New steps in the control of canine rabies in India.

In India, about 20,000 people die of rabies every year. The dog is the main reservoir and transmitter of the disease. A pilot rabies control programme was launched in five Indian federal states in February, 2007. This initiative is led by the Animal Welfare Board of India (AWBI) federating many animal welfare organizations and the Ministry of Agriculture. It aims at creating a "Rabies Free India." The programme combines parenteral vaccination of accessible owned and stray dogs, spaying/neutering followed by parenteral vaccination and oral vaccination of inaccessible dogs. The freeze-dried vaccine SAG2, including the bait casing, was registered in India following successful evaluation of vaccine-bait safety and efficacy (by survival after virulent challenge) in captive Indian stray dogs in the Bhopal High Security Animal Disease Laboratory. Furthermore, bait acceptance was tested under both experimental and field conditions.

The safety and efficacy of the oral rabies vaccine SAG2 in Indian stray dogs.

India is one of the countries with the highest prevalence of human rabies throughout the world. Dogs are primarily responsible for rabies transmission. Among them, stray dogs play a major role in that country. Parenteral vaccination programmes are insufficient to eliminate rabies partly due to difficulties in establishing satisfactory immunisation coverage in the dog population in view of the high proportion of stray dogs. Oral vaccination may be a useful adjunct to parenteral vaccination by increasing dog vaccination coverage. Safety, immunogenicity and efficacy of Rabidog SAG2 bait were evaluated in Indian stray dogs in captivity. Safety of SAG2 was demonstrated by the absence of adverse clinical sign, salivary excretion and absence of replication of the vaccine strain in brain and salivary glands of 21 vaccinated dogs, even when immunodepressed. Efficacy was shown 109 days post-vaccination after challenge with a highly virulent street rabies virus which killed all five controls whereas all nine vaccinated dogs survived, despite the fact that only five out of nine had seroconverted before challenge.

Kinetics of humoral immune response after rabies VR-G oral vaccination of captive fox cubs (Vulpes vulpes) with or without maternally derived antibodies against the vaccine.

In western Europe during the spring, the largest proportion of fox populations are cubs and the key to successful rabies oral vaccination campaigns is cub vaccination. In this paper we report on studies of the serology of 93 fox (Vulpes vulpes) cubs born to unvaccinated and orally vaccinated captive vixens, some of which were orally vaccinated at 30 or at 90 days of age with the vaccinia recombinant vaccine (VR-G) that expresses the rabies virus glycoprotein. The duration of cub passively acquired antibody, the development of immune responses to oral vaccination at either 30 or 90 days of age, possible interference between passive and active immunity to such vaccination and resistance to a potentially lethal rabies challenge dose when five months old were measured. The study showed that rabies neutralising antibody can be passed to their cubs by vixens orally vaccinated with VR-G during pregnancy. Maternally derived antibody titres in cubs declined with time and disappeared by 45-75 days after birth. Thirty days old cubs serologically responded to oral vaccination. No interference between antibody of maternal origin and active immunity conferred by VR-G oral vaccination or between antibody of maternal origin and protection was observed. Thus, very young cub immunisation against rabies with VR-G per os is possible whatever the immune status of their mothers. Provided a vaccine-bait suitable for such young cubs exists, oral vaccination at den entrances with VR-G is a feasibility.

Humoral and cell-mediated immune responses of foxes (Vulpes vulpes) after experimental primary and secondary oral vaccination using SAG2 and V-RG vaccines.

Humoral and cell-mediated immune responses of 36 captive foxes to two oral vaccines against rabies currently used for foxes in Europe were studied. The Street Alabama Dufferin (SAD) mutant Gif (SAG2) vaccine has been selected by double mutation from the SAD virus. The vaccinia recombinant virus (V-RG) expresses the rabies glycoprotein. Both vaccines induce similar humoral and cell-mediated responses after primary and secondary oral administration. We observed a typical anamnestic response, although of a limited duration, after the booster vaccination. Therefore, our results suggested that two successive oral vaccination campaigns should not significantly improve the immunisation of foxes. Lymphocyte in vitro proliferative response to the SAD antigen highlighted the presence in blood of a T-cell specific memory 6 months after vaccination. The synthesis of several vulpine cytokines was detected in peripheral blood mononuclear cells (PBMC) stimulated by SAD antigen via reverse transcription polymerase chain amplification. The data showed a concomitant expression of interleukin (IL)-4 and interferon-gamma in PBMC of vaccinated foxes. No change was detected in the level of IL-2, IL-10 and IL-12 synthesis, whereas the pro-inflammatory cytokine tumour necrosis factor-alpha seemed involved in the activation of naive T lymphocytes.

ELISA test for rabies antibody titration in orally vaccinated foxes sampled in the fields.

The assessment of the efficacy of rabies oral vaccination campaigns requires the titration of specific antibodies in the target species. Unfortunately, in Continental Europe, most fox serum samples are in fact "body fluids" taken from cadavers and the lack of a validated titration method for these poor quality sera made it impossible to survey and compare the efficacy of various oral vaccination protocols used by the different European teams. By using ready to use microplates sensitised with rabies virus glycoprotein purchased from a manufacturer and applying a simple and rapid ELISA technique on field fox sera, we obtained antibody quantitation highly correlated with seroneutralising antibody titres measured with a seroneutralisation test on cell culture. We obtained, with fox sera sampled in the same area, the same distribution of high, medium and low titres within all categories of serum quality (from high to very poor quality) and therefore conclude that this ELISA test allows a reliable titration even with highly contaminated body fluids. This test was shown to be equally capable of detecting rabies antibodies in serum samples taken from foxes vaccinated with an highly attenuated rabies virus (the SAG2 double mutant of the Street Alabama Dufferin strain) or with the VRG, the Vaccinia recombinant glycoprotein. Additionally, a strong correlation was demonstrated between titres given by this ELISA (or by the seroneutralisation test) and protection against challenge of foxes orally vaccinated with SAG2 vaccine baits. In view of this validation, this simple and reliable test is proposed for sero-surveying foxes following rabies oral vaccination campaigns.

Costs and benefits of rabies control in wildlife in France.

The author presents an evaluation of the cost of wildlife rabies in France. This study included the vaccination of domestic animals, the reinforcement of epidemiological surveillance networks and the support provided to diagnostic laboratories, the expenses associated with outbreaks of rabies (animal losses and associated economic losses), the clinical observation of those animals which had bitten humans and the preventive vaccination and post-exposure treatment of humans. A substantial percentage (72%) of this cost was the preventive vaccination of domestic animals. In France, as in other European countries in which the red fox (Vulpes vulpes) is the species most affected, to main strategies for controlling the disease at the reservoir level were evaluated, namely: fox depopulation and the oral vaccination of foxes. The combined costs and benefits of rabies and of both strategies were compared and included either the cost of fox culling or the cost of oral vaccination (baits, bait delivery and follow-up to ensure the efficiency of the vaccination). The cumulative annual costs of both strategies remained comparable until the fourth year, after which the oral vaccination strategy became beneficial. This forecast was made in 1988, readjusted in 1993 and confirmed by ex post analysis five years later. The expected benefits of oral vaccination have now been obtained. Fox depopulation has only ever resulted in a transient lull in the occurrence of the disease, while oral vaccination has proved to be capable of eliminating rabies even in situations in which fox populations were increasing.

Field evaluation of a dog owner, participation-based, bait delivery system for the oral immunization of dogs against rabies in Tunisia.

We evaluated a dog owner, participation-based, bait delivery system for the oral immunization of dogs against rabies. In a field study in a semirural area of northern Tunisia, dog owners were asked to come to temporary bait delivery sites. A total of 314 baits were given to 178 dog owners in four sites. The experimental baits used consisted of a freeze-dried core unit containing sulfadimethoxine (SDM) as a biological marker and an aromatized paraffin envelope. No vaccine was used. Preliminary tests had shown that by using a rapid commercial card test, positive SDM serum levels were detected in more than 95% of dogs up to two days after bait ingestion. During the two days following bait delivery, we visited more than 95% of all households in the study area and took blood samples from as many owned dogs as possible. Unconsumed baits were recovered and human contacts with the bait matrix were recorded. The campaign required 7.6 person-min per bait and 13.5 person-min per dog owner for providing baits, gloves, and instructions. The estimated average cost effectiveness ratio per dog accepting a bait was 1.7 US dollars. From the indications given by the dog owners and the results of the SDM test, it was concluded that 85-90% of the owned dogs in the study area had consumed a bait at least partially. Of 314 baits delivered, 78.7% were fully consumed by dogs and 4.1% were recovered during the household survey. The remaining baits (17.2%) that were not recovered were either not consumed or only partially consumed by the target dogs (3.7 baits per 100 inhabitants). These baits probably remained within the highly populated areas and were potentially accessible to other domestic animals and other nontarget species, including humans. Twenty-five unprotected human contacts with baits were recorded (1.7% of all inhabitants). Our study has demonstrated the potential of dog owner based bait delivery. This technique is simple and efficient, particularly if the human population is accustomed to mass immunization in defined centers. Before applying this method on a large scale with live vaccine loaded baits, further studies should focus on minimizing the number of human contacts with the vaccine bait, systematizing contact identification and establishing structures in ensuring proper treatment if exposure to vaccine should occur.

Field evaluation of two bait delivery systems for the oral immunization of dogs against rabies in Tunisia.

Two bait delivery systems for the oral immunization of dogs against rabies were tested in small scale field trials in a semi-rural area in Tunisia: bait delivery to owned dogs during door to door visits of households (door to door baiting) and distribution of baits on transect lines (transect line baiting). A prototype bait (DBL2) configured for industrial production and containing either sulfadimethoxine (SDM) as a systemic marker or Rhodamine B as a topical marker was used. The overall proportion of dogs which took a bait and presented topical marker staining after door to door baiting was 59.1%. The total time and costs spent per bait accepting dog averaged 34 person minutes and US$4, respectively. Unconsummated baits were readily recovered. No unprotected human contacts with baits were recorded. Door to door baiting is a very specific but time-consuming method that enables a safe administration of vaccine baits to owned dogs. For transect line baiting, baits were distributed at a density of ca 3000 baits per km2 along double transect lines. Baits were recovered after 20 h. According to the proportion of SDM positive serum samples, 24.1% of owned dogs in the baiting area had consumed baits. Of all owned and ownerless dogs, presumably free-roaming during transect line baiting, > 40% had consumed baits. The total time and costs spent per bait accepting dog averaged 48 person minutes and ca US$20, respectively. The household census revealed 32 direct human contacts with the bait matrix which corresponds to 1.4% of inhabitants. Placing baits on transect lines gives the possibility to vaccinate dogs not accessible by vaccination systems which base on dog owner participation. However, the method is not specific, less safe than other systems, not easily accepted by the human population, and costly.

Innocuity studies of SAG-2 oral rabies vaccine in various Zimbabwean wild non-target species.

The SAG-2 modified live rabies vaccine was tested for innocuity when administered by the oral route in several potential wild non-target bait-consuming species, as follows: ten chacma baboons (Papio ursinus), six African civets (Civettictis civetta), six slender mongooses (Galerella sanguinea), six honey badgers (Mellivora capensis), six large-spotted genets (Genetta tigrina), 39 multi-mammate mice (Mastomys natalensis), 26 bushveld gerbils (Tatera leucogaster) and six pied crows (Corvus albus). At least 9.0 log10 median tissue culture infectious doses (TCID50), given in a volume of 1 ml, was administered orally to each of the animals, except the rodents which received 8.0 log10 TCID50, given in 0.1 ml. All the animals were observed for not < 90 days for signs of vaccine-induced rabies. Most of the species were also tested for vaccine virus replication in the oral cavity and persistent virus infection in the brain, salivary gland and tonsil. None of the animals died of rabies and no persistent infection was found. Rabies virus which was pathologically and serotypically indistinguishable from the vaccinal strain was isolated from the saliva of one genet 1 day after vaccine administration. From this study it was concluded that SAG-2 rabies vaccine would be safe for use in most situations where oral vaccination campaigns for jackals are required in Zimbabwe.

[Transmission of rabies virus: importance of the species barrier]. / Transmission du virus de la rage: importance de la barrière d'espèce.

The authors describe experimental inoculations of different animal species with either the wild-type rabies virus or the modified one through in vivo or in vitro passages. With these experiments, it was possible to determine the 50% lethal dose of these viruses for these species, and to thus quantify the importance of the species barrier that opposes, in particular, the transmission of vulpine rabies to cats and dogs (respectively 10(5) and 10(6) times more resistant than foxes). Studies were also undertaken on the influence of the inoculation route and that of serial passages of the virus in vivo or in vitro on the importance of resistance to rabies. The epidemiological consequences of the existence of a species barrier, its nature and variability, are discussed.

[Human contamination by baits for vaccinating foxes against rabies in France]. / Contamination de personnes par des appâts vaccins après les campagnes de vaccination des renards contre la rage en France.

During 1992 and 1993, 4.4 million of baits have been distributed in France over 121,381 km2 for vaccinating foxes against rabies. Phone calls and visits addressed to the local veterinary authorities and to the centres for human antirabies treatment have been recorded according to an appropriate questionnaire. 70 persons declared to have found and sometimes to have touch a bait, 38 of them received a antirabies treatment. Only 9 children (less than 10 year old) handled a bait. Activities that led to find a bait have been: walking in the countryside or hunting (50% of cases), gardening or playing in the garden or near home (35%) and farming (13%). Often dogs were the first to discover the baits and led to a contact of humans with the vaccine in 54% of cases. No casualty occurred. The frequency of these reports decrease by 80% during the period which is considered to be the result of a better information and awareness of the public.

Field use of a vaccinia-rabies recombinant vaccine for the control of sylvatic rabies in Europe and North America.

During recent years, most research on the control of sylvatic rabies has concentrated on developing methods of oral vaccination of wild rabies vectors. To improve both the safety and the stability of the vaccine used, a recombinant vaccinia virus, which expresses the immunising glycoprotein of rabies virus (VRG), has been developed and tested extensively in the laboratory as well as in the field. From 1989 to 1995, approximately 8.5 million VRG vaccine doses were dispersed in Western Europe to vaccinate red foxes (Vulpes vulpes), and in the United States of America (USA) to vaccinate raccoons (Procyon lotor) and coyotes (Canis latrans). In Europe, the use of VRG has led to the elimination of sylvatic rabies from large areas of land, which have consequently been freed from the need for vaccination. Nevertheless, despite very good examples of cross-border cooperation, reinfections have occurred in some regions, due to the difficulty of co-ordinating vaccination plans among neighbouring countries. In the USA, preliminary data from field trails indicate a significant reduction in the incidence of rabies in vaccinated areas.

Comparison of the efficacy of the antirabies vaccines used for foxes in France.

Three manufactured antirabies vaccine baits made with the SAD B19, SAG 1 or VRG viruses have been used in a large scale programme in France since 1986. All three baits were found to be effective in achieving a significant decrease in the incidence of rabies, or even its local eradication. The VRG vaccine bait was found to be the most efficient, and resulted in rabies elimination in a non-alpine region after only two campaigns. The fact that the VRG-vaccinated bait was significantly better than the others was probably due to the better conservation of this vaccine and the bait casing in the environment even under summer conditions. The stability of the SAG 1 vaccine bait was improved in 1992, and it has since been giving good results. The choice of the pattern of the vaccination periods appeared to be a determining factor: distribution during spring, then autumn, then spring was found to be more efficient than a distribution during autumn, then spring, then autumn. A distribution of VRG bait during the summer of 1992 was far less efficient due to the lower percentage of foxes that take the baits during this period, despite the proven stability of the baits in such conditions.

A model for the control of Echinococcus multilocularis in France.

In some areas of France the prevalence of Echinococcus multilocularis in foxes is as high as 50%, whereas less than one in a thousand voles (principally Microtus arvalis) are infected. In these regions the control of rabies in foxes is achieved by using helicopters to spread bait containing oral vaccine in capsules. A mathematical model has been constructed in an attempt to determine if the addition of praziquantel to bait would be effective in eradicating E. multilocularis, or at least achieve a useful measure of control. It has been shown that the qualitative population dynamics of E. multilocularis are not affected by the detail of its epidemiology in the intermediate host population. The model is, however, sensitive to assumptions about the distribution and longevity of the adult worm in the definitive host. Given these assumptions, a method is provided that determines the feasibility of eradication conditional on the pre-control prevalence in foxes, or predicts the post-control prevalence if eradication is not feasible. If experiments could be designed to provide better information about the biological factors that determine the epidemiology of this parasite in the definitive host, a more reliable assessment of the feasibility of control would be achieved.

Assay of oral vaccination of dogs against rabies in Tunisia with the vaccinal strain SADBern.

The possibility of immunizing dogs orally against rabies, using SADBern, an attenuated strain, was tested on dogs in the field in Tunisia. This strain induced high neutralizing antibody titres and conferred to all vaccinated dogs total resistance against a challenge with a Maghrebian strain. However, an excretion of virus of vaccinal origin was observed in one dog, hampering the use of SADBern in dogs. Nevertheless, this work demonstrates for the first time that dogs in developing countries, especially those which are inaccessible to parenteral vaccination, could be efficiently immunized against rabies by the oral route.

Efficacy of three oral rabies vaccine-baits in the red fox: a comparison.

The three commercial vaccine-baits currently available in western Europe were fed to caged red foxes. The vaccines were: SADB19, Bundesforschunganstalt für Viruskrankheiten der Tiere-Tübingen, Germany: a live attenuated rabies strain; SAG1 VIRBAC, France: low pathogenicity mutant of the SAD strain and "Raboral", Rhône-Mérieux, France: a vaccinia rabies glycoprotein recombinant (V-RG). Protection rates conferred by the vaccine-baits were respectively 7 of 15 (SADB19), 5 of 9 (SAG1) and 6 of 12 (V-RG) when the vaccinated foxes were challenged intramuscularly with 10(4.7) Mice Intra-Cerebral Lethal Doses 50% (MICLD50) of rabies virus which killed all of 6 unvaccinated control foxes.

Current status of fox rabies in Europe.

There was a general decline in animal rabies in Europe in 1991 following the peak levels which occurred in 1989. This was ascribed, in France at least, to the normal decline in cases usually experienced following peak occurrence and also to oral immunization of foxes against rabies. European countries in which rabies occurs may be infected by fox, insectivorous bat or dog rabies. This paper makes a general summary of the rabies situation in Europe in 1991 and presents data obtained in 1991 from 15 European countries using oral vaccination against fox rabies.

SAG-2 oral rabies vaccine.

The live modified rabies virus vaccine strain SAG-2 was selected from SADBerne in a two step process employing anti-rabies glycoprotein monoclonal antibodies. The first two nucleotides coding for the amino acid in position 333 of the rabies glycoprotein are mutated. Arginine at position 333, which is associated with rabies pathogenicity, was substituted first by lysine and then by glutamic acid. The two nucleotide differences at position 333 in SAG-2 to any of six possible arginine triplets translated into excellent genetic stability and apathogenicity for adult mice, foxes, cats and dogs. The vaccination of foxes and dogs by the oral route provided protection against a lethal challenge with rabies virus.