PRACTICAL SIGNIFICANCE OF RABIES ANTIBODIES IN CATS AND DOGS*
AND
RESULTS OF A SURVEY ON RABIES VACCINATION AND QUARANTINE
FOR DOMESTIC CARNIVORA IN WESTERN EUROPE
M.F.A. Aubert
Centre national d'=E9tudes v=E9t=E9rinaires et alimentaires,
Laboratoire d'=E9tudes
sur la rage et la pathologie
des animaux sauvages, B.P. 9, 54220 Malz=E9ville, France
Note : Text figures have been omitted (SG)
Original: French
Summary: Doubt has sometimes been cast upon the protective
effect
of rabies antibodies in serum. Animals and humans suffering from fatal
rabies
often produce high antibody titres, while rabies cases are also observed
in
vaccinated animals. Cellular immunity is also largely involved in
protection.
Nevertheless, a large number of laboratory experiments and field
observations
clearly demonstrate that cats and dogs which develop antibodies after
vaccination and before challenge have a very high probability of
surviving any
challenge, no matter how strong the dose and which virus strain is
used.
Rabies antibody titration can, therefore, afford a strong
additional
guarantee to the vaccination certificates accompanying domestic
carnivores
during transportation between countries. Quarantine rules should also be
adapted
to the epidemiological features in the exporting country, e.g.
statistics of
vaccination failure in cats and dogs and host-virus adaptation of the
rabies
strains circulating in these countries.
1. INTRODUCTION
Cats and dogs can introduce rabies into disease-free countries if
they are
incubating the disease and are transported during the pre-symptomatic
phase. To
prevent such introduction, vaccination is recommended. The present
article
reviews publications dealing with rabies protection afforded to cats and
dogs by
vaccination. In addition, the Appendix presents the results of a survey
of the
current practices of OIE Member Countries in Western Europe with respect
to
rabies vaccination and quarantine of dogs and cats.
Only the parenteral route of vaccination will be considered, as the
oral
route is employed only for wandering and non-restrained carnivores;
extensive
results for individual cats and dogs are unavailable. Also, since oral
vaccination could mobilise immunity pathways other than those obtained
parenterally, the results with one procedure may not be transposable to
the
other.
Furthermore, no consideration will be given here to the results of
vaccination after exposure, which does little, if anything, to alter
disease
(20).
Emphasis will be given to the most common method for measuring rabies
immunisation: assays for rabies virus neutralising antibodies in
serum
(henceforth referred to as "neutralising antibodies"). The practical
significance and consequences of rabies virus neutralising antibodies in
cats
and dogs are considered; namely, to what extent do neutralising antibody
titres
confer protection against subsequent challenge?
No consideration will be given to the question of whether real
protection
against challenge is provided by neutralising antibodies and/or other
immunity
factors. Titres of neutralising antibodies in serum are simply viewed as
the
easiest means of evaluating the likelihood that a cat or dog will not
contract
rabies following exposure.
2. STUDIES IN DOGS
2.1. Neutralising antibodies after vaccination
General considerations
The kinetics obtained for neutralising antibodies after vaccination
have been
thoroughly described in the literature. The curve of neutralising
antibodies
after vaccination and boosters follows the pattern generally observed
with other
antigens: seroconversion and rapid rise of the level of neutralising
antibodies
after first vaccination, followed by a slow decrease, a new rise after
booster
to reach a higher level than previously observed, then a new decrease
leading to
a stabilised higher level (Fig. 1) (8, 51, 63). The decrease of
neutralising
antibody levels has been evaluated in domestic populations of owned dogs
in
several countries: in Canada, titres of neutralising antibodies in the
sera of
dogs showed a clear division between, on the one hand, dogs vaccinated
or
revaccinated one year before and, on the other hand, dogs revaccinated
three
weeks before (33). Data from Thailand and Java show that the
neutralising
antibody titre decreases very rapidly after 60 to 120 days to levels 5-
to
25-fold less than the highest point reached during the kinetics (Fig. 2)
(40,
65). The higher level of neutralising antibodies obtained when owned
dogs are
vaccinated several times has been described by Sasaki and colleagues
(55, 56).
Fig. 1
Kinetics of rabies neutralising antibodies in sera of
laboratory dogs vaccinated with a tissue culture vaccine
Variations according to vaccination route and antigenic value of the
vaccine
as measured by the NIH test
(63, 64)
Fig. 2
Kinetics of rabies neutralising antibodies in sera of owned dogs
of
various ages
in Thailand after one subcutaneous vaccination with a tissue
culture
vaccine
The number of dogs sampled was 54 at day 0 and 31 at day 360
(65)
With regard to the production of neutralising antibodies and the
relationship
of these antibodies to protection against challenge, a clear distinction
must be
made between live virus vaccines and inactivated virus vaccines. These
two types
of vaccine cannot be directly compared. The best relationship between
antibody
production and protection has always been obtained with inactivated
virus
vaccines and it is, therefore, the latter which will be considered in
greater
detail, especially as they currently represent the only type of vaccine
authorised in a great many countries.
High individual variability
In laboratory dogs bred and kept under the same conditions and in
comparable
health, neutralising antibody titres obtained after the same form of
vaccination
commonly range from zero to twenty international units per ml (IU) or
more (19,
51, 53).
Influence of vaccine types and potency
The first complete study of live virus vaccines was published by Dean
and
colleagues (32). This study established a correlation between antibody
production and resistance to challenge, which was confirmed by later
studies
(see below). As far as inactivated virus vaccines are concerned, besides
individual variation, the level of neutralising antibodies in serum
correlates
positively with the antigenic value of the vaccine as determined by the
American
National Institutes of Health (NIH) test. This observation is common in
the
course of vaccine production and control on laboratory dogs (Fig. 1)
(47, 63).
The influence of the antigenic value of the vaccine on the level of
neutralising
antibodies has also been demonstrated in domestic populations of owned
dogs; in
Switzerland, Engels and colleagues (35) showed that in owned dogs,
higher titres
were generally obtained with inactivated vaccines than with live (and
less
potent) vaccines.
However, when inactivated virus vaccines with an antigenic value (as
measured
by the NIH test) equal to or greater than 1.0 IU per dose are employed,
no
correlation can be shown between the level of neutralising antibodies in
individual dogs and the titre of the vaccine. This result was first
demonstrated
in owned dogs in France by Blancou and colleagues (13). In this
experiment, dogs
were sampled randomly from populations living under various conditions
and were
vaccinated with a range of commercially available vaccines. Chappuis and
colleagues (30) and Lazarowicz and colleagues (45) used laboratory dogs
to
investigate whether the administration of vaccines from the same
producer would
entail a correlation between the NIH titre of vaccines and the level of
neutralising antibody response. Even under standardised conditions, no
correlation was found. The same conclusion can be drawn from the results
of
Barth and colleagues (7).
In summary, a significant variation of neutralising antibody response
can be
shown only under a broad range of vaccine potencies (61). When the
potencies of
commercial inactivated virus vaccines are fairly high, the neutralising
antibody
response will be related only to the immune responses of individual
dogs.
Influence of the route of vaccination
Since Pasteur, the route of vaccination has been subcutaneous (s.c.).
Fuenzalida in 1967 demonstrated that the intramuscular (i.m.) route
resulted in
higher neutralising antibody titres in sera of dogs (37). Apart from
Merry (46),
who found no clear advantage for the i.m. over the s.c. route, the
results
obtained by Fuenzalida were largely confirmed (22, 25, 63). However, the
advantage of the i.m. route diminishes with high potency vaccines (Fig.
1) (63)
and the use of adjuvanted vaccines renders the i.m. route excessively
painful.
Adjuvants confer a longer lasting immunity, which can be obtained with a
smaller
quantity of antigen, as first demonstrated on laboratory dogs (52), then
on
owned dogs (43, 68, 69). Despite the use of smaller quantities of
antigen and a
reduced vaccination schedule (less frequent boosters), the neutralising
antibody
levels reached after one, two or three years with adjuvanted vaccines
were
equivalent to those reached with non-adjuvanted vaccines given according
to the
usual schedule (two injections of vaccine the first year, with annual
boosters).
The importance of the vaccination route was clearly demonstrated with
intradermal injection of vaccine in dogs (68). Unfortunately, the
advantages of
an enhanced response obtained with a minute dose of vaccine (2 x 0.1 ml)
were
offset by the fact that intradermal injection must be performed on the
inside of
the ear and, hence, this procedure must be conducted dangerously near
the mouth
of the animal.
Influence of age
It has been shown that dogs 11-16 weeks of age respond better to
Flury low
egg passage (LEP) or high egg passage (HEP) vaccine than dogs 5-10 weeks
of age
(81% vs. 38% protection from challenge, respectively) (41). The
relationship
between the age of animals and protection from challenge was confirmed
in a
laboratory study by Bunn in 3- to 5-month-old pups. Three months after
vaccination with Flury LEP vaccine, ten of forty pups had antibody
titres below
1/5 (24, 25).
A survey on owned dogs in France showed that even beyond three months
of age,
older dogs produced higher titres (Fig. 3) (13).
Fig. 3
Correlation between rabies antibody level reached
after one vaccine injection and age of dogs
Study conducted on 66 owned dogs in France
(13)
The influence of age on the neutralising antibody response in dogs
was also
clearly demonstrated on owned dogs in Thailand by Teepsumethanon and
colleagues
(65). These authors described the kinetics of neutralising antibodies in
three
age groups: 3 weeks to 3 months, 6 to 12 months, and more than 12
months.
Whenever the mean level of neutralising antibodies was evaluated after
vaccination, the older dogs had the highest levels of response. Given
the
difficult conditions prevailing in Thailand, the superior response of
older dogs
could also be related to the increased life expectancy of dogs with a
more
powerful immune system (Fig. 2).
The presence of specific neutralising antibodies transmitted to
puppies via
colostrum impedes development of active immunity. The interference
between
passive neutralising antibodies of maternal origin and active
immunisation has
been studied by Pr=E9causta (52, 53). Puppies of non-immune bitches
vaccinated at
the age of one month respond with the same neutralising antibody level
as
puppies vaccinated at seven months of age. Puppies of immune bitches
vaccinated
at one month of age show neutralising antibody levels which decrease
according
to the same kinetics as unvaccinated members of the same litter.
After ten weeks (44) to twelve weeks (52), no traces of maternal
neutralising
antibodies remain. Surveys in pet dog populations where systematic
vaccination
of adult dogs is practised (in France and elsewhere) have confirmed that
no
further interference between active and passive immunity occurs beyond
this age
(53).
Influence of the health and breeding status of dogs
Blancou and colleagues (19) compared the proportion of individuals
developing
neutralising antibodies in 64 dogs after the administration of
adjuvanted or
non-adjuvanted vaccines. This rate may vary considerably depending on
the
category of dog (bred for laboratories, belonging to individuals in
France or
uncontrolled in Tunisia). The rate drops from 100% to 59% in the case of
semi-stray dogs as compared to laboratory dogs (Fig. 4). Urban dogs in
Lima
(Peru) exhibited better rates than in Tunisia, but the rates were still
lower
than in dogs kept under laboratory conditions (31). Although the health
status
of these populations had not been measured in the previous studies, this
status
is probably responsible for the differences observed by Teepsumethanon
and
colleagues (65) in Thailand: Thai pet dogs which had received one s.c.
dose of
rabies vaccine exhibited a better neutralising antibody response when
they did
not suffer from anaemia (Fig. 5). In 440 pets under quarantine in
Hawaii, Sasaki
and colleagues (56) demonstrated that those with internal parasites had
significantly lower levels of neutralising antibodies than those without
parasites.
Fig. 4
Influence of the breeding standards of dogs on the level
of rabies antibody reached one year after vaccination
Comparison of laboratory dogs, pet dogs in France and stray dogs in
Tunisia
(19)
Fig. 5
Influence of the health status of Thai dogs on the level
of rabies antibody reached after one vaccination
Comparison of dogs with or without anaemia
(65)
2.2. Level of neutralising antibodies in sera and results of
challenge
Challenge under laboratory conditions
In view of the serious problem posed by rabies, challenge of
previously
vaccinated dogs has often been performed even when a large proportion of
the
dogs under experiment exhibited a seroconversion. Moreover, such
challenges are
performed in response to doubts which have sometimes been cast on the
significance of neutralising antibodies to rabies, due to the fact that
high
titres have been measured in human beings and animals dying of rabies.
In fact,
very few diseases show so clear a correlation as in rabies between
seroconversion before challenge and protection from challenge.
In the context of movement of dogs between countries, it is possible
to check
the efficiency of previous vaccinations. A large number of reports can
be
summarised by the simple comparison of the proportion of dogs surviving
challenge vs. the proportion of dogs with detectable neutralising
antibodies in
serum just before challenge (i.e. theoretically when the neutralising
antibody
level is lowest). These summaries are given in Tables 1 to 8.
Table 1
Laboratory dogs: one intramuscular vaccination with various
vaccines,
challenge with rabies virus NYC-Ga strain, one year after
vaccination
(61)
Vaccine Dogs with antibodies
Dogs surviving challenge
just before challenge
(%)
LEP tissue culture 87 29/30
LEP tissue culture 69 26/29
ERA tissue culture 57 27/30
LEP chicken embryo 54 28/30
Suckling mouse brain 48 27/27
HEP tissue culture 42 27/29
Suckling mouse brain 28 23/29
CVS adjuvanted 0 17/29
None 0 3/30
Results of challenge Antibodies before
challenge
yes no
Rabid 3* 26
Surviving 157 47
* titres of 1/2, 1/3 and 1/5 (end point neutralising dilution of the
serum)
Table 3
Laboratory dogs: one vaccination with HEP vaccine, challenge with
rabies
virus NYC-Ga strain
three years after vaccination
(22)
Antigenic value Dogs with detectable antibodies
Dogs surviving challenge
of vaccine 12 months after vaccination
Vaccinated Controls
0.6 8/8 8/8
0/7
1.7 19/20 17/18**
3/12
2.3 6/9 8/9***
4/12
4.6 10/10 9/9
-
* measured by the NIH test, expressed in IU/dose
** The dog which died of rabies had always had the lowest antibody
titre in
the group
*** The dog which died of rabies had never seroconverted
Table 5
Laboratory dogs: subcutaneous vaccination with tissue culture
vaccine,
challenge with rabies virus NYC-Ga strain, three years after
vaccination
(52)
Vaccination Dogs with detectable antibodies
Dogs surviving
just before challenge
challenge
Yes 14/25 23/25*
No 0/10 2/10
* One of the two dogs which died following challenge was
seronegative, the
other had a titre of 1/4 (endpoint neutralising dilution of the serum)
Table 7
Laboratory dogs: intramuscular vaccination with ERA vaccine,
challenge with rabies virus fox strain four or five years after
vaccination
(44)
Antigenic value Dogs with detectable
Dogs surviving challenge
of vaccine* antibodies just before challenge
Vaccinated
Controls
4.2 9/10** 10/10
0/5
* measured by the NIH test, expressed in IU/dose
** two dogs with an antibody titre <0.5 IU/ml
Sikes and colleagues (62) employed several types of vaccine on dogs
and
challenged them one or three years after vaccination (Tables 1 and 2).
Sikes
(61) commented on the three-year experiment as follows: "In this study,
as in
many others, presence of neutralising antibodies to rabies at the time
of
challenge did not indicate protection for all of the animals. Likewise,
absence
of neutralising antibodies in serum at the time of challenge did not
mean the
animals were unprotected. However, there was strong statistical
significance
(P < 0.1) that animals with neutralising antibodies at the
time of
challenge were better protected than those with no detectable
neutralising
antibodies."
Sikes employed the NYC-Ga (New York City-Georgia) dog salivary gland
strain
of rabies virus. The same strain has also been used for challenge in
other
experiments (Tables 3 to 6) and the results confirm each point of the
statements
made by Sikes (60) regarding vaccination of dogs:
a) generally, groups of dogs with a high percentage of seroconversion
will
have the highest probability of surviving challenge;
b) on an individual basis:
- a dog with neutralising antibodies just before challenge
will have
the best chance of surviving a severe challenge;
- a dog with no detectable neutralising antibodies just before
challenge will
have a high chance of surviving a severe challenge if it seroconverted
after
vaccination;
- some dogs will not survive a severe challenge even if they have
detectable
neutralising antibody titres before challenge; generally these
titres are
the lowest of the group.
In studies of fox strains of rabies virus (Tables 7 and 8), the
possibility
of procuring a strong immunity as long as four to five years after
vaccination,
and of enhancing protection by the use of adjuvanted vaccines, has been
demonstrated. These studies also confirmed the correlation between
neutralising
antibodies and protection against a fox strain.
Bunn and colleagues (28, 29) gathered pre-challenge neutralising
antibody
titres and challenge results obtained on dogs by the United States
National
Veterinary Services Laboratories and by vaccine manufacturers. Most of
the dogs
were challenged with the NYC-Ga strain, but results obtained with fox or
skunk
strains were also added. Sera were titrated either by the virus
neutralisation
test in mice (MNT) (5) or the rapid fluorescent focus inhibition test
(RFFIT)
(62). Data on neutralising antibodies originally expressed in
arithmetical
dilutions by Bunn (26, 27) have been converted into IU in Figure 6.
Beyond 0.03
IU/ml with the MNT or 0.05 IU/ml with the RFFIT, the expected survival
to
challenge by a dog strain reaches 95%. With 288 dogs having RFFIT titres
above
0.1 IU/ml, a 100% survival rate was obtained. The maximum survival rate
observed
among animals with the highest neutralising antibody titres measured by
MNT was
99.5%.
Fig. 6
Survival rate after challenge of laboratory dogs correlated
with the level of rabies antibody reached before challenge
Dogs were vaccinated with various vaccines and challenged one year
after
vaccination with NYC-Ga,
fox or skunk strains; the number of dogs in each class is written at
the top
of the bars
(65)
Given the higher susceptibility of dogs to dog strains (e.g. NYC-Ga),
which
was proven by cross challenge of dogs with homologous and heterologous
(fox)
strains (15, 17), the challenge with fox strains could be expected to be
less
severe. Unfortunately, the data are too scarce to permit a definitive
conclusion.
Natural infection of vaccinated dogs
The number of vaccinated dogs which become naturally infected is
related to
several factors other than vaccine potency, such as probability of
encountering
an infected animal, severity of bites, health status and immune
efficiency of
the vaccinated dogs, and host-virus adaptation. Such considerations
could
explain why vaccinated dogs suffer rabies more often in the course of
dog rabies
enzootics than during fox rabies enzootics. In Thailand, 9% of the dogs
found
positive upon laboratory diagnosis had been vaccinated within the
previous two
years (39). In Nigeria, a survey of 2,500 dogs vaccinated over two
years, showed
that at least four died of rabies three to eight months after
vaccination (1,
2).
The following reasons (16) for the failure of immunity may be
suggested:
- inappropriate vaccination with inadequately stored or improperly
injected
vaccine
- vaccination during the incubation of rabies or before the onset of
an
immunological response
- a heavy challenge overwhelming host defences
- intrinsic incapacity in the host.
Whatever the origins of rabies cases recorded in vaccinated dogs,
their
number seems relatively low in areas contaminated with fox rabies (e.g.
in
Europe) (20). In France, only ten cases of so-called vaccination
failures in
dogs (and four among cats) have been registered over a period of
twenty-three
years (6). This number should be compared with the 4,250,000 cats and
dogs
vaccinated annually in France (this figure is based on the annual number
of
vaccine doses sold for domestic carnivores). The probability of a cat or
dog
becoming rabid if vaccinated can be estimated as 14/(23 x 4,250,000),
which is
less than 1/6,980,000. In France, dogs in contact with a rabid animal in
an
enzootic area are not sacrificed and can be kept alive if, prior to
contamination, they have been properly vaccinated (with certificate and
identification). In such cases, the animals are immediately
revaccinated. A
study of more than 3,500 dogs which had close contacts (bites in 36% of
cases)
with foxes (mainly) or other carnivores which were diagnosed as rabid by
laboratory examination, revealed that only three dogs developed rabies
(50). The
failure rate in animals which were definitely contaminated can be
estimated as 3/3,500, given that injection of vaccine after
contamination has
been shown to provide no protection (20). It must be emphasised that
these
failures were recorded before 1984 and that failure is now less
probable, given
the generalisation of adjuvanted vaccines for dogs. In the United States
of
America, four rabies vaccine failures were recorded in cats and dogs in
1988
with 33,182,575 vaccinated domestic carnivores the same year (rate ="
1/8,296,000) (34).
Such evaluations could be useful in comparing the risks of
vaccination with
those of quarantine. For even when they are strictly managed,
quarantines still
entail a risk. For instance, in many countries, the quarantine period is
six
months. However, longer incubation periods have been reported in dogs
(8.5
months after challenge) (67) and in other carnivores (12 months or more
for
foxes) (57). According to Sasaki and colleagues (55), Beynon determined
that a
quarantine period of nine months would be necessary to detect all cases
of
incubating rabies with a 95% degree of confidence.
3. STUDIES IN CATS
3.1. Neutralising antibodies after vaccination
Although fewer studies have been conducted on vaccination of cats
against
rabies, several of the characteristics observed in dogs were also
observed in
cats:
- the kinetics of neutralising antibodies follow the same profile in
the two
species (8, 23, 64)
- the relationship between the potency of vaccines and the level of
neutralising antibodies: Lawson and colleagues (44) have shown that the
less
diluted modified live vaccines induced the highest rate of
seroconversion in
vaccinated cats (Table 9), but Lazarowicz and colleagues (45) obtained
no
correlation of the antigenic value of inactivated virus vaccines as
determined
by the NIH test and mean neutralising antibody titres in vaccinated
cats.
However, as for dogs, it is necessary to take account of the fact that
the
production of antibodies (and protection against challenge) obtained
after
administration of live virus vaccine (44) and inactivated virus vaccine
(45) can
show great divergence and are not readily comparable.
- intramuscular vaccination provides longer lasting protection than
subcutaneous vaccination (Table 10) (59).
Table 9
Laboratory cats: intramuscular vaccination with various
vaccines,
challenge with rabies virus fox strain five weeks and four years
after
vaccination
(44)
Vaccination Cats with detectable
Cats surviving
antibodies just before challenge
challenge
One year before
challenge
Yes (subcutaneous) 5/5 5/5
No 0/4 0/4
Three years before
challenge
Yes (intramuscular) 25/25 24/25*
No 0/10 1/10
* Prior to challenge the cat which died of rabies had an antibody
titre of
1/2 (endpoint neutralising dilution)
Table 11
Laboratory cats: subcutaneous vaccination with tissue culture
vaccine,
challenge with rabies virus NYC-Ga strain, 3.4 to 3.7 years after
vaccination
(64)
Vaccination Cats with antibodies
>0.5 Cats surviving challenge
IU/ml just before challenge
Vaccinated Controls
Inactivated virus in cell
culture:
antigenic value: 0.9* 8/8 7/8**
antigenic value: 1.8 5/8 8/8
0/16
Modified live virus:
ERA 2/8 3/8***
* Measured by the NIH test, expressed in IU/dose
** The cat which died of rabies had a pre-challenge titre of 5.34
IU/ml
*** The cats which died of rabies had the lowest antibody titres
ERA: Elizabeth (Gaynor) Rokitniki Abelseth
Challenge was performed with a dog strain (NYC-Ga) mimicking the
situation of
canine street rabies (42, 61, 64) or, in other experiments, a fox strain
mimicking the situation of sylvatic fox rabies in continental Europe
(38, 44).
With both strains, the general conclusion was the same as for dogs: the
probability of a cat surviving challenge can be predicted by the level
of
neutralising antibodies. Of course, unexpected deaths can occur: Kihm
and
colleagues (42) reported a rabies death in a cat which had a
pre-challenge titre
of 5.34 IU, and Blancou and colleagues (18) in another cat with a
pre-challenge
titre of 0.87 IU/ml.
The cumulative challenge results on cats reported by Bunn (26, 27)
are
described in Figure 8 and Tables 13 and 14. With a neutralising antibody
level
of more than 0.1 IU (measured by MNT) or more than 0.2 IU (measured by
RFFIT),
all of the cats survived challenge.
Fig. 8
Survival rate after challenge of laboratory cats correlated with
the level of rabies antibody reached before challenge
Cats were vaccinated with various vaccines and challenged one year
after
vaccination with NYC-Ga,
fox or skunk strains; the number of cats in each class is written at
the top
of the bars
(27)
Table 13
Challenge results from rabies immonogenicity tests conducted in
dogs and
cats
with vaccines approved for use in the United States of America
(27)
Animals Antibody test
Antibody titre*
<5 5-9 10-19
20-39 >40
Dogs MNT 56/251** 9/100 9/92
1/63 0/171
RFFIT 84/241 13/112 9/119 0/87
0/201
Total 140/492 22/212
18/211 1/150 0/372
Cats MNT 25/155 5/57 5/94
0/33 0/144
RFFIT 17/87 3/59 1/62 1/49
1/187
Total 42/242 8/116
6/156 1/82 1/331
* antibody titres expressed as 50% endpoint dilutions established by
either
the virus neutralisation test in mice (MNT) or the rapid fluorescent
focus
inhibition test (RFFIT)
** challenge results are expressed as number of animals which
died/number of
animals challenged
Natural infection of vaccinated cats
The safety problem associated with the receptivity of cats to live
virus
vaccines such as Flury LEP and HEP or Street Alabama Dufferin (SAD)
strain
vaccines will not be reviewed here (11). But it should be remembered
that while
cats are the species with the largest number of rabies cases directly
induced by
the inoculation of live modified virus strains, other species such as
dogs and
foxes are also receptive (72).
Inactivated virus vaccines are employed on cats as they are more
efficient in
protecting the species against natural challenge. However, considering
the
results of challenge experiments on vaccinated cats, natural infection
among
vaccinated pet cats is suspected to be as frequent as for vaccinated
dogs. But
investigations on rabies cases in vaccinated cats are scarce: apart from
the
four cases reported in France (6) there appear to be no other reports.
This
discrepancy is due to the fact that dogs have been studied considerably
more
than cats.
4. THE SIGNIFICANCE OF NEUTRALISING ANTIBODIES
IN NON-VACCINATED CARNIVORES
4.1. Non-specific and specific neutralising factors
Sekine and colleagues (58) found that sera of normal rabbits and
guinea-pigs
contained non-specific inhibitors capable of neutralising the virus in
the
presence of complement. In a well-conducted seroneutralisation on mice,
therefore, inactivation of sera is performed for 30 minutes at 56=B0C.
Virus
inhibition by other substances was described in infected skunks and
foxes (74).
Infection by mycobacteria, e.g. Bacillus Calmette-Gu=E9rin (BCG), can
also induce
the production of rabies neutralising antibodies in mice and provide
protection
against rabies in a number of animals (70). Since more specific
immunological
tests (such as enzyme-linked immunorescent assay: ELISA) have become
widespread,
non-specific neutralising factors have not generated further scientific
reports.
In endemic areas, serosurveys in wild carnivores demonstrated a high
proportion of apparently healthy individuals with neutralising
antibodies in
serum (54, 71) and it has been suggested that these antibodies may have
been
produced following contact with virus from other species, which was
therefore
immunising but rarely fatal (12). However, the same observations have
also been
reported for dog populations in areas where dog rabies is endemic: in
Thailand,
in areas where no canine vaccination programme has ever been conducted,
15-20%
of dogs had neutralising antibodies, yet remained perfectly normal when
observed
for prolonged periods (75); similar results had also been reported
previously in
other countries of Asia and in Africa (3, 36). These observations
correlate with
the high probability of inter-individual contamination within the
reservoir
species, which is not the case for pet populations in areas where rabies
is
endemic. The possibility of non-fatal contamination of dogs by
non-canine
strains (e.g. those from wild animals living in the region) has also
been
proposed (20). Several questions thus arise regarding:
a) the specificity of serum titrations and the threshold level for
protection
against rabies;
b) the possibility of rabies outbreaks in naturally seroconverted
dogs, and
the interval between seroconversion and the onset of clinical symptoms.
4.2. Rabies infections
The viral infection triggers the production of neutralising
antibodies. When
a high dose of rabies virus reaches the central nervous system,
neutralising
antibodies are not detectable before or at the onset of clinical signs;
they are
usually induced by longer incubation periods. This phenomenon has been
studied
mainly in laboratory rodents, which supply the chief model of rabies
immunopathology (49, 73). Unfortunately (but not surprisingly,
considering the
difficulty of handling rabid carnivores), there appears to be no
literature on
the frequency and intensity of neutralising antibody production in
non-vaccinated infected cats and dogs. Some data can be found in
articles by
Artois and colleagues (4), Blancou and colleagues (17) and Fekadu (36)
regarding
latent or abortive rabies.
Bell and colleagues (10) proved that dogs which recovered from rabies
after
intracerebral inoculation of homologous strains, had high titres of
neutralising
antibody in the cerebrospinal fluid as well as in serum and retained
these
titres for several months, whereas vaccinated dogs did not have high
cerebrospinal fluid titres. Murphy and colleagues (48) demonstrated the
same
phenomenon in cats.
Bell and colleagues (9) were the first to apply cerebrospinal fluid
titration
for an epidemiological survey. Of 120 dogs sampled in an area where
rabies was
enzootic (Buenos Aires), none was found to be positive; thus, it cannot
be
concluded that non-fatal rabies is common.
Blenden and colleagues (21) have suggested that the kinetics of
antibody
levels in blood and cerebrospinal fluid should be compared, to determine
whether
specific antibodies have been produced by infection or by immunisation.
Without
a booster after a first blood and cerebrospinal sampling, the antibody
level
should remain stable in cases of immunisation, or increase in cases of
infection. In fact, such procedures have never been routinely used
anywhere.
Indeed, given the variability of the titration test, the constancy of an
antibody titre over time is difficult to verify even in a vaccinated
animal.
Given the lack of easily-performed experimental methods, the only
basis for
considering that an individual dog or cat possessing rabies neutralising
antibodies has been vaccinated is good individual identification and
certification.
5. DISCUSSION
Laboratory conditions described in the challenge of vaccinated cats
and dogs
generally appear more severe than natural conditions of challenge in the
field.
In normal practice, experimenters use extremely long intervals between
vaccination and challenge (three to five years) and high virus doses
involving
100% mortality in controls. In areas contaminated by fox rabies, natural
challenge is not as severe for dogs and this could compensate for the
fact that
the health status of pets may be lower than that of dogs bred in the
laboratory.
Epidemiological observation is by far the more important evidence; in
continental Europe, rabies vaccination of cats and dogs is so efficient
that
where the annual risk of a fatal case of rabies has been evaluated for a
vaccinated pet, this risk is minute (1/6,980,000). It is also noteworthy
that in
continental Europe, fox rabies has never been propagated by domestic
animals
from an enzootic area to a free one - even if administrative rules
concerning
compulsory confinment, leashing or vaccination have sometimes been
broken either
deliberately or by the simple fact that rabid pets have escaped from
their
owners.
If a neutralising antibody titration were required for certifying the
immunological capacity of vaccinated animals, two questions would arise
regarding:
a) the choice of techniques for antibody titration
b) the definition and acceptance of a minimum antibody titre
considered as
providing protection against rabies.
A general analysis of challenge experiments leads to the conclusion
that
neutralising antibody titres enable prediction of survival more often on
a
qualitative basis (i.e. Do the animals have detectable neutralising
antibodies
or not?) than on a quantitative basis. This fact becomes apparent when
one tries
to determine a "protective" threshold. For this purpose, either method
of
seroneutralisation (RFFIT or MNT) can be employed, provided a
correlation
between the two methods has been demonstrated in the same laboratory
(14, 66).
Agreements on the international transfer of dogs and cats could be
formulated, therefore, based on a designated minimum level of
neutralising
antibodies, and could be proposed as an alternative to quarantine
measures. The
designated threshold could be based on the results presented in this
study. The
security of the protection constituted by this threshold would be
increased by
the extent to which it excedes the level recognised as effective against
experimental challenge in cats and dogs (0.1 IU/ml and 0.2 IU/ml,
respectively,
measured by RFFIT).
6. ACKNOWLEDGEMENT
The author wishes to express his gratitude to Dr J. Blancou for
kindly
revising the manuscript of this paper.
REFERENCES
1. Aghomo H.O. & Rupprecht C.E. (1990). - Antigenic
characterisation of
virus isolates from vaccinated dogs dying of rabies. Trop. Anim. Hlth
Prod., 22, 275-280.
2. Aghomo H.O. & Rupprecht C.E. (1990). - Further studies on
rabies virus
isolated from healthy dogs in Nigeria. Vet. Microbiol.,
22, 17-22.
3. Andral L. & Serie C. (1965). - Etude exp=E9rimentale sur la
rage en
Ethiopie. Ann. Inst. Pasteur, 108, 442-450.
4. Artois M., Aubert M.F.A., Blancou J. & Pericard M. (1984). -
Rage
exp=E9rimentale du chat : sensibilit=E9 - sympt=F4mes - excr=E9tion du
virus. Rev.
M=E9d. V=E9t., 135 (5), 281-287.
5. Atanasiu P. (1973). - Quantitative assay and potency test of
antirabies
serum and immunoglobulin. In Laboratory technique in rabies, 3rd
Ed. WHO,
Geneva, 314-318.
6. Aubert M.F.A. & Barrat J. (1991). - Les =E9checs de
vaccinations chez
les animaux domestiques. Bull. Epid=E9miol. Mensuel Rage Anim.
France,
21 (5), 1.
7. Barth R. & Jaeger O. (1977). - Zur Pr=FCfung der Immunit=E4ts
Dauer von
Tollwutkombinationsvaccinen am Hund. Die blauen Hefte f=FCr den
Tierarzt,
57, 337-346.
8. Barth R., Gruschkau H. & Jaeger O. (1985). - Chick embryo cell
inactivated rabies vaccine for veterinary use. Laboratory and field
experience.
In Rabies in the tropics (E. Kuwert, C. M=E9rieux, H. Koprowski
& K.
B=F6gel, eds). Springer Verlag, Berlin, 241-248.
9. Bell J.F., Gonzalez A.M., Diaz B. & Moore G.J. (1971). -
Nonfatal
rabies in dogs. Experimental studies and results of a survey. Am.
J.
Vet. Res., 32 (12), 2049-2058.
10. Bell J.F., Sancho M.I., Diaz A.M. & Moore G.J. (1972). -
Nonfatal
rabies in an enzootic area. Results of a survey and evaluation of
techniques.
Am. J. Epidemiol., 95 (2), 190-198.
11. Bellinger D.A., Chang J., Bunn T.O., Pick J.R., Murphy M. &
Rahija R.
(1983). - Rabies induced in a cat by high-egg passage Flury strain
vaccine.
J. Am. Vet. Med. Assoc., 183, 997.
12. Blancou J. (1988). - Ecology and epidemiology of fox rabies.
Rev.
Infect. Dis., 10 (Suppl. 4), S606-S609.
13. Blancou J., Aubert M.F.A., Toma B. & Andral L. (1980). -
Immunit=E9
humorale du chien apr=E8s primovaccination contre la rage : =E9tude dans
les
conditions de la pratique. Recl M=E9d. V=E9t., 150 (4),
313-318.
14. Blancou J., Aubert M.F.A. & Cain E. (1983). - Comparaison de
quatre
techniques de titrages s=E9rologiques des anticorps contre le virus de
la rage
chez le chien. J. Biol. Standard, 11, 271-277.
15. Blancou J., Aubert M.F.A. & Soulebot J.P. (1983). -
Diff=E9rences dans
le pouvoir pathog=E8ne de souches de virus rabique adapt=E9es au renard
ou au chien.
Ann. Inst. Pasteur Virol., 134E, 523-531.
16. Blancou J., Firon J.P. & Firon P.E. (1983). - D=E9faut de
r=E9action
immunitaire du chien apr=E8s vaccination contre la rage. Etude d'un cas.
Cons=E9quence. Rec. M=E9d. V=E9t., 159 (10), 789-793.
17. Blancou J., Aubert M.F.A. & Perrin G. (1985). - Rage
exp=E9rimentale du
chien. Sensibilit=E9, sympt=F4mes, excr=E9tion du virus. R=E9action
immunitaire et
r=E9sistance trois ans apr=E8s vaccination. Rev. M=E9d. V=E9t.,
136 (2),
147-152.
18. Blancou J., Artois M., Barrat J. & Prave M. (1986). -
Vaccination du
chat contre la rage : taux d'anticorps et r=E9sistance =E0 l'=E9preuve
un an apr=E8s
vaccination. Rev. M=E9d. V=E9t., 137 (17), 29-36.
19. Blancou J., Aubert M.F.A., Prave M. & Haddad N. (1986). -
Influence
du statut sanitaire des carnivores sur leur capacit=E9 =E0 s'immuniser
contre la
rage. Sci. Tech. Anim. Lab., 11 (3), 237-242.
20. Blancou J., Soria Baltazar R., Artois M., Toma B. & Rollin P.
(1989).
- Rabies despite pre- or post-exposure vaccination. In Progress
in rabies
control (O. Thraenart, H. Koprowski, K. B=F6gel & P. Sureau, eds).
Wells
Medical, Kent, 441-447.
21. Blenden C.D., Torres Anjel M.J. & Satalowitch F.T. (1985). -
Applications of laboratory technology in the evaluation of the risk of
rabies
transmissions by biting dogs and cats. Adv. Anim. Welfare Sci.,
221-246.
22. Brown A.L., Merry D.L. & Beckenhauer W.H. (1973). - Modified
live-virus rabies vaccine produced from Flury high egg-passage virus
grown on an
established canine-kidney cell line: three-year duration-of-immunity
study in
dogs. Am. J. Vet. Res., 34 (11), 1427-1432.
23. Brun A., Chappuis G., Precausta P. & Terre J. (1976). -
Immunisation
des chats contre la panleucop=E9nie et la rage. Rev. M=E9d.
V=E9t., 127
(11), 1575-1580.
24. Bunn T.O. (1983). - Rabies vaccine for use in dogs. In
Rabies in
the tropics (E. Kuwert, C. M=E9rieux, M. Koprowski & K. B=F6gel,
eds). Springer
Verlag, Berlin, 262-273.
25. Bunn T.O. (1985). - Rabies vaccine for use in dogs. In
Rabies in
the tropics (E. Kuwert, C. M=E9rieux, M. Koprowski & K. B=F6gel,
eds). Springer
Verlag, Berlin, 221-226.
26. Bunn T.O. (1991). - Cat rabies. In The natural history of
rabies,
2nd Ed. (G. Baer, ed.). CRC Press, 379-387.
27. Bunn T.O. (1991). - Canine and feline vaccines, past and present.
In The natural history of rabies, 2nd Ed. (G. Baer, ed.). CRC
Press,
415-425.
28. Bunn T.O. & Ridpath M.D. (1983). - The relationship between
rabies
antibody titers in dogs and protection from challenge. Rabies Info.
Exch., 8, 43-45.
29. Bunn T.O., Ridpath H.D. & Beard P.D. (1984). - The
relationship
between rabies antibody titers in dogs and cats and protection from
challenge.
Rabies Info. Exch., 11, 8-13.
30. Chappuis G. & Tixier G. (1982). - Etude de la relation
existant entre
le titre NIH et les anticorps s=E9roneutralisants obtenus apr=E8s
vaccination chez
les chiens. Comp. Immun. Microbiol. Infect. Dis., 5 (1-3),
151157.
31. Chomel B., Chappuis G., Bullon F., Cardenas E., de Beublain T.D.,
Maufrais M.C. & Giambruno E. (1987). - Serological results of a dog
vaccination campaign against rabies in Peru. Rev. Sci. Tech. Off.
Int.
Epiz., 6 (1), 97113.
32. Dean D.J., Evans W.M. & Thompson W.R. (1964). - Studies on
the low
egg passage Flury strain of modified live rabies virus produced in
embryonating
eggs and tissue culture. Am. J. Vet. Res., 25 756-763.
33. Derbyshire J.B. & Matthews K.A. (1984). - Rabies antibody
titres in
vaccinated dogs. Can. Vet. J., 25, 383-385.
34. Eng T.R. & Fishbein D.B. (1990). - Epidemiological factors,
clinical
findings and vaccination status of rabies in cats and dogs in the United
States
in 1988. J. Am. Vet. Med. Assoc., 197 (2), 201-209.
35. Engels M., Fluckiger M., Knusli K. & Wyler R. (1982). - Der
Immunstatus gegen Tollwut bei 200 geimpften Hunden aus dem Kanton
Zurich.
Schweizer Arch. Tierheilk., 124, 149-156.
36. Fekadu M. (1991). - Latency and aborted rabies. In The
natural
history of rabies, 2nd Ed. (G. Baer, ed.). CRC Press, 191-198.
37. Fuenzalida E. (1967). - Estado actual de desarrollo de la vacuna
antirr=E1bica preparada de cerebros de ratones lactantes en
Latinoam=E9rica.
In First International Seminar on Rabies, Buenos Aires, 417.
38. Gani=E8re J.P., Andr=E9-Fontaine G., Blancou J., Artois M. &
Aubert A.
(1989). - Vaccination antirabique du chien et du chat : taux d'anticorps
et
r=E9sistance =E0 l'=E9preuve virulente deux ans apr=E8s l'injection de
rappel d'un
vaccin additionn=E9 d'adjuvant. Rev. M=E9d. V=E9t., 140
(4), 281-285.
39. Hemachudha T. (1989). - Rabies. In Handbook of clinical
neurology.
Viral diseases (P.J. Vinken, G.W. Bruyn & H.L. Klawans, eds).
Elsevier,
383-404.
40. Hirayama N., Raharjo Jusa E., Aeny Rochman Noor M., Sakaki K.
& Ogata
M. (1990). - Immune state of dogs injected with rabies vaccines in the
West
Java, Indonesia. Jpn. J. Vet. Sci., 52 (5),
1099-1101.
41. Kaeberlee M.L. (1958). - Newer tools for the prevention of rabies
in
domestic animals. Ann. N.Y. Acad. Sci., 70 (3), 467-477.
42. Kihm U., Lazarowicz M., Bommeli W. & Zutter R. (1982). -
Potency of
two rabies vaccines in cats as determined by antibody assay and virulent
virus
challenge. Comp. Immun. Microbiol. Infect. Dis., 5 (1-3),
227-232.
43. Koutchoukali M.A., Blancou J., Chappuis G., Tixier G., Eloit M.,
Gani=E8re
J.P., Chantal J., Simon S., Berthier A. & Toma B. (1985). -
R=E9ponse
s=E9rologique du chien apr=E8s primovaccination antirabique =E0 l'aide
de vaccins
adjuv=E9s ou non. Ann. Rech. V=E9t., 16, 345-349.
44. Lawson K.F. & Crawley J.F. (1972). - The ERA strain of rabies
vaccine. Rev. Can. M=E9d. Comp., 36, 339-344.
45. Lazarowicz M., Kihm V., Bommeli W. & Zutter R. (1982). -
Potency
testing of inactivated rabies vaccines in mice, dogs and cats. Comp.
Immun.
Microbiol. Infect. Dis., 5 (1-3), 233-235.
46. Merry D.L., Brown A.L. & Beckenhauer W.H. (1970). -
Subcutaneous vs.
intramuscular inoculation of dogs. Vet. Bull., 40, 190.
47. Merry D.L. & Kolar J.R. (1984). - A comparative study of four
rabies
vaccines. Vet. Med. Small Anim. Clin., 79 (5),
661-664.
48. Murphy A.F., Bell F.J., Bauer S.D., Gardner J.J., Moore G.J.,
Harrison
A.K. & Coe E.J. (1980). - Experimental chronic-rabies in the cat.
43
(3), 231-241.
49. Nathanson N. & Gonzales Scarano F. (1991). - Immune response
to
rabies virus. In The natural history of rabies, 2nd Ed. (G. Baer,
ed.).
CRC Press, 145-161.
50. Prave M. (1985). - Mesures conservatoires l=E9gales appliqu=E9es
aux animaux
contamin=E9s de rage en France. Bilan apr=E8s 8 ans (1976-1984). In
Pasteur
et la rage (R. Rosset, ed.). Infos Tech. Serv. V=E9t.,
92-95,
264S-269S.
51. Precausta P. (1972). - Vaccin antirabique inactiv=E9 =E0 usage
v=E9t=E9rinaire
pr=E9par=E9 =E0 partir de culture cellulaire. Symp. Ser. Immunobiol.
Stand.,
21, 162-178.
52. Precausta P., Soulebot J.P., Bugand M., Brun A. & Chappuis G.
(1982).
- Modalit=E9s de production et immunit=E9 conf=E9r=E9e par un vaccin
antirabique
inactiv=E9 provenant de culture cellulaire. Comp. Immun. Microbiol.
Infect.
Dis., 5, 217-226.
53. Precausta P., Soulebot J.P., Chappuis G., Brun A., Bugand M.
&
Petermann M.G. (1985). - Nil cell inactivated tissue culture vaccine
against
rabies. Immunisation of Carnivores. In Rabies in the tropics (E.
Kuwert,
C. M=E9rieux, H. Koprowski & K. B=F6gel, eds). Springer Verlag,
Berlin, 227-240.
54. Rosatte R.C. & Gunson J.R. (1984). - Presence of neutralizing
antibodies to rabies virus in striped skunks from areas free of skunk
rabies in
Alberta. J. Wildl. Dis., 20 (3), 171-176.
55. Sasaki D.M. & Gooch J.M. (1983). - Cost effectiveness of
Hawaii's
anti-rabies quarantine program. Hawaii Med. J., 42,
157-160.
56. Sasaki D.M. & Gooch J.M. (1992). - Rabies serosurvey of
quarantine
pets and mongooses. Report presented to the 16th State Legislature,
75pp.
57. Schmidt R.C. & Sikes R.K. (1968). - Immunisation of foxes
with
inactivated-virus rabies vaccine. Am. J. Vet. Res., 29
(9),
1843-1849.
58. Sekine N. & Yoshino K. (1974). - Inhibitors against rabies
virus
present in normal rabbit sera. Arch. Ges. Virusforsch.,
45, 89-98.
59. Sharpee R.L., Nelson L.O. & Beckenhauer W.H. (1985). -
Inactivated
tissue culture rabies vaccine with three years immunogenicity in dogs
and cats.
In Rabies in the tropics (E. Kuwert, C. M=E9rieux, H. Koprowski
& K.
B=F6gel, eds). Springer Verlag, Berlin, 262-273.
60. Sikes R.K. (1971). - Evaluation of canine rabies vaccine.
In
Rabies (Y. Nagano & F.M. Davenport, eds). University Park Press,
Baltimore,
London & Tokyo, 343-361.
61. Sikes R.K., Peacock G.V., Acha P.L., Arko R.J. & Dierks R.
(1971). -
Rabies vaccines: duration of immunity. Study in dogs. J. Am. Vet.
Med.
Ass., 1491-1499.
62. Smith J.S., Yager P.A. & Baer G.M. (1973). - A rapid
reproducible
test for determining rabies neutralizing antibody. Bull. Wld Hlth
Org.,
48, 535-541.
63. Soulebot J.P., Stellmann Ch., Bornarel P., Petermann H.G., Lang
R. &
Branche R. (1970). - Influence de la voie d'inoculation des vaccins
antirabiques
chez le chien. Bull. Soc. Sci. V=E9t. M=E9d., 72, 409-417.
64. Soulebot J.P., Brun A., Chappuis G., Guillemin F., Petermann
H.G.,
Precausta P. & Terre J. (1981). - Experimental rabies in cats:
immune
response and persistence of immunity. Cornell Vet., 71
(3),
311-325.
65. Teepsumethanon W., Polsuwan C., Lumlertdaecha B., Khawplod P.,
Hemachudha
T., Chutivonsge S., Wilde H., Chiewbamrungkiat M. & Phanuphak P.
(1991). -
Immune response to rabies vaccine in Thai dogs: a preliminary report.
Vaccine, 9, 627-630.
66. Thraenart O., Ramakrishnan K., J=E4ger O. & Marcus I. (1989).
-
Antibody induction determined by the mouse neutralization test, rapid
fluorescent focus inhibition test, and Essen-enzyme linked
immunoadsorbent assay
is correlated. In Progress in Rabies Control (O. Thraenart, H.
Koprowski,
K. B=F6gel & P. Sureau, eds). Wells Medical, Kent, 384-402.
67. Tierkel E.S., Koprowski H., Black J. & Gorrie R.H. (1949). -
Preliminary observations in the comparative prophylactic vaccination of
dogs
against rabies with living virus vaccines and phenolized vaccine. Am.
J. Vet.
Res., 10, 361.
68. Toma B., Koutchoukali M.A., Blancou J., Chappuis G., Tixier G.
&
Eloit M. (1985). - Vaccination of dogs against rabies. Comparison of
serological
responses one year after intradermal or subcutaneous vaccination. In
Rabies in the tropics (E. Kuwert, C. M=E9rieux, H. Koprowski &
K. B=F6gel,
eds). Springer Verlag, Berlin, 255-261.
69. Toma B., Koutchoukali M.A., Blancou J., Eloit M., Ganiere J.P.
&
Chantal J. (1987). - Vaccination antirabique du chien : r=E9ponse
s=E9rologique
compar=E9e un an apr=E8s premier rappel =E0 l'aide de vaccin contenant
un adjuvant.
Rev. M=E9d. V=E9t., 138, 905-911.
70. Tsiang H., Blancou J. & Lagrange P.H. (1981). - BCG
modulation of
delayed type hepersensitivity, humoral response and acquired resistance
after
rabies vaccination. Arch. Virol., 69, 167-176.
71. Wandeler A., Wachendorfer G., Forster U., Krekel H., Muller J.
&
Steck F. (1974). - Rabies in wild carnivores in Central Europe.
Zentbl.
VetMed., B, 21, 757-764.
72. Whetsone C.A., Bunn T.O., Emmons R.W. & Wiktor T.J. (1984). -
Use of
monoclonal antibodies to confirm vaccine-induced rabies in ten dogs, two
cats
and one fox. J. Am. Vet. Med. Assoc., 185, 285.
73. Wiktor T.J. (1978). - Cell mediated immunity and post exposure
protection
from rabies by inactivated vaccines of tissue culture origin. Dev.
Biol.
Stand., 40, 255-264.
74. Wilsnack R.E. & Parker R.I. (1966). - Pathogenesis of skunk
rabies
virus: rabies inhibiting substance as related to rabies diagnosis.
Am. J.
Vet. Res., 27 (116), 39-43.
75. Yasmuth C., Nelson K.E., Laima T., Supawadee J. & Thaiyanant
P.
(1983). - Prevalence of abortive canine rabies in Chiang Mai, Thailand.
J.
Med. Ass. Thai., 66, 169-175.