1stClinical diseases in "infected" foxes and results of pathogenicity study
Experimental infection was conducted in several groups of foxes (n = 5) 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 1.
EBLV-1 and -2 IC pathogenicity experiment
- For the intracranial experiments using EBLV-1 and EBLV-2, two trials were undertaken. In the first one, two groups of two animals received 4.7 logs MIC LD50 of EBLV-1 and 3.2 logs of EBLV-2 respectively (group 2). In the second trial, two animals were infected with 4.4 logs of EBLV-1 and 2.8 logs of EBLV-2 (group 5). (Additional file 1) One negative control was included in that IC experiment.
All foxes in group 2, IC inoculated with either EBLV-1 or EBLV-2, died between 8 and 10 days post-inoculation (p.i.) and between 12 and 22 days p.i. respectively.
The animals showed clinical signs between 2 and 4 days before euthanasia following EBLV-2 inoculation and between 1 and 5 days following EBLV-1 inoculation. The observed clinical signs were the following:
• EBLV-1 (fox 5FD-68AA receiving 4.7 logs MIC LD50), dead 8 days p.i., dazed;
• EBLV-1 (fox 5FD-5F3C, 4.7 logs), dead 10 days p.i., loss of appetite, anorexia, hypersalivation;
• EBLV-2 (fox 601-15A3, 3.2 logs), dead 12 days p.i., loss of appetite, anorexia, excitement, crisis;
• EBLV-2 (fox 601-E233, 3.2 logs), dead 22 days p.i., excitement, biting and very aggressive behaviour.
- In group 5, fox 5FE-5AEC inoculated with EBLV-1 (4.4 logs) died 8 days p.i following a phase of paralysis, fox 5FE-58DA inoculated with EBLV-2 (2.8 logs) died 282 days later, showing paralysis and alopecia.
Both antigen and infectious particles were detected by FAT and by RTCIT in brain samples (cortex, hippocampus, cerebellum and medulla oblongata) from all infected animals (5FD-68AA, 5FD-5F3C, 601-15A3 and 601-E233) belonging to the group 2. Viral RNA was detected in the same infected tissues for each animal. Salivary glands were diagnosed rabies positive in foxes 601-E233, 5FE-58DA and 5FD-5F3C inoculated respectively with 3.2 logs and 2.8 logs of EBLV-2 and 4.7 logs of EBLV-1.
IC EBLV-1 and 2 inoculated foxes (5FE-5AEC and 5FE-58DA) belonging to the group 5 were diagnosed positive by FAT (Additional file 1).
EBLV-1 and -2 IM pathogenicity experiment
- None of the 5 foxes intramuscularly inoculated with 3.5 logs MIC LD50 of EBLV-2 (group 1) 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 (except fox numbered 126-DF42 that died of a non-rabid undetermined cause 168 days p.i). All of the controls were lyssavirus negative using referenced diagnosis techniques.
- The doses of EBLV-1 intramuscularly inoculated to the next group of foxes (group 3; n = 6) were distributed as described in Additional file 1.
All six EBLV-1 inoculated foxes, except one (fox 634-8FE1 receiving a viral dose of 3.7 logs MIC LD50), survived the challenge till 20 months p.i. and did not develop any clinical signs indicative of rabies or suggestive of encephalitis. The clinical rabies signs observed in fox 634-8FE1 that died 14 days p.i. were: difficulty to swallow, balance problems, dazed appearance. The second fox inoculated with the same dose (fox 633-6AD2) did not exhibit any clinical signs and remained healthy during the 19-month of the observation period. At necropsy of fox 634-8FE1, all specimens of brain (hippocampus, medulla oblongata, thalamus, cortex and cerebellum) were shown to be positive by FAT, RTCIT and RT-PCR. The sub maxillary salivary glands (left and right) were tested negative by FAT. Fox 633-6AD2 was diagnosed negative on brain by FAT, RTCIT and RT-PCR.
- In the third IM experiment (group 4), 3 groups of five animals were infected with serial dilutions of EBLV-1 (4.4, 3.7 and 3 logs MIC LD50) as described in Additional file 1. One negative control was included in the assay.
All inoculated foxes, except fox 5FE-45F8 (receiving 3.7 logs of virus), 5FE-60DD (3.0 logs) and 5FD-ACB1 (3.0 logs) survived the challenge and did not develop clinical signs of rabies or signs suggesting encephalitis, during the 13 months of observation. Fox 5FE-45F8 (3.7 logs), had an infected wound on the back, it was euthanized on humane grounds 61 days p.i. It was diagnosed negative by FAT, RTCIT, and RT-PCR.
The clinical signs observed for the two animals 5FE-60DD (3.0 logs) and 5FD-ACB1 (3.0 logs), moribund at 17 and 24 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-ACB1 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-ACB1. The sub maxillary salivary glands (left and right) of the two dead animals were rabies negative by FAT and by RT-PCR.
Virus was not isolated from control foxes 126-C920 and 601-F206 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-1 and EBLV-2 viruses were negative both by RTCIT and by RT-PCR. The fox numbered 5FD68AA, inoculated intracranially with EBLV-1 (EBLV-1 IC group) died 8 days after infection, neither viral RNA nor infectious virus were detected in the oral swab taken one day before the mortality (D7).
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% (ED50). The comparison of this ED50 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.
Figure 1 shows the dynamics of rabies virus neutralizing antibodies of all experimented foxes. Each figure expresses FAVNt results with the neutralising response obtained against either CVS-11 or EBLV-1.
For these serological tests, the log (ED50) value of 1.55 corresponds to the specific EBLV-1 titre of 1 UI/ml measured with the OIE dog reference serum that is calibrated against CVS-11. As there are no specific reference serum for EBLVs with a recognized titre (in IU/ml), we have chosen to express the serological results as the log(ED50) 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 0.24 UI/ml could be adopted [28, 29]. Based on these previous studies, we assume for this serological study, that animals seroconverted above the value of 1.00 log for ED50 in both EBLV-1 mFAVN and CVS-11 FAVNt.
EBLV-1 serology
At day 0, all experimented foxes were negative for rabies virus antibody by using both EBLV-1 mFAVNt and CVS-11 FAVNt.
In group 1 (Additional file 1, figure 1A1), all the 5 intramuscularly inoculated animals developed EBLV-2 antibodies. Thirty five days p.i., animal 13C-A15A showed a log(ED50) = 1.79. The four remaining animals ranged between 0.24 and 0.36 IU/ml. Four days later (i.e. 39 days p.i.), 4 out of the five inoculated animals showed a rabies EBLV antibody peak (geometric mean = 1.00) with a maximum of 1.67. At the end of the trial (1097 days p.i.) one surviving animal presented a high log(ED50) of 1.19 while the 4 others ranged between 0.24 and 0.48.
In group 3 (Additional file 1, figure 1B1), different doses of EBLV-1 were used. Four animals out of the five seroconverted 12 days post infection with log(ED50)> 1.00, the mean of log(ED50) was 1.85. This value peaked at 2.20 on day 26 p.i. and remained stable up to 81 days p.i. (mean = 1.65). At the end of trial (651 days p.i.), the mean log(ED50) was 1.25. Two of the 4 surviving animals demonstrated a log(ED50) still positive: 2.15 and 1.91.
In group 4 (Additional file 1, figure 1C1), 14 out of the fifteen animals IM infected with EBLV-1 developed rabies antibody between 11 and 24 days post infection with a mean log(ED50) of 0.96 and 1.58 respectively. This value decreased down to 0.44 at the end of trial. One animal (5FD-8E03), receiving 3.7 log of MIC LD50 of infectious virus did not develop antibodies after inoculation, all along the experiment, the log(ED50) was under 0.24.
Considering that the cut off value for log(ED50) is 1.00, 13 animals seroconverted (log(ED50) ranging between 1.08 and 2.63) between 11 and 48 days p.i. The 2 foxes that died after inoculation (5FE-60DD and 5FD-ACB1) developed detectable EBLV-1 antibodies before succumbing: log(ED50) = 0.96 and 1.91 respectively 14 and 17 days p.i. At the end of the experiment, the mean log(ED50) was 1.01 in surviving animals.
Five days after intracranial inoculation with pure EBLV-1 the log(ED50) of fox 5FE-5AEC, peaked at 1.08 and then decreased to 0.72 at day 7. The animal was euthanised on day 8 p.i. The other animal intracranially inoculated with EBLV-2 (5FE-58DA) did not develop detectable antibody during the entire experiment, the log(ED50) varied between 0.24 and 0.36 between inoculation and 136 days p.i. The animal was euthanased 282 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(ED50)<0.24 throughout the experiments.
EBLV-2 serology
Foxes inoculated via the IC route did not seroconvert prior to clinical disease during the 22 days of observation (log(ED50) = 0.95).
The log(ED50) of EBLV-2 specific antibodies of animals inoculated via the IM route generally fluctuated between 1.55 and 2.39, i.e. above the positive threshold (1.43) between days 30 and 330 Of the 5 animals, three had substantially higher antibody levels than the other two.
There were substantial differences between the specific serological responses of the EBLV-2 inoculated animals depending on whether the sera were tested against a homologous genotype (i.e. EBLV-2) or a heterologous one (EBLV-1 or CVS).
CVS serology
In group 1 (Additional file 1, figure 1A2), three animals out of the five animals infected with 3.5 log MIC LD50 of EBLV-2, seroconverted, log(ED50)>1.00 between 28 (13C-A15A: 1.08) and 49 days p.i. (1E2-CB6E: 1.43 and 1E2-D586: 1.67, which is the maximal value observed in this study). The log(ED50) of two other animals (13C-F633, 1DF-7D9E) was 0.24 and 0.84. At the end of the trial, all surviving animals (n = 5) presented a log(ED50) between 0.24 and 0.48.
Five foxes intramuscularly inoculated with EBLV-1 (group 3, Additional file 1, figure 1B2) seroconverted with log(ED50) between 1.08 and 2.03, 12–29 days p.i. The mean value peaked at 18 days p.i. (1.24) and decreased down to 0.65 21 months p.i.
In the final IM EBLV-1 experiment (group 4, Additional file 1, figure 1C2), only 6 animals out of the 15 infected seroconverted above 1.00 between 11 and 39 days post inoculation, with a mean log(ED50) varying between 0.38 and 0.47. The mean log(ED50) for FAVNt conducted with CVS-11 increased from 0.29 to 0.62 (14-days p.i), then decreased to 0.44 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-2 infected foxes than EBLV-1 infected animals (Figure 2, panels E, F, H and I) similar to that seen in EBLV-2 infected experimental mice [30]. The distribution and intensity of lyssavirus antigen detection varied between viruses and inoculation route. There was substantially more immunostaining on brain tissue from EBLV-1 infected animals than that from EBLV-2 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-1 as no animals peripherally inoculated with EBLV-2 succumbed to disease. In the case of EBLV-1 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-1 and EBLV-2 infected animals with distinct signal in many (40–50%) motor neuron cell bodies. Thoracic and cervical spinal sections were negative as confirmed by PCR and virus re-isolation (RTCIT). 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.