration of immunity in dogs following vaccination with a core combination vaccine against canine distemper virus, canine adenovirus type-1, canine parvovirus, and rabies virus

Thirty-two seronegative pups were vaccinated at 8 weeks of age with modified-live canine distemper virus (CDV), canine adenovirus type-2 (CAV-2), and canine parvovirus (CPV) vaccine and at 12 weeks with a modified-live CDV, CAV-2, CPV, and killed rabies virus vaccine. An additional 31 seronegative pups served as age-matched, nonvaccinated controls. All test dogs were strictly isolated for 3 years after receiving the second vaccination and then were challenged with virulent rabies virus. Clinical signs of rabies were prevented in 28 (88%) of the 32 vaccinated dogs. In contrast, 97% (30 of 31) of the control dogs died of rabies infection. These study results indicated that no immunogenic interference occurred between the modified-live vaccine components and the killed rabies virus component. Furthermore, these results indicated that the rabies component in the test vaccine provided protection against virulent rabies challenge in dogs 12 weeks of age or older for a minimum of 3 years following vaccination.     

Prevalence and intensity of Echinococcus multilocularis in red foxes (Vulpes vulpes schrencki) and raccoon dogs (Nyctereutes procyonoides albus) in Otaru City, Hokkaido, Japan

A survey was done in an attempt to investigate the epidemiological status of Echinococcus multilocularis in red foxes and raccoon dogs in Otaru city from June to September 1999. Sixty-seven red foxes (Vulpes vulpes schrencki) and 13 raccoon dogs (Nyctereutes procyonoides albus) were captured, and postmortem examinations were conducted with them. Thirty-eight red foxes (56.7%) and 3 raccoon dogs (23.1%) were found to be infected with E. multilocularis. The total biomass of E. multilocularis in all infected red foxes and raccoon dogs were 2,817,000 and 1,515 worms, respectively. Nine of the infected red foxes harboring more than 100,000 worms accounted for 90.6% of the total biomass. No significant differences in the prevalence were observed between male and female, and juvenile and adult. However, the worm burden was higher in juvenile than in adult foxes. In one of the infected raccoon dogs, mature worms and eggs of E. multilocularis were found in the intestine and fecal sample, respectively. This result suggested that the raccoon dogs are probably playing a small role in the egg contamination of the environment. The validity of coproantigen ELISA for diagnosis of foxes was confirmed by comparing the results of autopsy, egg examination and coproantigen ELISA using rectal fecal samples.     

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.     

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.     

Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies.     

Studies on efficacy and stability of a vaccine bait containing ERA strain of rabies virus propagated in a BHK-21 cell line.

In a dose response study in foxes, the median protective dose of ERA BHK21 vaccine in a blister pack bait was 10(6.0) tissue culture infective doses (TCID)/mL, while artificially aged baits with titers of 10(6.3) TCID/mL induced seroconversion in 78% of foxes. There was no significant difference in the development of antibodies in foxes receiving 1, 2 or 3 mL volumes of vaccine in the bait. When baits were exposed to the elements and fed to foxes over a 21 day period, 85% of the animals seroconverted. Age, sex and the way in which the vaccine container was contacted did not appear to be factors in the responses of these animals. Juvenile foxes, approximately six months of age, were marked more readily with the tetracycline bait marker than older animals. Approximately 25% of foxes did not appear to respond well to vaccination and the titer of the vaccine was a critical factor in producing seroconversion in these animals.