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The SAG2 vaccine (RABIGEN® SAG2) is a modified live attenuated rabies virus vaccine, selected from the SAD Bern strain in a two-step process of amino acid mutation using neutralizing monoclonal antibodies. The strain is genetically stable and does not spread in vivo or induce a persistent infection. Its absence of residual pathogenicity was extensively demonstrated in multiple target and non target species (such as wild carnivores and rodent species), including non-human primates. The efficacy of SAG2 baits was demonstrated according to the EU requirements for the red fox and raccoon dog. The use of safe and potent rabies vaccines such as SAG2 largely contributed to the elimination of rabies in Estonia, France, Italy and Switzerland. Importantly, these countries were declared free of rabies after few years of oral vaccination campaigns with SAG2 baits distributed with an appropriate strategy. The excellent tolerance of the SAG2 vaccine has been confirmed in the field since its first use in 1993. No safety issues have been reported, and in particular no vaccine-induced rabies cases were diagnosed, after the distribution of more than 20 million SAG2 baits in Europe.

Since the late 1930s, the red fox (Vulpes vulpes) has been the main vector of rabies in Europe. Practically, decimation of fox population did not prevent the spread of the disease. The only efficient method to control wildlife rabies consisted in using oral vaccination by depositing vaccine baits containing a capsule or a plastic sachet filled with an attenuated anti-rabies liquid vaccine throughout fox habitats. Several live virus vaccines have been and are currently being used: the SAD B19 and SAD P5/88 are rabies strains attenuated in cell culture, the SAG1 and SAG2 strains are apathogenic mutants of an already attenuated rabies strain, and the VRG vaccine is an attenuated vaccinia virus recombinant coding for the rabies glycoprotein gene. These vaccines have different residual pathogenicity. SAG1 and SAG2 are pathogenic only for suckling mice inoculated by intracerebral and oral routes. VRG presents absolutely no rabies risk to humans and the environment and the residual pathogenicity of the vaccinia vector virus is very low even for humans. Other parameters such as the thermostability of the vaccine and the melting point of the bait casing are of utmost importance to guarantee the success of oral vaccination campaigns. Additionally, VRG is the only vaccine that did not interfere with maternal immunity in fox cubs, an important issue for spring campaigns. Successes and failure of national programmes confirm that whatever the ecological conditions, the same rules must be strictly followed to ensure the success of rabies elimination programmes: (i) considering the strategy, any rabies vaccination programme must be organised with the support of a national scientific team specially designated for the task that will have to apply the only methods that have already been proved successful elsewhere in Europe, including rabies surveillance, bait distribution calendar and pattern, and monitoring of this distribution; (ii) vaccination must be pursued for at least two years after the last reported case of rabies in the area; (iii) the choice of a low cost but poorly efficient and poorly stable vaccine does not prove to be cost-beneficial for successful elimination of rabies. Several European countries have become rabies-free: Belgium, Luxembourg, France, Italy, Switzerland, Finland and the Netherlands. Since the European Union is going comprise 25 countries from May 2004, all the scientific knowledge is available for establishing efficient and adapted oral programmes aimed at eliminating terrestrial rabies from this area.

This paper describes the role of fox population level on Echinococcus multilocularis infection in foxes in a highly endemic area in eastern France. Fox population level was monitored by spotlight survey at Le Souillot from 1989 to 2000, and from 1992 to 2000 at Chemin, a control site located in a low endemic area. The infection level of the fox population was estimated at Le Souillot from winter 1995 to winter 1999 using a coproantigen ELISA performed on faeces collected in the field. Population biomass of intermediate hosts (Microtus arvalis and Arvicola terrestris) was monitored using index methods from 1995 to 1999. At Le Souillot, a significant decline in the fox population level was recorded after spring 1997 (P<0.001), and the population level remained low until 2000. The decline occurred when 31% of the grassland area was treated with bromadiolone, an anticoagulant used at a large scale for the control of A. terrestris population outbreaks. No decline of population was recorded at Chemin, where bromadiolone was not used for rodent control. Significant differences among ELISA OD distributions in fox faeces were recorded for the five winters under study at Le Souillot (P=0.0004). The median of ELISA OD distribution was 0.209 and 0.207 before the population decline (winter 1995 and 1996, respectively), significantly increased to 0.306 just after the decline (winter 1997), and then significantly decreased to 0.099 and 0.104 afterwards (winter 1998 and 1999, respectively). Therefore, the decrease in infection level occurred during winter 1998, 1 year after the population decline, when the intermediate host biomass in the field was at its highest. These results suggest a complex dependence between the fox population level and E. multilocularis infection in a high endemicity area. Alternative ways to control fox population as a way to reduce E. multilocularis transmission in a given area are discussed.

A simple and rapid enzyme linked immunosorbent assay (ELISA) to detect rabies antibodies in field fox sera has been standardised and established in several European laboratories. The same panels of 100 coded sera were investigated by four laboratories using this ELISA assay and reference serum neutralisation techniques (fluorescent antibody virus neutralisation (FAVN) test and rapid fluorescent focus inhibition test (RFFIT)). This indirect ELISA technique is highly correlated with conventional seroneutralisation test on cell culture. Results obtained with this ELISA test provide an almost perfect agreement between laboratories while the agreement of FAVN test and RFFIT results is substantial and very satisfactory. In view of this study, this simple and rapid ELISA test is a suitable tool for evaluating the sero-conversion rate in fox populations following oral rabies vaccination campaigns in European countries.

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 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.

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.

During the spring of 1997, various protocols of rabies vaccine bait (SAG2) distribution for foxes were compared: in the first test zone, a first distribution was organised at the end of April, followed by a second distribution two weeks later; in the second test zone, there was a first distribution at the same period as for the previous zone, followed by a second distribution four weeks later, at the end of May. In two control zones, a classical single bait distribution was organised during the same periods as for the second distribution in the respective test zones. No statistical differences were observed for adult foxes or fox cubs sampled in the test and control zones neither for baits uptake nor for seroconversion rate. However, seroconversion rates observed in fox cubs population were significantly higher (P < 0.01) in areas vaccinated at the end of May (43 and 56%) compared with those vaccinated at mid-May (24 and 20%). The vaccinal efficacy of baits was also significantly (P < 0.05) increased for the fox cubs in the areas vaccinated at the end of May (46 and 57%) compared with those vaccinated at mid-May (24 and 25%). This increase in immunological response by fox cubs when vaccinating in late spring must be related to their development. In the early spring, fox cubs are generally too young to have access to baits or to be vaccinated when eating them. For most of these fox cubs, a second distribution will not constitute a booster. Therefore, in order to increase the efficient access of fox cubs to vaccine baits, Spring distribution of baits should preferably be organised during May or June rather than in April.

The vaccination of foxes by distributing vaccine baits in the environment was initiated in France in 1986. Two campaigns per year were carried out: one in the spring and one in the autumn. After the spring campaigns, only 22-52% of fox cubs consumed vaccine baits compared to 75% of the adults and 70-80% of the adults or fox cubs after autumn campaigns. In order to reduce the period of time during which fox cubs do not have access to baits and are not immunised, a vaccination campaign was organised during the summer of 1992 over a contaminated area of 25,748 km2 where vaccines had never previously been given. Vaccine bait stability was assessed during the same summer in the field and their appetence tested on captive foxes. The efficacy of the campaign was evaluated by the relative decrease in rabies incidence and the rate of bait uptake by foxes compared to those from neighbouring areas vaccinated for the first time with the same vaccine during the spring or autumn. Summer vaccination significantly increased (P < 0.01) bait uptake by fox cubs (71%) compared with spring vaccination (39%), but no significant difference was observed for adult foxes. Moreover, the decrease in rabies incidence, measured during the 6-month period following the campaigns was less pronounced after summer vaccination (49% decrease) than when the first vaccination was carried out during the spring or autumn (79 and 72% decrease, respectively). Three campaigns led to an apparent elimination of rabies when the first campaign was performed in the spring or autumn, but only to a 76% decrease in rabies incidence density index when the first campaign was performed during the summer. The high thermostability of the Raboral VRG bait permits its use during the summer for an emergency campaign. For routine vaccination plans, however, the classical calendar of spring and autumn vaccination campaigns should continue to be preferred.

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.

In a plateau and hill region of France (the Doubs), two protocols of rabies vaccine bait distribution targeted at foxes were compared: helicopter distribution of vaccine baits alone (control zone) and a combined aerial distribution by helicopter with an additional deposit of vaccine baits at fox den entrances by foot (test zone). In the test zone covering an area of 436 km2, baits were distributed by helicopter at a rate of 13.4 baits/km2. Additionally, an average of 11.4 vaccine baits at the entrances of 871 fox dens were terrestrially distributed by 110 persons (9,964 baits). In this test zone, 90% of the young foxes were marked with tetracycline which permitted estimation of the bait consumption; however, only 38% had significant titre of rabies antibodies and less than one fox cub per 2.4 of those having consumed at least one bait were immunized. In the control zone, these percentages were significantly lower: respectively, 35 and 17% and one fox cub per 4.2. The relative lack of benefit between bait uptake and rate of immunological response may be due to maternal immunity which could have interfered with fox cub active immunization. A booster effect following a second distribution of baits by foot may be suggested in both adult foxes and their offspring. That these baits needed to be terrestrially distributed in order to obtain a booster effect is uncertain. Terrestrial distribution at fox den entrances is difficult to do and entails additional expenses not incurred in aerial distribution. The cost of terrestrial vaccination is 3.5 times higher than classical aerial vaccination and takes 63.5 times longer. A cost effective analysis of this type of supplementary terrestrial intervention determined that bait deposit at den entrances can be recommended for restricted areas, where residual focii exist, as a complement to the aerial distribution of baits.

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.

The main host reservoir and vector of rabies in Western Europe is the red fox (Vulpes vulpes). A vaccination strategy for this species has been developed and tested in Switzerland since 1978. Results indicate that the vaccine which is administered in spring and autumn for at least two consecutive years is more effective than destruction by shooting or gassing the animals. The same approach using bait containing increasingly effective and safe vaccines has been in use in France since 1986. By creating an immunological barrier from the English channel to the Swiss border, it has been possible to stop the southern progression of the disease. In the following years the vaccination program was extended to all contaminated areas in France (141,700 km2). From 1989 to 1996 rabies decreased in incidence by 99.7% and disappeared from 95% of the previously contaminated area. Although no case of rabies involving a non-flying mammal has been reported since October 1996, rabies cannot be considered as eradicated as long as places of active disease subsist in neighboring areas of Belgium and the Sarreland. Bat rabies in Europe is caused by two viral genotypes that have never been isolated in any species other than bats and man. A total of four cases of bat rabies have been diagnosed in France since 1989. All four cases occurred in the Serotine community including one in 1997. All cases of canine rabies reported in the last 20 years have been observed in imported animals. The last was in 1995 and could have been prevented by stricter border control.

A polymerase chain reaction (PCR) method was developed in order to permit a sensitive and specific identification of Echinococcus multilocularis DNA from fox faecal specimens. In an attempt to overcome problems related to the presence of endogenous PCR inhibitors in faecal extracts, we investigated a DNA extraction technique using an anion binding resin (Gene-Fizz). This simple and rapid procedure was applied to 61 faecal samples. Compared with the traditional microscopic examination, the sensitivity of PCR using Gene-Fizz was 82.3% and the specificity was 95.5%. No PCR signal was obtained for 20 Echinococcus granulosus isolates, showing that this method allowed discrimination between E. multilocularis and E. granulosus. Large-scale epidemiological surveys of fox excrements may be possible by using Gene-Fizz treatment prior to PCR amplification reactions.

The safety of the SAG2 virus, a low virulence mutant of the SAD strain, was investigated in ten species of mammals and seven species of birds liable to consume vaccine baits. These species are the western hedgehog (Erinaceus europaeus), the meadow vole (Microtus arvalis), the bank vole (Clethrionomys glareolus), the water vole (Arvicola terrestris), the field mouse (Apodemus flavicollis or A. sylvaticus), the Norway rat (Rattus norvegicus), the european badger (Meles meles), the domestic ferret (Mustela putorius furo), the wild boar (Sus scrofa), the domestic goat (Capra hircus), the carrion crow (Corvus corone), the rook (Corvus frugilegus), the buzzard (Buteo buteo), the red kite (Milvus milvus), the tawny owl (Strix aluco), the long-eared owl (Asio otus) and the barn owl (Tyto alba). The vaccine was administered orally to each species, by an intramuscular (i.m.) route to the rodents and ferret, and by an intracerebral route to the field mouse. No pathogenicity was observed in the 169 animals vaccinated throughout an observation period of over 30 days. After euthanasia, no rabies virus could be detected either in the brain or in the salivary glands of any of the animals. The SAG2 virus administered orally, triggered a specific seroconversion in the field mouse, wild boar, ferret and most of the raptors. Following administration by the i.m. route, specific antibody titres were observed in most of the rodents, as well as in the ferrets.

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.

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.

Until recent years, the depopulation of reservoir species was the only feasible measure to break the transmission chain of rabies in wildlife. In Europe at the beginning of this century, when applied with compulsory muzzling and restrictions on movement, dog depopulation was shown to be effective before any large scale vaccination of dogs. Some examples of the success of the application of depopulation measures on wild carnivores in the past are described. But in the case of enzootics of rabies in the fox, the control measures applied to fox populations sometimes decreased the prevalence or postponed the invasion of an area but did not free or protect an area permanently. Some examples taken from France illustrate the difficulties of drawing clear conclusions from the results of control measures. Conversely, the oral vaccination of foxes was demonstrably successful in preventing the spread of rabies. However, mathematical modelling, historical considerations and first observations in areas in which foxes have been vaccinated strongly suggest that the 'traditional' methods of controlling fox populations, at least by hunting, should not be restricted because they complement the effects of vaccination.

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.

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.

Two rabies virus strains collected from naturally infected foxes in France in 1976 and 1986 were inoculated in 2 groups each consisting of 10 foxes (approximately 50 lethal doses50 mouse intracerebral per fox). Another 20 healthy foxes were kept in the same cages as the inoculated animals in order to study the transmission of both strains. All the inoculated foxes became rabid and transmitted rabies to their cage companion. The intervals between death of the inoculated foxes and death of the cage companion were significantly more variable (P less than 0.001) with the earlier strain, from 1 to 33 days compared to the range obtained with the later strain, 10-14 days. However, the mean intervals did not differ. Epidemiological consequences of such a decrease in variability, if confirmed in the field could decrease the epidemiological extent of rabies. Viremia was demonstrated in 2 foxes inoculated with the later strain but we cannot assume this to be a characteristic of this strain compared with the earlier strain. Rabies virus (both strains) was isolated from the saliva of foxes 29 days or less before the onset of clinical symptoms. This last result raises the question of the time limit as regards security before onset of clinical symptoms in animals involved in human contamination.

Red fox behavioural ecology was studied in a rabies-enzootic area in order to determine how population size is balanced despite rabies-induced mortality. The results suggest that the red fox rabies virus equilibrium evolves, and is due to the solitary behaviour pattern of the fox which reduces the risk of virus transmission from on territory to another; and to the subsequent autumn dispersal, which allows the local fox population to recover in the space of under a year. The hypothesis is put forward that rabies does not seem to regulate fox population size. There would therefore be no reason to fear a population explosion after oral immunisation of foxes against rabies.

Post mortem analysis of 1259 foxes from all over France, between 1976 and 1980, allowed comparison of age-structure of fox populations from long-time invaded areas (enzootic situation) or rabies-free areas. Heads received for rabies diagnosis have been aged by the cementum annulation technique, the sex of 650 of these foxes was known or fixed by examination of cells of the brain tissue for sex chromatin (found only in females). Results led to the generalization that young foxes (5-13 months) contracted rabies less frequently than did other ones. Consequently in rabies enzootic areas the age-structure of the fox population was lowered. The effect of 'gazage des terriers' (i.e. shooting foxes bolted from their holes with tear gas), which killed four females for each male, probably reduced the proportion of vixen in the population. No other obvious differences existed between sex-ratios. Consequently rabies does not seem to exercise any influence on this second parameter. The consequence of these results on the fox-culling is discussed.

We have investigated the influence of anti-rabies vaccination on the onset of the disease as well as the delay of death in foxes previously infected with rabies virus. A live vaccinia recombinant virus expressing the rabies virus glycoprotein (VVTGgRAB) was used as vaccine. Foxes were divided into six experimental groups of four animals. On day 0, each fox was experimentally infected with a rabies virus suspension. VVTGgRAB was administered by the oral route to each animal of three groups on day 0, 3 or 14. Foxes of other groups were used as unvaccinated controls or received, on day 0, the parental Copenhagen strain of vaccinia virus. The length of post-challenge survival and duration of clinical disease were recorded for each animal tested. All foxes, vaccinated or not, died from rabies as confirmed by fluorescent antibody tests. Animals vaccinated on days 0 and 3 died after a shorter period of incubation than unvaccinated controls. On the other hand, animals vaccinated on day 14 post-challenge died later than control animals. Foxes administered vaccinia virus died at the same time as unvaccinated controls. These results demonstrate that early and late death phenomena, as consequences of interactions between oral vaccination with VVTGgRAB and rabies infection, can occur in foxes.

Three wild foxes were trapped, inoculated with rabid fox salivary glands virus and radio-tracked straightaway for about three weeks until the death of the animal. Comparison of movements between incubation and clinical period indicate principally no obvious alteration of the activity area; an important increase in movement, especially during the day-time and lastly some variable changes in the activity pattern according to the individual. A common characteristic was a long period of immobility, before death, near the border of the usual activity area. In connection with these remarks, many sub-cutaneous lesions were observed from bites on the body of one of these three foxes, after necropsy. Epidemiological consequences of these first results were argued, especially as far as transmission of rabies from one fox to another is concerned.

After studying more than 1,000 siphonaptera tera collected from about 300 wild carnivora (with a majority of foxes, Vulpes vulpes), in the North-east of France, the authors state more precisely the biology and chorology of the main fleas collected: Pulex irritans, Ctenocephalides felis felis, C, canis, Chaetopsylla globiceps, Ch. rothschildi, Ch. trichosa, Paraceras melis melis. 9 other species are also noted.

In Western Europe the fox and other carnivora are parasitized by two specific ticks. The study of host-parasite relationships and knowledge of rabies epidemiology led us to investigate for a possible part played by ticks as reservoir of rabies virus. Several hundreds of ticks (Pholeoixodes hexagonus and Pholeoixodes canisuga) were collected on wild foxes. Ticks collected from naturally infected rabid foxes were set on healthy foxes in order to contaminate them. Ticks collected from non-rabid foxes were set on healthy foxes in order to contaminate them. Ticks collected from non-rabid foxes were inoculated with rabies virus (fox salivary glands), and put on other healthy foxes. In all these experiments, ticks, inoculated or not, had usual feeding behaviour. No fox was rabid following infestation by ticks inoculated or collected from rabid foxes. Specific ticks of foxes do not appear to be a passive vector nor a reservoir of rabies virus in Western Europe.

The best conditions for performing a lymphocyte transformation test have been studied in the red fox with three phytomitogens: phytohaemagglutinin, concanavalin A and Pokeweed (phytolacca). These assays were carried out with varying different parameters of the reaction, among which concentration of mitogens, nature and duration of the lymphocyte cultures. Isolation of fox lymphocytes gave lower yields than human lymphocytes, but were similar in number to those from other domestic animal species. The best mitogenetic reactivity was observed with concanavalin A (optimal concentration being not very different from one mitogen to another) and the maximum transformation ratio was usually observed after four days of lymphocyte cultivation in round bottom plates. Mean value of stimulation ratio is about ten times less that in man, but not very different from dogs. These results allowed the use of this technique a second time to explore specific stimulations by rabies antigen. These last were observed only in 33% of foxes recently vaccinated against rabies, either with live or inactivated virus vaccine.