BACKGROUND
rabies is a viral zoonosis that causes progressive and incurable encephalitis.
rabies infection is caused by neurotropic rna viruses belonging to the rhabdoviridae family, lyssavirus genus.
there are seven classified members of lyssavirus  <cit> , the classical rabies virus  and the rabies-related viruses , with four additional viruses identified more recently in bat species from europe and asia: aravan, khujand, irkut and west caucasian bat viruses  <cit> -these  <dig> isolates are proposed as new genotypes.
genotypes  are associated in two immunopathologically and genetically distinct phylogroups  <cit> .
phylogroup  <dig> comprises five genotypes: duvenhage virus , european bat lyssaviruses , eblv- <dig> , australian bat lyssavirus   and the classical rabies virus .
classical rabies viruses circulate in carnivora world-wide and specifically in the americas in chiroptera.
phylogroup  <dig> includes two african genotypes, mokola virus  and lagos bat virus  isolated from shrews, cats and frugivorous and insectivorous bats.
members of classical rabies virus are found worldwide in carnivora  and in chiroptera in the americas), ablv in frugivorous and insectivorous bats  while the following genotypes duvenhage , eblv- <dig> and  <dig>  are isolated in insectivorous bats.
it has been shown  <cit> , that genotypes of phylogroup  <dig> are pathogenic for mice when injected by intracranial and intramuscular routes, while lyssaviruses from phylogroup  <dig> are less pathogenic by the intramuscular route.
in europe, bats are important lyssavirus reservoirs, with more than  <dig> reported cases since  <dig>  out of these  <dig> european rabid bats,  <dig> cases have been reported in the netherlands from  <dig> to  <dig>  <cit>  and  <dig> in germany from  <dig> to  <dig>  <cit> .
bats infected with eblv- <dig> and eblv- <dig> have been reported in several european countries, from russia to spain, particularly in coastal regions and more than 95% of the rabid bats are identified as eptesicus serotinus.
all infections in eptesicus serotinus are due to the two eblv- <dig> subtypes   <cit> , while the eblv- <dig> subtypes are host-restricted to myotis species.
despite four fatal infections in man , european bat lyssaviruses have rarely been reported to cross the species barrier: dead-end infections have been reported in denmark in sheep  <cit>  and in a stone marten in germany  <cit> .
antibodies have also been reported from a cat in denmark  <cit> , suggesting evidence of infection and more recently a cat from northwestern france was reported with rabies caused by infection with eblv- <dig>  <cit> .
since the late 1930's, in europe, red foxes have been the main reservoir and vector of classical rabies.
rabies virus was shown to be highly pathogenic in the fox with an incubation period varying from  <dig> days to  <dig> months depending on the dose and on the route of inoculation  <cit> .
comparative experimental studies on the pathogenicity and on the transmission of rabies have been undertaken using classical rabies virus and eblvs on various animal models: bats  <cit> , mice  <cit> , sheep and fox  <cit> , and ferrets  <cit> .
the knowledge on the susceptibility of terrestrial, wild and domestic animals to eblv- <dig> and eblv- <dig> remains limited.
in this study, we have undertaken experimental infections of silver foxes  by different routes and with different viral doses.
the sensitivity of silver foxes to rabies virus isolated from naturally infected foxes has been experimentally shown to be similar for red foxes .
the presence of the virus in different organs was assessed using the fluorescent antibody test  and the rabies tissue-culture infection test   <cit>  and reverse transcriptase – polymerase chain reaction   <cit> .
the presence of viral rna on oral swabs was assessed by rt-pcr.
specific neutralizing antibodies in serum samples were measured with classical and modified favnt.
RESULTS
1st clinical diseases in "infected" foxes and results of pathogenicity study
experimental infection was conducted in several groups of foxes  according to virus strains, doses and routes of infection.
the outcome of infection for all infected foxes in the five groups is presented in table  <dig>
eblv- <dig> and - <dig> ic pathogenicity experiment
- for the intracranial experiments using eblv- <dig> and eblv- <dig>  two trials were undertaken.
in the first one, two groups of two animals received  <dig>  logs mic ld <dig> of eblv- <dig> and  <dig>  logs of eblv- <dig> respectively .
in the second trial, two animals were infected with  <dig>  logs of eblv- <dig> and  <dig>  logs of eblv- <dig> .
one negative control was included in that ic experiment.
all foxes in group  <dig>  ic inoculated with either eblv- <dig> or eblv- <dig>  died between  <dig> and  <dig> days post-inoculation  and between  <dig> and  <dig> days p.i.
respectively.
the animals showed clinical signs between  <dig> and  <dig> days before euthanasia following eblv- <dig> inoculation and between  <dig> and  <dig> days following eblv- <dig> inoculation.
the observed clinical signs were the following:
• eblv- <dig> , dead  <dig> days p.i., dazed;
• eblv- <dig> , dead  <dig> days p.i., loss of appetite, anorexia, hypersalivation;
• eblv- <dig> , dead  <dig> days p.i., loss of appetite, anorexia, excitement, crisis;
• eblv- <dig> , dead  <dig> days p.i., excitement, biting and very aggressive behaviour.
- in group  <dig>  fox 5fe-5aec inoculated with eblv- <dig>  died  <dig> days p.i following a phase of paralysis, fox 5fe-58da inoculated with eblv- <dig>  died  <dig> days later, showing paralysis and alopecia.
both antigen and infectious particles were detected by fat and by rtcit in brain samples  from all infected animals  belonging to the group  <dig>  viral rna was detected in the same infected tissues for each animal.
salivary glands were diagnosed rabies positive in foxes 601-e <dig>  5fe-58da and 5fd-5f3c inoculated respectively with  <dig>  logs and  <dig>  logs of eblv- <dig> and  <dig>  logs of eblv- <dig>
ic eblv- <dig> and  <dig> inoculated foxes  belonging to the group  <dig> were diagnosed positive by fat .
eblv- <dig> and - <dig> im pathogenicity experiment
- none of the  <dig> foxes intramuscularly inoculated with  <dig>  logs mic ld <dig> of eblv- <dig>  died.
all foxes remained healthy during the 3-year observation period with no signs indicative of rabies.
following euthanasia of these five animals, no rabies virus antigen, no infectious particles and no viral rna were detected in brain or salivary glands samples.
none of the five negative controls died during the experiment .
all of the controls were lyssavirus negative using referenced diagnosis techniques.
- the doses of eblv- <dig> intramuscularly inoculated to the next group of foxes  were distributed as described in additional file  <dig>
all six eblv- <dig> inoculated foxes, except one , survived the challenge till  <dig> months p.i.
and did not develop any clinical signs indicative of rabies or suggestive of encephalitis.
the clinical rabies signs observed in fox 634-8fe <dig> that died  <dig> days p.i.
were: difficulty to swallow, balance problems, dazed appearance.
the second fox inoculated with the same dose  did not exhibit any clinical signs and remained healthy during the 19-month of the observation period.
at necropsy of fox 634-8fe <dig>  all specimens of brain  were shown to be positive by fat, rtcit and rt-pcr.
the sub maxillary salivary glands  were tested negative by fat.
fox 633-6ad <dig> was diagnosed negative on brain by fat, rtcit and rt-pcr.
- in the third im experiment ,  <dig> groups of five animals were infected with serial dilutions of eblv- <dig>  as described in additional file  <dig>  one negative control was included in the assay.
all inoculated foxes, except fox 5fe-45f <dig> , 5fe-60dd  and 5fd-acb <dig>  survived the challenge and did not develop clinical signs of rabies or signs suggesting encephalitis, during the  <dig> months of observation.
fox 5fe-45f <dig> , had an infected wound on the back, it was euthanized on humane grounds  <dig> days p.i.
it was diagnosed negative by fat, rtcit, and rt-pcr.
the clinical signs observed for the two animals 5fe-60dd  and 5fd-acb <dig> , moribund at  <dig> and  <dig> days p.i.
were: loss of appetite, anorexia, "strange behaviour evocative of classical rabies" and hypersalivation, prostration, excitement, tendency of biting the cage respectively.
comparative analysis of fat and rt-pcr was undertaken on different parts of brain and sub maxillary salivary glands samples collected from foxes 5fe-60dd and 5fd-acb <dig> and showed respectively the presence of antigen in medulla oblongata and thalamus, and cortex and thalamus.
viral rna was shown in the same infected brain tissues and in the following additional tissues: hippocampus, cerebellum and cortex for fox 5fe-60dd and in hippocampus, medulla oblongata, and cerebellum for fox 5fd-acb <dig>  the sub maxillary salivary glands  of the two dead animals were rabies negative by fat and by rt-pcr.
virus was not isolated from control foxes 126-c <dig> and 601-f <dig> that were located in adjacent cages to challenged animals.
following necropsy of these control animals, viral rna and rabies virus antigen was not detected from brain samples.
detection of viral rna or infectious virus on oral swabs
the confidence of negative pcr results was established by checking for each test the amplification of internal control, 18s rrna.
all oral swabs taken all over the experiments from foxes inoculated intramuscularly and intracranially with undiluted stock or serially diluted eblv- <dig> and eblv- <dig> viruses were negative both by rtcit and by rt-pcr.
the fox numbered 5fd68aa, inoculated intracranially with eblv- <dig>  died  <dig> days after infection, neither viral rna nor infectious virus were detected in the oral swab taken one day before the mortality .
neither viral rna nor infectious particles were detected on oral swabs taken from "uninoculated" animals during all experiments.
rabies virus neutralizing antibodies
the measurement of seroneutralising antibodies has been commonly used for many years to assess the level of protective immunity against rabies in animals.
serological monitoring is used as vna is the principal modality of protection following rabies infection, which can be correlated with the dose.
in seroneutralisation tests, the dilution of the serum that neutralises 50% of the challenge virus is the efficient dilution 50% .
the comparison of this ed <dig> with the one of a reference serum whose titre in iu/ml is established gives the titre of the tested serum.
in all the favn tests published here, the reference serum used was the oie international serum of dog origin.
for these serological tests, the log  value of  <dig>  corresponds to the specific eblv- <dig> titre of  <dig> ui/ml measured with the oie dog reference serum that is calibrated against cvs- <dig>  as there are no specific reference serum for eblvs with a recognized titre , we have chosen to express the serological results as the log that corresponds to the neutralization of 50% of the challenge virus.
several studies on the sensitivity of favn test have shown that the threshold of positivity of  <dig>  ui/ml could be adopted  <cit> .
based on these previous studies, we assume for this serological study, that animals seroconverted above the value of  <dig>  log for ed <dig> in both eblv- <dig> mfavn and cvs- <dig> favnt.
eblv- <dig> serology
at day  <dig>  all experimented foxes were negative for rabies virus antibody by using both eblv- <dig> mfavnt and cvs- <dig> favnt.
in group  <dig> , all the  <dig> intramuscularly inoculated animals developed eblv- <dig> antibodies.
thirty five days p.i., animal 13c-a15a showed a log =  <dig> .
the four remaining animals ranged between  <dig>  and  <dig>  iu/ml.
four days later ,  <dig> out of the five inoculated animals showed a rabies eblv antibody peak  with a maximum of  <dig> .
at the end of the trial  one surviving animal presented a high log of  <dig>  while the  <dig> others ranged between  <dig>  and  <dig> .
in group  <dig> , different doses of eblv- <dig> were used.
four animals out of the five seroconverted  <dig> days post infection with log>  <dig> , the mean of log was  <dig> .
this value peaked at  <dig>  on day  <dig> p.i.
and remained stable up to  <dig> days p.i.
.
at the end of trial , the mean log was  <dig> .
two of the  <dig> surviving animals demonstrated a log still positive:  <dig>  and  <dig> .
in group  <dig> ,  <dig> out of the fifteen animals im infected with eblv- <dig> developed rabies antibody between  <dig> and  <dig> days post infection with a mean log of  <dig>  and  <dig>  respectively.
this value decreased down to  <dig>  at the end of trial.
one animal , receiving  <dig>  log of mic ld <dig> of infectious virus did not develop antibodies after inoculation, all along the experiment, the log was under  <dig> .
considering that the cut off value for log is  <dig> ,  <dig> animals seroconverted  ranging between  <dig>  and  <dig> ) between  <dig> and  <dig> days p.i.
the  <dig> foxes that died after inoculation  developed detectable eblv- <dig> antibodies before succumbing: log =  <dig>  and  <dig>  respectively  <dig> and  <dig> days p.i.
at the end of the experiment, the mean log was  <dig>  in surviving animals.
five days after intracranial inoculation with pure eblv- <dig> the log of fox 5fe-5aec, peaked at  <dig>  and then decreased to  <dig>  at day  <dig>  the animal was euthanised on day  <dig> p.i.
the other animal intracranially inoculated with eblv- <dig>  did not develop detectable antibody during the entire experiment, the log varied between  <dig>  and  <dig>  between inoculation and  <dig> days p.i.
the animal was euthanased  <dig> days p.i.
with symptoms, without serological analysis of blood samples by favnt.
all negative control animals belonging to the five groups remained seronegative with log< <dig>  throughout the experiments.
eblv- <dig> serology
foxes inoculated via the ic route did not seroconvert prior to clinical disease during the  <dig> days of observation  =  <dig> ).
the log of eblv- <dig> specific antibodies of animals inoculated via the im route generally fluctuated between  <dig>  and  <dig> , i.e.
above the positive threshold  between days  <dig> and  <dig> of the  <dig> animals, three had substantially higher antibody levels than the other two.
there were substantial differences between the specific serological responses of the eblv- <dig> inoculated animals depending on whether the sera were tested against a homologous genotype  or a heterologous one .
cvs serology
in group  <dig> , three animals out of the five animals infected with  <dig>  log mic ld <dig> of eblv- <dig>  seroconverted, log> <dig>  between  <dig>  and  <dig> days p.i.
.
the log of two other animals  was  <dig>  and  <dig> .
at the end of the trial, all surviving animals  presented a log between  <dig>  and  <dig> .
five foxes intramuscularly inoculated with eblv- <dig>  seroconverted with log between  <dig>  and  <dig> , 12– <dig> days p.i.
the mean value peaked at  <dig> days p.i.
and decreased down to  <dig>   <dig> months p.i.
in the final im eblv- <dig> experiment , only  <dig> animals out of the  <dig> infected seroconverted above  <dig>  between  <dig> and  <dig> days post inoculation, with a mean log varying between  <dig>  and  <dig> .
the mean log for favnt conducted with cvs- <dig> increased from  <dig>  to  <dig>  , then decreased to  <dig>  at the end of the trial.
histopathology
eblv infected animals were euthanased at a time of advanced disease after which necropsy took place.
both neural and extra-neural tissues were collected for pathology studies.
it was observed that brain tissue from ic inoculated foxes demonstrated greater meningoencephalitis or gliosis in the form of generalised and focal cellular infiltrate including peri-vascular cuffing in eblv- <dig> infected foxes than eblv- <dig> infected animals  similar to that seen in eblv- <dig> infected experimental mice  <cit> .
the distribution and intensity of lyssavirus antigen detection varied between viruses and inoculation route.
there was substantially more immunostaining on brain tissue from eblv- <dig> infected animals than that from eblv- <dig> infected animals following ic inoculation.
regional differences were most prominent in the hippocampus and cerebellum.
comparison of results between ic and im routes was only possible in the case of eblv- <dig> as no animals peripherally inoculated with eblv- <dig> succumbed to disease.
in the case of eblv- <dig> both antigen detection and inflammatory infiltrate varied in regions of the brain.
the most obvious differences included the rarity of infected neurons in the hippocampus in im inoculated animals and enhanced inflammatory infiltrate in the thalamus and medulla.
cervical spinal cord sections were positive in both ic inoculated eblv- <dig> and eblv- <dig> infected animals with distinct signal in many  motor neuron cell bodies.
thoracic and cervical spinal sections were negative as confirmed by pcr and virus re-isolation .
isolated neurons in the brachial plexi were also positive but this could not be confirmed by other methods.
other extra-neural tissues contained a small number of non-specifically stained cells.
discussion
red foxes are the principal reservoir and vector of classical rabies virus  in western europe  <cit> .
thanks to the campaigns of oral vaccination of foxes, france and other western european countries have been rabies free for many years  <cit> .
this study was undertaken to assess the risk of spillover of a bat lyssavirus strain from a bat to a fox and causing rabies infection with eblv- <dig> and eblv- <dig>  and to investigate whether or not foxes can transmit eblvs.
these experiments were conducted with high doses of virus using two different routes of infection: intramuscular and intracranial.
in our study, we have shown that all foxes infected experimentally intracranially with undiluted stock or diluted batches of eblv- <dig> and  <dig> exhibited clinical signs of rabies and succumbed between  <dig> and  <dig> days post infection.
all clinically ill foxes developed neutralising antibodies and contained viral antigen, infectious particles and viral rna in the brain.
salivary glands were positive in three out of six animals, but none of the oral swabs collected from these animals were positive for viral rna or infectious particles.
the two successive intramuscular trials undertaken with eblv- <dig>  did not give reproducible results, and surprisingly, some foxes challenged with the minimal doses of eblv- <dig> succumbed and presented neurological clinical signs indicative of rabies.
surprisingly, in both trials, none of the foxes infected with undiluted stock succumbed, and all but two, developed a high neutralising antibody response suggesting an abortive peripheral infection.
similarly to intracranial experiments, neither viral rna nor infectious particles were detected in any of the saliva obtained with oral swabs collected regularly from all infected animals and kept dry on ice until analysis.
in our study, we have shown the influence of the strain of challenge virus used in favn assays .
similar observations have been previously reported by moore et al  <cit> .
favnt assays using homologous virus  reported higher neutralising titres for all foxes infected intramuscularly with eblv- <dig> than when the assay was conducted against cvs- <dig>  this differential neutralising response has also been observed from a qualitative point of view.
with the eblv- <dig> modified favnt,  <dig> of the  <dig> experimented animals seroconverted while only  <dig> seroconverted with favnt conducted against cvs- <dig>
rabies pathogenesis has been assessed by several groups by using different variants of lyssavirus including the classical genotype  <dig> isolated from infected american bats, dogs or foxes  <cit> , and eblv- <dig> and eblv- <dig> on ferrets, sheep, foxes, cats and mice  <cit> .
more recently, experimental studies in bats using eblv- <dig> and eblv- <dig> have shown that bats are likely to transmit the virus following a shallow bite to peripheral tissue  <cit> .
infection on mice with eblv- <dig> in our experiments involved agitation, hyperactivity and aggressiveness before death .
clinical signs observed in mice after infection with eblv- <dig> were different from the ones following eblv- <dig> infection: prostration and paralysis before death are frequent .
the same variable clinical signs have also been observed in foxes after infection with rabies viruses: experimentally infected foxes first exhibit loss of appetite, hyperactivity, tremor, hypersensitivity, and sudden aggressiveness, convulsions and paralysis are common late signs  <cit> .
in our study, we observed that the clinical phase is short and varies between  <dig> to  <dig> days.
similar results were shown by some rabies pathologists and reviewed by wandeler in  <dig>  <cit> , the clinical phase lasted one to seventeen days  <cit> .
in this study, foxes have been infected intracranially  and only six animals have been inoculated.
the two facts make the comparison of observed clinical signs between eblvs and genotype  <dig> virus inconclusive.
the only reliable observations undertaken on these foxes are the following: death occurs within one week post inoculation and usual clinical signs of rabies have been observed.
red foxes have been shown to be highly susceptible to classical rabies by the intramuscular route, for example, the im ld <dig> of a street rabies virus isolated in north america intramuscularly was less than  <dig> mic ld <dig>  <cit> .
in the same way, blancou et al.
<cit>  showed that the intramuscular route is  <dig>  ×  <dig> to  <dig>  ×  <dig> times more efficient to infect red foxes than the oral route.
the dose of virus, the route of inoculation and entry into the central nervous system are of the utmost importance.
it has been suggested that both virus and host factors including strain, route of inoculation, distance of inoculation site from the brain and the immunologic status of the host, influence the incubation periods, and whether the resulting infection is abortive or fatal  <cit> .
in our study, we have shown that fatal clinical diseases can be induced by intracranial route, while the intramuscular route and a high dose of eblv result in a non-fatal disease, accompanied by a serological neutralising response.
similar to our study, it has been shown by blancou et al.
<cit>  that foxes were susceptible to low doses of canine rabies virus from north africa, and resisted the inoculation of a high dose and became immune, reflecting a possible abortive peripheral infection or a failure in virus replication.
spill over of rabies virus  has been reported to occur from bats to red foxes in newfoundland, canada  <cit>  and from bats to skunks in arizona, usa  <cit> .
these observations suggest that north american bat strains of rabies virus more readily spill over and adapt to a new host when compared with eblvs.
we speculate that these observations suggest that 'old world' bat strains of rabies virus have evolved over thousands of years and are fully-adapted to their chiropteran host.
in contrast, we hypothesise that 'new world' bat strains of rabies virus are in evolutionary terms 'younger' viruses, are less adapted to their chiropteran host and are therefore more prone to spill over in search of a new mammalian host.
because of the narrow co-adaptation between a rabies strain and its usual normal host, spill overs into other species have a very low probability in relation to establishing a new rabies cycle.
it means that specific virus variants within a single genotype  tend to be maintained in their specific hosts without mixing.
as for carnivora, the compartmentalisation of bat rabies seems to be the epidemiological rule, although spill-over infection to other mammals has been documented  <cit> .
natural transmission of eblv- <dig> to terrestrial animals has been previously reported in danish sheep  <cit>  and in a stone marten in germany  <cit> , the first case of transmission in terrestrial wildlife.
ferrets have been reported by vos et al.
<cit>  as being susceptible to infection with a high dose of eblv- <dig> , resulting in an incubation period of between  <dig> and  <dig> days.
these animals are dead-end hosts and are unable to transmit the infectious virus to a new host.
similarly to our results, all ferrets that became clinically ill were positive by fat in brain, all oral swabs were negative and viral rna could only be detected in salivary glands of infected animals  <cit> .
it has rarely been reported that infection of mammals with eblv has resulted in fatal disease  <cit> .
soria balthazar et al.
<cit>  infected sheep with an eblv- <dig> virus isolated from a rabid bat  in denmark in  <dig> , with doses ranging between  <dig>  and  <dig>  mic ld <dig>  only one animal infected with the highest dose succumbed to disease  <dig> days post inoculation, the incubation period lasted  <dig> days.
no antigen was detected in the brain while infectious particles were detected by mouse inoculation test.
additionally, it has been shown in the same study, that  <dig> out of  <dig> foxes intramuscularly inoculated into the masseter with different doses of "duvenhage like" virus, died between  <dig> and  <dig> days post infection  <cit> .
rabies diagnosis  undertaken on the brains of these  <dig> animals showed the presence of rabies antigen in two and infectious particles have been detected in one.
all of these experiments, including our results, strongly suggest that the outcome of disease after exposure to eblvs may be dependent on the host species, the inoculation site and the type of infecting virus.
most natural rabies cases result from the bite of a rabid animal  <cit> , although infection through aerosols under experimental conditions in mice has been reported  <cit> .
the other routes of infection are rare.
few studies have been undertaken to monitor the viral shedding of naturally or experimentally infected animals.
bats naturally or experimentally infected with genotype  <dig> lyssavirus have been shown to harbour virus in the nasal mucosa  <cit>  and in the saliva  respectively, leading to the supposition that a transmission between bats living in the same colony is possible.
recently hughes et al.
<cit>  reported the presence of viral rna in oral swabs of experimented bats infected with khujand and irkut new lyssaviruses, between  <dig> and  <dig> days before the onset of illness.
in  <dig>  the presence of viral rna has been detected by molecular tools on an oral swab collected from a common serotine naturally infected with eblv- <dig> three days before death; no infectious virus was detected.
our experiments have not detected viral rna or virus infectious particles in any of the oral swabs collected throughout the experiments.
as observed in other infection models, shedding of virus may be intermittent and can explain the absence of detection of viral rna in oral swabs.
comparative studies should be undertaken in order to further ameliorate our understanding of rabies virus excretion in saliva and salivary gland infection.
the diseased brain tissue was characterised by diffuse meningioencephalitis with gliosis and perivascular cuffing as observed in eblv- <dig> infected cats and dogs  <cit>  and viral antigen is seen in neurons throughout the various regions of the brain.
there was a suggestion of antigen distribution differences in the cerebellum and hippocampus; the signal appeared less intense and more focal in eblv- <dig> compared to that in eblv- <dig> infected tissue.
in general the number of infected cells and intensity of staining was greater for eblv- <dig> than eblv- <dig>  inclusion bodies similar but less distinct than rabv negri bodies were observed in eblv- <dig> infected cells but rarely in eblv- <dig> infected tissue, and inflammation was more prevalent in eblv- <dig> than eblv- <dig>  inflammation of the brain in the form of gliosis and perivascular cuffing were also observed in those foxes that succumb to clinical disease; eblv- <dig> ic and im and eblv- <dig> ic inoculations.
similar observations recently conducted by brookes et al.
<cit>  on ic and im sheep experiments with eblv- <dig> and- <dig>  showed that as the previous study conducted on ferrets  <cit> , eblvs show a limited capacity to induce lethal disease when inoculated at a peripheral site .
the pathogenicity of eblv- <dig> for foxes appears to be low using the intramuscular route, which closely mimics the classical natural transmission route of rabies virus to new hosts.
furthermore, the absence of detection of infectious particles or of viral rna in saliva throughout the intracranial and intramuscular experiments undertaken in our study suggests that foxes may not be able to transmit eblv- <dig> as virus was not detected in the saliva of clinically infected animals.
the chance of eblv spill over from bat to fox is low, there is a minimal chance of contact between insectivorous bats and foxes which seem to be dead-end hosts for eblvs viruses.
CONCLUSIONS
the susceptibility of foxes to experimental infection with eblvs appears to be very low and thus the virus represents a low risk to red foxes in the population.
this does not, however, negate the fact that red foxes are the main host and vector of classical rabies in europe.
further studies, including the use of different eblv isolates and doses, different routes and sites of infection and the use of other potential host species as experimental models are required in order to ameliorate our understanding of pathogen adaptation and of the host response, which can result in an abortive infection, survival with clinical sequelae or lethal infection.
further in vitro studies on the eblv- <dig> neurotropism and similar fox experiments with the subcutaneous route to mimic superficial exposure should be undertaken to assess the risk of eblv spillover.

 rabies is a viral zoonosis that causes progressive and incurable encephalitis. rabies infection is caused by neurotropic rna viruses belonging to the rhabdoviridae family, lyssavirus genus. there are seven classified members of lyssavirus, the classical rabies virus and the rabies-related viruses, with four additional viruses identified more recently in bat species from europe and asia: aravan, khujand, irkut and west caucasian bat viruses -these isolates are proposed as new genotypes. genotypes are associated in two immunopathologically and genetically distinct phylogroups. phylogroup comprises five genotypes: duvenhage virus, european bat lyssaviruses, eblv-, australian bat lyssavirus and the classical rabies virus. classical rabies viruses circulate in carnivora world-wide and specifically in the americas in chiroptera. phylogroup includes two african genotypes, mokola virus and lagos bat virus isolated from shrews, cats and frugivorous and insectivorous bats. members of classical rabies virus are found worldwide in carnivora and in chiroptera in the americas), ablv in frugivorous and insectivorous bats while the following genotypes duvenhage, eblv- and are isolated in insectivorous bats. it has been shown, that genotypes of phylogroup are pathogenic for mice when injected by intracranial and intramuscular routes, while lyssaviruses from phylogroup are less pathogenic by the intramuscular route. in europe, bats are important lyssavirus reservoirs, with more than reported cases since out of these european rabid bats, cases have been reported in the netherlands from to and in germany from to. bats infected with eblv- and eblv- have been reported in several european countries, from russia to spain, particularly in coastal regions and more than 95% of the rabid bats are identified as eptesicus serotinus. all infections in eptesicus serotinus are due to the two eblv- subtypes, while the eblv- subtypes are host-restricted to myotis species. despite four fatal infections in man, european bat lyssaviruses have rarely been reported to cross the species barrier: dead-end infections have been reported in denmark in sheep and in a stone marten in germany. antibodies have also been reported from a cat in denmark, suggesting evidence of infection and more recently a cat from northwestern france was reported with rabies caused by infection with eblv-. since the late 1930's, in europe, red foxes have been the main reservoir and vector of classical rabies. rabies virus was shown to be highly pathogenic in the fox with an incubation period varying from days to months depending on the dose and on the route of inoculation. comparative experimental studies on the pathogenicity and on the transmission of rabies have been undertaken using classical rabies virus and eblvs on various animal models: bats, mice, sheep and fox, and ferrets. the knowledge on the susceptibility of terrestrial, wild and domestic animals to eblv- and eblv- remains limited. in this study, we have undertaken experimental infections of silver foxes by different routes and with different viral doses. the sensitivity of silver foxes to rabies virus isolated from naturally infected foxes has been experimentally shown to be similar for red foxes. the presence of the virus in different organs was assessed using the fluorescent antibody test and the rabies tissue-culture infection test and reverse transcriptase – polymerase chain reaction. the presence of viral rna on oral swabs was assessed by rt-pcr. specific neutralizing antibodies in serum samples were measured with classical and modified favnt.  1st clinical diseases in "infected" foxes and results of pathogenicity study experimental infection was conducted in several groups of foxes according to virus strains, doses and routes of infection. the outcome of infection for all infected foxes in the five groups is presented in table eblv- and - ic pathogenicity experiment - for the intracranial experiments using eblv- and eblv- two trials were undertaken. in the first one, two groups of two animals received logs mic ld of eblv- and logs of eblv- respectively. in the second trial, two animals were infected with logs of eblv- and logs of eblv-. one negative control was included in that ic experiment. all foxes in group ic inoculated with either eblv- or eblv- died between and days post-inoculation and between and days p.i. respectively. the animals showed clinical signs between and days before euthanasia following eblv- inoculation and between and days following eblv- inoculation. the observed clinical signs were the following: • eblv-, dead days p.i., dazed; • eblv-, dead days p.i., loss of appetite, anorexia, hypersalivation; • eblv-, dead days p.i., loss of appetite, anorexia, excitement, crisis; • eblv-, dead days p.i., excitement, biting and very aggressive behaviour. - in group fox 5fe-5aec inoculated with eblv- died days p.i following a phase of paralysis, fox 5fe-58da inoculated with eblv- died days later, showing paralysis and alopecia. both antigen and infectious particles were detected by fat and by rtcit in brain samples from all infected animals belonging to the group viral rna was detected in the same infected tissues for each animal. salivary glands were diagnosed rabies positive in foxes 601-e 5fe-58da and 5fd-5f3c inoculated respectively with logs and logs of eblv- and logs of eblv- ic eblv- and inoculated foxes belonging to the group were diagnosed positive by fat. eblv- and - im pathogenicity experiment - none of the foxes intramuscularly inoculated with logs mic ld of eblv- died. all foxes remained healthy during the 3-year observation period with no signs indicative of rabies. following euthanasia of these five animals, no rabies virus antigen, no infectious particles and no viral rna were detected in brain or salivary glands samples. none of the five negative controls died during the experiment. all of the controls were lyssavirus negative using referenced diagnosis techniques. - the doses of eblv- intramuscularly inoculated to the next group of foxes were distributed as described in additional file all six eblv- inoculated foxes, except one, survived the challenge till months p.i. and did not develop any clinical signs indicative of rabies or suggestive of encephalitis. the clinical rabies signs observed in fox 634-8fe that died days p.i. were: difficulty to swallow, balance problems, dazed appearance. the second fox inoculated with the same dose did not exhibit any clinical signs and remained healthy during the 19-month of the observation period. at necropsy of fox 634-8fe all specimens of brain were shown to be positive by fat, rtcit and rt-pcr. the sub maxillary salivary glands were tested negative by fat. fox 633-6ad was diagnosed negative on brain by fat, rtcit and rt-pcr. - in the third im experiment, groups of five animals were infected with serial dilutions of eblv- as described in additional file one negative control was included in the assay. all inoculated foxes, except fox 5fe-45f, 5fe-60dd and 5fd-acb survived the challenge and did not develop clinical signs of rabies or signs suggesting encephalitis, during the months of observation. fox 5fe-45f, had an infected wound on the back, it was euthanized on humane grounds days p.i. it was diagnosed negative by fat, rtcit, and rt-pcr. the clinical signs observed for the two animals 5fe-60dd and 5fd-acb, moribund at and days p.i. were: loss of appetite, anorexia, "strange behaviour evocative of classical rabies" and hypersalivation, prostration, excitement, tendency of biting the cage respectively. comparative analysis of fat and rt-pcr was undertaken on different parts of brain and sub maxillary salivary glands samples collected from foxes 5fe-60dd and 5fd-acb and showed respectively the presence of antigen in medulla oblongata and thalamus, and cortex and thalamus. viral rna was shown in the same infected brain tissues and in the following additional tissues: hippocampus, cerebellum and cortex for fox 5fe-60dd and in hippocampus, medulla oblongata, and cerebellum for fox 5fd-acb the sub maxillary salivary glands of the two dead animals were rabies negative by fat and by rt-pcr. virus was not isolated from control foxes 126-c and 601-f that were located in adjacent cages to challenged animals. following necropsy of these control animals, viral rna and rabies virus antigen was not detected from brain samples. detection of viral rna or infectious virus on oral swabs the confidence of negative pcr results was established by checking for each test the amplification of internal control, 18s rrna. all oral swabs taken all over the experiments from foxes inoculated intramuscularly and intracranially with undiluted stock or serially diluted eblv- and eblv- viruses were negative both by rtcit and by rt-pcr. the fox numbered 5fd68aa, inoculated intracranially with eblv- died days after infection, neither viral rna nor infectious virus were detected in the oral swab taken one day before the mortality. neither viral rna nor infectious particles were detected on oral swabs taken from "uninoculated" animals during all experiments. rabies virus neutralizing antibodies the measurement of seroneutralising antibodies has been commonly used for many years to assess the level of protective immunity against rabies in animals. serological monitoring is used as vna is the principal modality of protection following rabies infection, which can be correlated with the dose. in seroneutralisation tests, the dilution of the serum that neutralises 50% of the challenge virus is the efficient dilution 50%. the comparison of this ed with the one of a reference serum whose titre in iu/ml is established gives the titre of the tested serum. in all the favn tests published here, the reference serum used was the oie international serum of dog origin. for these serological tests, the log value of corresponds to the specific eblv- titre of ui/ml measured with the oie dog reference serum that is calibrated against cvs- as there are no specific reference serum for eblvs with a recognized titre, we have chosen to express the serological results as the log that corresponds to the neutralization of 50% of the challenge virus. several studies on the sensitivity of favn test have shown that the threshold of positivity of ui/ml could be adopted. based on these previous studies, we assume for this serological study, that animals seroconverted above the value of log for ed in both eblv- mfavn and cvs- favnt. eblv- serology at day all experimented foxes were negative for rabies virus antibody by using both eblv- mfavnt and cvs- favnt. in group, all the intramuscularly inoculated animals developed eblv- antibodies. thirty five days p.i., animal 13c-a15a showed a log =. the four remaining animals ranged between and iu/ml. four days later, out of the five inoculated animals showed a rabies eblv antibody peak with a maximum of. at the end of the trial one surviving animal presented a high log of while the others ranged between and. in group, different doses of eblv- were used. four animals out of the five seroconverted days post infection with log>, the mean of log was. this value peaked at on day p.i. and remained stable up to days p.i.. at the end of trial, the mean log was. two of the surviving animals demonstrated a log still positive: and. in group, out of the fifteen animals im infected with eblv- developed rabies antibody between and days post infection with a mean log of and respectively. this value decreased down to at the end of trial. one animal, receiving log of mic ld of infectious virus did not develop antibodies after inoculation, all along the experiment, the log was under. considering that the cut off value for log is, animals seroconverted ranging between and ) between and days p.i. the foxes that died after inoculation developed detectable eblv- antibodies before succumbing: log = and respectively and days p.i. at the end of the experiment, the mean log was in surviving animals. five days after intracranial inoculation with pure eblv- the log of fox 5fe-5aec, peaked at and then decreased to at day the animal was euthanised on day p.i. the other animal intracranially inoculated with eblv- did not develop detectable antibody during the entire experiment, the log varied between and between inoculation and days p.i. the animal was euthanased days p.i. with symptoms, without serological analysis of blood samples by favnt. all negative control animals belonging to the five groups remained seronegative with log< throughout the experiments. eblv- serology foxes inoculated via the ic route did not seroconvert prior to clinical disease during the days of observation = ). the log of eblv- specific antibodies of animals inoculated via the im route generally fluctuated between and, i.e. above the positive threshold between days and of the animals, three had substantially higher antibody levels than the other two. there were substantial differences between the specific serological responses of the eblv- inoculated animals depending on whether the sera were tested against a homologous genotype or a heterologous one. cvs serology in group, three animals out of the five animals infected with log mic ld of eblv- seroconverted, log> between and days p.i.. the log of two other animals was and. at the end of the trial, all surviving animals presented a log between and. five foxes intramuscularly inoculated with eblv- seroconverted with log between and, 12– days p.i. the mean value peaked at days p.i. and decreased down to months p.i. in the final im eblv- experiment, only animals out of the infected seroconverted above between and days post inoculation, with a mean log varying between and. the mean log for favnt conducted with cvs- increased from to, then decreased to at the end of the trial. histopathology eblv infected animals were euthanased at a time of advanced disease after which necropsy took place. both neural and extra-neural tissues were collected for pathology studies. it was observed that brain tissue from ic inoculated foxes demonstrated greater meningoencephalitis or gliosis in the form of generalised and focal cellular infiltrate including peri-vascular cuffing in eblv- infected foxes than eblv- infected animals similar to that seen in eblv- infected experimental mice. the distribution and intensity of lyssavirus antigen detection varied between viruses and inoculation route. there was substantially more immunostaining on brain tissue from eblv- infected animals than that from eblv- infected animals following ic inoculation. regional differences were most prominent in the hippocampus and cerebellum. comparison of results between ic and im routes was only possible in the case of eblv- as no animals peripherally inoculated with eblv- succumbed to disease. in the case of eblv- both antigen detection and inflammatory infiltrate varied in regions of the brain. the most obvious differences included the rarity of infected neurons in the hippocampus in im inoculated animals and enhanced inflammatory infiltrate in the thalamus and medulla. cervical spinal cord sections were positive in both ic inoculated eblv- and eblv- infected animals with distinct signal in many motor neuron cell bodies. thoracic and cervical spinal sections were negative as confirmed by pcr and virus re-isolation. isolated neurons in the brachial plexi were also positive but this could not be confirmed by other methods. other extra-neural tissues contained a small number of non-specifically stained cells. discussion red foxes are the principal reservoir and vector of classical rabies virus in western europe. thanks to the campaigns of oral vaccination of foxes, france and other western european countries have been rabies free for many years. this study was undertaken to assess the risk of spillover of a bat lyssavirus strain from a bat to a fox and causing rabies infection with eblv- and eblv- and to investigate whether or not foxes can transmit eblvs. these experiments were conducted with high doses of virus using two different routes of infection: intramuscular and intracranial. in our study, we have shown that all foxes infected experimentally intracranially with undiluted stock or diluted batches of eblv- and exhibited clinical signs of rabies and succumbed between and days post infection. all clinically ill foxes developed neutralising antibodies and contained viral antigen, infectious particles and viral rna in the brain. salivary glands were positive in three out of six animals, but none of the oral swabs collected from these animals were positive for viral rna or infectious particles. the two successive intramuscular trials undertaken with eblv- did not give reproducible results, and surprisingly, some foxes challenged with the minimal doses of eblv- succumbed and presented neurological clinical signs indicative of rabies. surprisingly, in both trials, none of the foxes infected with undiluted stock succumbed, and all but two, developed a high neutralising antibody response suggesting an abortive peripheral infection. similarly to intracranial experiments, neither viral rna nor infectious particles were detected in any of the saliva obtained with oral swabs collected regularly from all infected animals and kept dry on ice until analysis. in our study, we have shown the influence of the strain of challenge virus used in favn assays. similar observations have been previously reported by moore et al. favnt assays using homologous virus reported higher neutralising titres for all foxes infected intramuscularly with eblv- than when the assay was conducted against cvs- this differential neutralising response has also been observed from a qualitative point of view. with the eblv- modified favnt, of the experimented animals seroconverted while only seroconverted with favnt conducted against cvs- rabies pathogenesis has been assessed by several groups by using different variants of lyssavirus including the classical genotype isolated from infected american bats, dogs or foxes, and eblv- and eblv- on ferrets, sheep, foxes, cats and mice. more recently, experimental studies in bats using eblv- and eblv- have shown that bats are likely to transmit the virus following a shallow bite to peripheral tissue. infection on mice with eblv- in our experiments involved agitation, hyperactivity and aggressiveness before death. clinical signs observed in mice after infection with eblv- were different from the ones following eblv- infection: prostration and paralysis before death are frequent. the same variable clinical signs have also been observed in foxes after infection with rabies viruses: experimentally infected foxes first exhibit loss of appetite, hyperactivity, tremor, hypersensitivity, and sudden aggressiveness, convulsions and paralysis are common late signs. in our study, we observed that the clinical phase is short and varies between to days. similar results were shown by some rabies pathologists and reviewed by wandeler in, the clinical phase lasted one to seventeen days. in this study, foxes have been infected intracranially and only six animals have been inoculated. the two facts make the comparison of observed clinical signs between eblvs and genotype virus inconclusive. the only reliable observations undertaken on these foxes are the following: death occurs within one week post inoculation and usual clinical signs of rabies have been observed. red foxes have been shown to be highly susceptible to classical rabies by the intramuscular route, for example, the im ld of a street rabies virus isolated in north america intramuscularly was less than mic ld. in the same way, blancou et al. showed that the intramuscular route is × to × times more efficient to infect red foxes than the oral route. the dose of virus, the route of inoculation and entry into the central nervous system are of the utmost importance. it has been suggested that both virus and host factors including strain, route of inoculation, distance of inoculation site from the brain and the immunologic status of the host, influence the incubation periods, and whether the resulting infection is abortive or fatal. in our study, we have shown that fatal clinical diseases can be induced by intracranial route, while the intramuscular route and a high dose of eblv result in a non-fatal disease, accompanied by a serological neutralising response. similar to our study, it has been shown by blancou et al. that foxes were susceptible to low doses of canine rabies virus from north africa, and resisted the inoculation of a high dose and became immune, reflecting a possible abortive peripheral infection or a failure in virus replication. spill over of rabies virus has been reported to occur from bats to red foxes in newfoundland, canada and from bats to skunks in arizona, usa. these observations suggest that north american bat strains of rabies virus more readily spill over and adapt to a new host when compared with eblvs. we speculate that these observations suggest that'old world' bat strains of rabies virus have evolved over thousands of years and are fully-adapted to their chiropteran host. in contrast, we hypothesise that'new world' bat strains of rabies virus are in evolutionary terms'younger' viruses, are less adapted to their chiropteran host and are therefore more prone to spill over in search of a new mammalian host. because of the narrow co-adaptation between a rabies strain and its usual normal host, spill overs into other species have a very low probability in relation to establishing a new rabies cycle. it means that specific virus variants within a single genotype tend to be maintained in their specific hosts without mixing. as for carnivora, the compartmentalisation of bat rabies seems to be the epidemiological rule, although spill-over infection to other mammals has been documented. natural transmission of eblv- to terrestrial animals has been previously reported in danish sheep and in a stone marten in germany, the first case of transmission in terrestrial wildlife. ferrets have been reported by vos et al. as being susceptible to infection with a high dose of eblv-, resulting in an incubation period of between and days. these animals are dead-end hosts and are unable to transmit the infectious virus to a new host. similarly to our results, all ferrets that became clinically ill were positive by fat in brain, all oral swabs were negative and viral rna could only be detected in salivary glands of infected animals. it has rarely been reported that infection of mammals with eblv has resulted in fatal disease. soria balthazar et al. infected sheep with an eblv- virus isolated from a rabid bat in denmark in, with doses ranging between and mic ld only one animal infected with the highest dose succumbed to disease days post inoculation, the incubation period lasted days. no antigen was detected in the brain while infectious particles were detected by mouse inoculation test. additionally, it has been shown in the same study, that out of foxes intramuscularly inoculated into the masseter with different doses of "duvenhage like" virus, died between and days post infection. rabies diagnosis undertaken on the brains of these animals showed the presence of rabies antigen in two and infectious particles have been detected in one. all of these experiments, including our results, strongly suggest that the outcome of disease after exposure to eblvs may be dependent on the host species, the inoculation site and the type of infecting virus. most natural rabies cases result from the bite of a rabid animal, although infection through aerosols under experimental conditions in mice has been reported. the other routes of infection are rare. few studies have been undertaken to monitor the viral shedding of naturally or experimentally infected animals. bats naturally or experimentally infected with genotype lyssavirus have been shown to harbour virus in the nasal mucosa and in the saliva respectively, leading to the supposition that a transmission between bats living in the same colony is possible. recently hughes et al. reported the presence of viral rna in oral swabs of experimented bats infected with khujand and irkut new lyssaviruses, between and days before the onset of illness. in the presence of viral rna has been detected by molecular tools on an oral swab collected from a common serotine naturally infected with eblv- three days before death; no infectious virus was detected. our experiments have not detected viral rna or virus infectious particles in any of the oral swabs collected throughout the experiments. as observed in other infection models, shedding of virus may be intermittent and can explain the absence of detection of viral rna in oral swabs. comparative studies should be undertaken in order to further ameliorate our understanding of rabies virus excretion in saliva and salivary gland infection. the diseased brain tissue was characterised by diffuse meningioencephalitis with gliosis and perivascular cuffing as observed in eblv- infected cats and dogs and viral antigen is seen in neurons throughout the various regions of the brain. there was a suggestion of antigen distribution differences in the cerebellum and hippocampus; the signal appeared less intense and more focal in eblv- compared to that in eblv- infected tissue. in general the number of infected cells and intensity of staining was greater for eblv- than eblv- inclusion bodies similar but less distinct than rabv negri bodies were observed in eblv- infected cells but rarely in eblv- infected tissue, and inflammation was more prevalent in eblv- than eblv- inflammation of the brain in the form of gliosis and perivascular cuffing were also observed in those foxes that succumb to clinical disease; eblv- ic and im and eblv- ic inoculations. similar observations recently conducted by brookes et al. on ic and im sheep experiments with eblv- and- showed that as the previous study conducted on ferrets, eblvs show a limited capacity to induce lethal disease when inoculated at a peripheral site. the pathogenicity of eblv- for foxes appears to be low using the intramuscular route, which closely mimics the classical natural transmission route of rabies virus to new hosts. furthermore, the absence of detection of infectious particles or of viral rna in saliva throughout the intracranial and intramuscular experiments undertaken in our study suggests that foxes may not be able to transmit eblv- as virus was not detected in the saliva of clinically infected animals. the chance of eblv spill over from bat to fox is low, there is a minimal chance of contact between insectivorous bats and foxes which seem to be dead-end hosts for eblvs viruses.  the susceptibility of foxes to experimental infection with eblvs appears to be very low and thus the virus represents a low risk to red foxes in the population. this does not, however, negate the fact that red foxes are the main host and vector of classical rabies in europe. further studies, including the use of different eblv isolates and doses, different routes and sites of infection and the use of other potential host species as experimental models are required in order to ameliorate our understanding of pathogen adaptation and of the host response, which can result in an abortive infection, survival with clinical sequelae or lethal infection. further in vitro studies on the eblv- neurotropism and similar fox experiments with the subcutaneous route to mimic superficial exposure should be undertaken to assess the risk of eblv spillover. 
these viruses have rarely been reported to infect humans and terrestrial mammals, as the only exceptions are sheep in denmark, a stone marten in germany and a cat in france. these data suggest that the chance of a eblv spill-over from bat to fox is low, but with a greater probability for eblv- than for eblv- and that foxes seem to be able to clear the virus before it reaches the brain and cause a lethal infection. none of the animals given eblv- developed clinical disease. the purpose of this study was to investigate the susceptibility of foxes to eblvs using silver foxes as a model. virus dissemination and inflammatory infiltrate in the brain were demonstrated but virus specific neutralising antibody was limited = – and – respectively for specific eblv- and eblv-2). our experimental studies have shown that the susceptibility of foxes to eblvs is low by the intramuscular route, however, animals were sensitive to intracranial inoculation. foxes were also susceptible, at a low level, to peripheral infection with eblv- but not eblv- three out of foxes developed clinical signs between and days post-eblv- infection. mortality was 100% for both eblv- and eblv- delivered by the ic route.
 since there have been in excess of cases of rabies as a result of european bat lyssaviruses types and infection, mainly in serotine and myotis bats respectively. these viruses have rarely been reported to infect humans and terrestrial mammals, as the only exceptions are sheep in denmark, a stone marten in germany and a cat in france. the purpose of this study was to investigate the susceptibility of foxes to eblvs using silver foxes as a model.  our experimental studies have shown that the susceptibility of foxes to eblvs is low by the intramuscular route, however, animals were sensitive to intracranial inoculation. mortality was 100% for both eblv- and eblv- delivered by the ic route. virus dissemination and inflammatory infiltrate in the brain were demonstrated but virus specific neutralising antibody was limited = – and – respectively for specific eblv- and eblv-2). foxes were also susceptible, at a low level, to peripheral infection with eblv- but not eblv- three out of foxes developed clinical signs between and days post-eblv- infection. none of the animals given eblv- developed clinical disease.  these data suggest that the chance of a eblv spill-over from bat to fox is low, but with a greater probability for eblv- than for eblv- and that foxes seem to be able to clear the virus before it reaches the brain and cause a lethal infection. 