BRIEF REPORT

Responses of mice to inoculation with low doses of a bat rabiesvirus variant

Christina Ndaluka • Richard Bowen

Received: 11 May 2012 / Accepted: 7 December 2012 / Published online: 5 February 2013

� Springer-Verlag Wien 2013

Abstract Rabies is generally considered a fatal disease,

yet neutralizing antibodies to rabies virus (RV) have fre-

quently been found in sera from healthy, insectivorous

bats, and mark-recapture studies have demonstrated bats

that are still alive years after the first detection of anti-RV

antibodies. To explore this phenomenon, we exposed mice

to a big brown bat variant of RV, using three routes of

inoculation, two doses of virus and two frequencies of

exposure. We found the highest rate of seroconversion

without mortality in mice that received repeated intra-

muscular inoculation of the higher dose of virus, and mice

inoculated intranasally experienced the highest mortality.

Rabies is almost always fatal once symptoms appear [1],

although recently, there have been a few cases of human

survival of clinical rabies [2, 3]. Interestingly, there are

multiple reports of apparently healthy bats captured in the

wild having anti-RV neutralizing antibodies [4–10]. Both

natural and experimental infections have shown that bats

are susceptible and can become sick and die of rabies;

however, some bats survive [4, 10–12].

Although the bite route is the major mode of RV

transmission, bats are also likely exposed to RV through

non-bite routes, particularly in colonial species that live at

high densities, where exposure to aerosolized RV may

occur [13]. Considering the very high population densities

in maternity colonies [14] and the perpetual although low-

level presence of rabid bats in such colonies, exposure to

small quantities of RV may be routinely experienced.

The objective of this study was to determine the neu-

tralizing antibody response of mice to inoculation with low

doses of a big brown bat variant of RV in order to gain

insight into possible mechanisms by which some wild adult

bats acquire neutralizing rabies antibodies but fail to

become rabid.

Big brown bat RV variant #20708 Denver was originally

isolated from the brain of a dead big brown bat (Epetiscus

fuscus) and propagated an additional two times in NB cells

cultured in medium containing 1 % mouse serum to pre-

clude the complicating factor of inoculating mice with

heterologous serum proteins. Immunostaining for RV

antigens in cultured cells or mouse brain was accomplished

using the direct fluorescent antibody technique [15] with a

mixture of FITC-labeled mouse monoclonal antibodies that

bind to RV nucleocapsid protein (Light Diagnostics Rabies

DFA Reagent, EMD Millipore Corp., Billerica, MA).

Stock RV was titrated on mouse neuroblastoma cells using

a standard quantal assay in 96-well plates. The 50 % tissue

culture infective dose (TCID50) was calculated for each

sample by the Spearman–Karber method.

Young adult, female ICR mice were purchased from

Charles River Laboratories, housed in groups of five and

maintained under ABSL-3 conditions with free access to

food and water throughout the experiment. Mice were

4 months of age at the time of the first RV inoculation.

Groups of five mice were inoculated according to a fac-

torial design in which the variables evaluated were route of

inoculation (intramuscular vs. intradermal vs. intranasal),

dose of virus (50 vs. 500 TCID50) and number of inocu-

lations (twice, on days 0 and 28, vs. twice weekly for

6 weeks). In all cases, the volume of the inoculum was

C. Ndaluka

Department of Microbiology, Immunology and Pathology,

Colorado State University, Fort Collins, CO 80523, USA

R. Bowen (&)

Department of Biomedical Sciences, Colorado State University,

1680 Campus Delivery, Fort Collins, CO 80523, USA

e-mail: rbowen@colostate.edu; Richard.Bowen@colostate.edu

123

Arch Virol (2013) 158:1355–1359

DOI 10.1007/s00705-013-1605-y

25 ll. Intramuscular inoculations were delivered in the

hind leg (quadriceps muscle), and intradermal inoculations

were delivered in the inner thigh, in both cases using an

insulin syringe with a 28-gauge needle and without anes-

thesia. Intranasal inoculations were conducted under keta-

mine–xylazine anesthesia, with the inoculum delivered into

the nares while the mice were held vertically. Following

inoculation, mice were monitored twice daily for clinical

signs of disease. When definitive signs of rabies were

observed, the mice were anesthetized, bled by cardiac

puncture and euthanized by cervical dislocation. Slip

smears of brainstem were prepared on standard microscope

slides, fixed in acetone and stained for DFA detection of

RV antigens as described above. Mice that survived the

low-dose inoculations were re-challenged intramuscularly

in the right triceps muscle with a dose of 104 TCID50 of the

#20708 Denver RV three months post-inoculation. RV

isolation from mouse brain was carried out as described

above, and samples were considered negative if viral

antigens were not detected after three passages.

Serologic testing was performed using a variation of the

RFFIT originally described by Smith and colleagues [16].

Each assay incorporated a positive control consisting of the

WHO rabies immunoglobulin standard (RIG). The CVS-11

strain of RV and BHK-21 cells were used in the assay;

DFA staining was as described above, and 20 fields were

classified as having or not having positive cells. Samples

were considered positive if the neutralization titer was C5.

Statistical testing was calculated using SAS/STAT

software (SAS Institute). Comparison of mortality and

antibody response for all 12 groups was done using the

Proc GLIMMIX with a binary distribution given dichoto-

mous dependent variables. Mortality and antibody response

analyses had independent variables of inoculation route,

viral dose and frequency of inoculation and dependent

binomial variables of death versus survival and antibody

response verses no antibody response. Antibody responses

were compared at different time points throughout the

study. Mortality and antibody response were re-evaluated

pairwise based on their percent differences using McNe-

mars Chi-square or Pearson’s Chi-square tests.

Weekly virus titrations were performed on the putative

500 TCID50 virus inoculum to confirm that the dose

delivered was less than fivefold different from the target.

The mean over the six weeks was 352 TCID50/25 ll with a

range of 158 TCID50/25 ll to 601 TCID50/25 ll, which

was considered acceptable with respect to reproducibility.

The fraction of mice in each treatment group that

developed confirmed clinical rabies is presented in Table 1.

Across all other factors, mice that were inoculated by the

intranasal route experienced higher mortality than mice

inoculated intramuscularly or intradermally (p = 0.0013).

Those inoculated with the 500 TCID50 dose, independent

of inoculation route or schedule, were more likely to suc-

cumb to clinical rabies than mice inoculated at the 50

TCID50 dose (p = 0.0025). Mice inoculated 12 times over

the course of the study, independent of inoculation route or

dose, were more likely to experience mortality than mice

inoculated only twice (p = 0.001).

All mice inoculated via the intramuscular or intradermal

routes survived until the end of the study. Seven mice from

three different groups inoculated intranasally succumbed to

clinical rabies. Four of these mice were from the same

group that was inoculated 12 times with the 500 TCID50

dose (Table 1). The majority of deaths occurred in the

group that received 500 TCID50 12 times by the intranasal

route. All mice that were euthanized due to clinical

symptoms of rabies were found to have RV in their brain

by DFA. Mouse 7-1 was removed from the study due to

non-study complications and was negative via brain smear

DFA.

Three months after initiating low-dose inoculations of

RV, all surviving mice were challenged intramuscularly

with 104 TCID50 of the same virus. Seven naı̈ve mice of the

same age were also inoculated as positive controls. None of

the mice, including the naı̈ve group, succumbed to clinical

rabies within 4 weeks of the re-challenge. At the time of

termination, brain smears were made from all mice, and of

61 mice tested, brain smear results were negative for 46

mice but were inconclusive due to high background for 15

mice. RV isolation with brain homogenates was attempted

for these 15 mice and was found to be negative for virus for

all but one mouse in the group that was inoculated at the

lower dose, intramuscularly, twice. Surprisingly, that

mouse was found to have RV in the brain by RV isolation;

this animal failed to have a detectable antibody response

until the terminal bleed at 1 month after the re-challenge. It

seems likely that the study was terminated while this

mouse was in the prodromal stage and that it would have

developed clinical symptoms.

Comparison of inoculation routes independent of inoc-

ulation schedule or dose revealed a significantly lower

fraction of mice that developed neutralizing antibody fol-

lowing intradermal inoculation compared to inoculation by

intramuscular or intranasal routes (p \ 0.025, Table 2).

Among mice that were inoculated by the intranasal route,

neutralizing anti-rabies antibody was detected in all seven

mice that succumbed to clinical infection and in two of the

14 mice that survived to the end of the study. Neutralizing

anti-rabies antibody was detected only in the terminal

serum from mice that developed clinical disease.

When the influence of RV dose was assessed indepen-

dently of inoculation route or schedule using chi square

statistics, more mice inoculated with the 500 TCID50 dose

had RV neutralizing antibody one month (p = 0.04), two

months (p = 0.001) and three months after inoculation

1356 C. Ndaluka, R. Bowen

123

(p = 0.014). Evaluation of the influence of inoculation

schedule independently of inoculation route or dose

revealed that at both two and three months after inocula-

tion, more mice inoculated 12 times had neutralizing RV

antibody than mice inoculated only twice at two months

(p \ 0.025).

None of the naı̈ve or previously inoculated mice suc-

cumbed to RV infection within one month following the re-

challenge. Analysis of the serologic responses of these

mice across all treatment groups revealed a significant

increase between three and three and a half months in the

number of mice with detectable RV-neutralizing antibody

titer (p = 0.03) and between three to four months

(p = 0.007) (Table 2). This increase in the fraction of mice

with antibody at two weeks after re-challenge could be an

indication of immune-system priming to RV from the

previous exposures to RV; this was particularly evident in

the group that received 12 intramuscular inoculations of the

low dose. The increase in antibody prevalence four weeks

after re-challenge was also observed in a majority of con-

trol mice.

Among the reservoir hosts for rabies viruses, bats appear

to be unique in that neutralizing antibodies are frequently

detected in wild, healthy bats multiple years in a row [4, 6],

Table 1 Morbidity and

mortality summary for each

group

Group Inoculation dose

(TCID50)

Inoculation

route

Frequency of

inoculation

Incubation period

(days)

Mortality

rate

1 50 IM 29/week, 6 weeks NA 0/5

2 50 IM Days 0 and 28 NA 0/5

3 50 ID 29/week, 6 weeks NA 0/5

4 50 ID Days 0 and 28 NA 0/5

5 50 IN 29/week, 6 weeks NA 0/5

6 50 IN Days 0 and 28 27 1/5

7 500 IM 29/week, 6 weeks NA 0/5

8 500 IM Days 0 and 28 NA 0/5

9 500 ID 29/week, 6 weeks NA 0/5

10 500 ID Days 0 and 28 NA 0/5

11 500 IN 29/week, 6 weeks 37 (27–45) 4/5

12 500 IN Days 0 and 28 27 1/5

Table 2 Neutralizing anti-rabies-virus antibody responses over the course of the study

Group Inoculation dose

(TCID50)

Inoculation

route

Inoculation

schedule

Fraction of mice that were seropositivea, b

0.5 month 1 month 2 months 3 months 3.5 months 4 months

1 50 IM Multiple 0/5 0/5 1/5 1/5 4/5 1/5

2 50 IM 29 0/5 0/5 0/5 1/5 1/5 2/5

3 50 ID Multiple 0/5 0/5 0/5 0/5 1/5 1/5

4 50 ID 29 0/5 0/5 0/5 0/5 0/4c 2/5

5 50 IN Multiple 0/5 0/5 0/5 0/5 1/5 1/5

6 50 IN 29 0/5 1/5 0/4 0/4 1/4 2/4

7 500 IM Multiple 0/5 4/5 4/5 4/4d 4/4 3/4

8 500 IM 29 0/5 0/5 1/5 0/5 0/5 1/5

9 500 ID Multiple 0/5 0/5 1/5 2/5 2/5 2/5

10 500 ID 29 0/5 0/5 0/5 0/5 1/5 3/5

11 500 IN Multiple 0/5 1/5 4/4 1/1 1/1 1/1

12 500 IN 29 0/5 1/5 1/4 1/4 2/4 3/4

Controle 104 IM 19 NA NA NA 0/7 1/7 4/7

a Mice were rechallenged at 3 months after initial virus inoculationb Number of mice with RFFIT titers C5/number of mice in groupc One mouse not bled due to barbaring injuryd One mouse was removed from the study due to non-study complicationse Control mice were challenged at 3 months, coincident with the second challenge of other mice

Responses of mice to inoculation with low doses of a bat rabies virus variant 1357

123

suggesting that infection frequently is not fatal. An

important question that remains to be answered, and is the

focus of the study presented here, is how wild bats

apparently are frequently exposed to a sufficient dose of

RV to induce formation of anti-RV antibodies yet fail to

progress to develop clinical disease. Multiple hypotheses

have been proposed to explain this observation, including

repeated exposure to low and sub-infectious doses of RV

such that they become immunized but not productively

infected, or that exposures other than through deep intra-

muscular bites may immunize but not lead to clinical

rabies. These alternative hypotheses are by no means

mutually exclusive.

In the current study, we used a mouse model to investi-

gate the effects of different routes of exposure, frequencies

of exposures and doses of virus on seroconversion and

development of clinical disease. Mice inoculated intrana-

sally had the highest mortality, which precluded evaluation

of immunogenicity following exposure by this route. The

high mortality following intranasal inoculation was not

unexpected, since inoculation by this route provides a direct

pathway for the virus to gain access to the central nervous

system. Infection by intranasal inoculation with rabies virus

has been demonstrated by some investigators in bats, mice

and skunks [17–19] but has been unsuccessful in other

studies using bats [20–22]. The numerically highest rate of

seroconversion was observed in mice inoculated intramus-

cularly, and this occurred in the absence of mortality, even

at the highest dose of virus. Finally, exposure to RV via

intradermal inoculation was significantly less effective than

either intramuscular or intranasal inoculation in eliciting

development of anti-rabies-virus antibodies and failed to

result in disease. Based on previous studies in which various

species of bats were experimentally infected with different

lyssaviruses by the same routes used here, it appears that

there is considerable variability in the efficiency with which

infection occurs [17, 20–22].

It is unknown if the anti-rabies antibody found in wild

bat populations is actually protective again RV infection.

To determine whether inoculation of mice with low doses

of RV leads to protection from a higher dose challenge or

evidence of immune priming, all mice that survived the

initial inoculations were re-challenged with 104 TCID50 of

the same virus used originally. Previous studies in our

laboratory demonstrated that this virus was lethal in 9 of 10

mice that had no previous exposure to RV. However, none

of the mice in this experiment, including naı̈ve controls,

that were challenged with this dose of virus succumbed to

the re-challenge. There was, however, a significant increase

in the number of mice that seroconverted in response to the

re-challenge, which could be an indication that their

immune systems had been primed to respond in a protec-

tive manner to an exposure to RV. At the end of the study,

the number of mice with neutralizing antibody was

increasing. The fact that none of the mice, particularly the

naı̈ve controls, succumbed to rabies following the re-

challenge was surprising. This could be due in part to the

age of the mice [23–25].

This study demonstrates that inoculation of mice with

very low doses of RV resulted in a level of seroconversion

without mortality, similar to what has been observed in

wild bats living in an environment where RV is endemic.

Although rates of seroconversion varied among treatment

groups, some mice in each category of route, frequency and

dose developed a detectable humoral immune response to

RV, providing some support for the speculation that non-

lethal RV infections can be initiated by many different

types of exposure. These results shed some light on how

natural bat populations may be able to acquire a humoral

immune response to rabies infection, and the next logical

step is to repeat some of this work in bats.

Conflict of interest The authors declare that they have no conflict

of interest.

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