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Antibody Titres Against Canine Distemper Virus in Vaccinated and

Unvaccinated Dogs

Article  in  Journal of Veterinary Medicine Series B · May 2004

DOI: 10.1111/j.1439-0450.2004.00738.x · Source: PubMed

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J. Vet. Med. B 51, 99–103 (2004)
 2004 Blackwell Verlag, Berlin
ISSN 0931–1793
Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland
Antibody Titres Against Canine Distemper Virus in Vaccinated and Unvaccinated
Dogs
A. Jóźwik1,3, T. Frymus1, B. Mizak2 and A. RzeZ_ utka2
Addresses of authors: 1Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University,
Warsaw, Poland; 2Department of Carnivores and Fur Animal Diseases, National Veterinary Research Institute, Pulawy, Poland;
3
Corresponding author: Tel.: +48 22 8475818; fax: +48 22 8475818; E-mail: jozwika@alpha.sggw.waw.pl
With 3 figures and 1 table
Summary
In 248 urban dogs the serum antibody titre against canine
distemper virus (CDV) was determined by the immunoper-
oxidase monolayer assay. Among them 72 animals had been
routinely vaccinated at least once against distemper, 20 dogs
had never been immunized and the vaccination history of 156
was unknown. The mean antibody titre in vaccinated dogs
(114 ± 8.2) indicated immunity (‡100) and was significantly
higher when compared with unvaccinated animals (29 ± 8.8,
P < 0.05) or with dogs with unknown vaccination history
(29 ± 5.0, P < 0.001). Among 156 dogs with unknown vac-
cination history only 26 (19%) had antibody titres indicating
immunity against CDV (‡100). Vaccinated dogs were divided
into five groups according to the time since the most recent
distemper vaccination: group A: up to 1 month (n ¼ 13);
group B: 1–2 months (n ¼ 6); group C: 2–12 months (n ¼ 20);
group D: 12–24 months (n ¼ 4); group E: 25–36 months
(n ¼ 9) and group F: 36 or more months (n ¼ 18). The mean
titre in group A was 490 ± 4.5, in B 359 ± 3.8, in C
204 ± 4.6, in D 269 ± 1.9 and the differences between these
groups were not significant. However, dogs in groups E and F
had significantly lower mean antibody titres (21 ± 8.3,
P < 0.01 and 39 ± 7.6, P < 0.001, respectively) when com-
pared with group A. Nine dogs suffering from distemper
confirmed by the immunofluorescence test or nested RT-PCR
were also included in this study. In five of them high titres of
anti-CDV antibody were detected (160–1280). All these ani-
mals recovered (in two localized muscle twitching persisted).
On the contrary, the remaining four patients with low anti-
body titre to CDV (5–40) died.
Introduction
Outbreaks of canine distemper have been recently documented
in several countries, such as Denmark (Blixenkrone-Møller
et al., 1993), the US (Patronek et al., 1995), Japan (Gemma
et al., 1996), Finland (Ek-Kommonen et al., 1997) and
Germany (Haas et al., 1997). In a previous report we demon-
strated that distemper is still an important infection also in
Poland and that vaccination significantly reduces the risk of
this disease (Jóźwik and Frymus, 2002). However, the duration
of the post-vaccinal immunity against the canine distemper
virus (CDV) and also the required frequency of revaccinations
of adult dogs remain controversial aspects of the immuno-
prophylaxis of distemper. The first studies by Baker in 1962
showed that 1 year after vaccination the antibody titre
U. S. Copyright Clearance Center Code Statement: 0931–1793/2004/5103–0099$15.00/0
Received for publication October 27, 2003
decreased to low levels in 33% of dogs, and after 2 years in
the remaining animals (cit. after Carmichael, 1999). Probably
according to these data, all vaccine manufacturers recommen-
ded annual revaccinations against distemper, what has been
established as a common practice in many countries including
Poland. However, some recent data indicate that the post-
vaccinal immunity against CDV persists much longer than
1 year. Olson et al. (1997) examined serologically 30 dogs
exported after the vaccination to Iceland, which is considered
to be free from CDV and where vaccination against distemper
is not permitted. They demonstrated in 73.3% of these animals
neutralizing titres ‡16 of anti-CDV antibody after a mean time
of 5 years and 5 months since the most recent immunization
(range from 4 years and 2 months to 9 years and 10 months).
Similarly Carmichael (1999) in nine vaccinated beagles kept in
strict isolation found even after 6 years neutralizing antibody
titre ‡80 against distemper. Also the studies of Schultz (1999)
revealed that after a vaccination against distemper 95% of
dogs were protected from clinical disease for at least 5 years. It
has been also pointed out that the recommended vaccination
schedules for adult dogs are contrary with those widely
accepted in the human medicine (Paul et al., 2003). Well-
documented epidemiological studies indicate that immunity
against many diseases induced in human beings by vaccination
is extremely long lasting, even life-long. For example, vaccin-
ation against measles, a Morbillivirus infection with several
similarities to distemper, is carried out only in children,
without revaccination of adults (Collier and Oxford, 1993).
Based on these data the need for annual revaccination of adult
dogs against the most important diseases including distemper
has been questioned recently by the American Animal
Hospital Association Canine Vaccine Task Force (Paul et al.,
2003).
However, the post-vaccinal immunity developed in experi-
mental conditions in isolated dogs may last longer than that
obtained in a natural environment. It is well known that many
factors can suppress the immune response to vaccination. The
most important of them are the maternal immunity of a
puppy, helminth infestation and stress because of other
reasons (Greene, 1998; Jarrett and Ramsey, 2001). Also,
improper handling and storage of the vaccine may affect its
efficacy, specially in the case of immunization against distem-
per (Chappuis, 1995). Finally, commercially available vaccines
may have different immunogenicity. A major distemper
epidemic has been described in Finland in dogs vaccinated
with a product of poor efficacy (Ek-Kommonen et al., 1997;
www.blackwell-synergy.com
100 A. Jóźwik et al.

Rikula et al., 2000). Because of these factors and little evidence
on the duration of protective immunity in dogs vaccinated
under natural conditions many authors still recommend
annual booster in adult animals (McCaw et al., 1998; Jarrett
and Ramsey, 2001).
Taking into account these discrepancies the aim of the study
was to investigate in natural conditions the anti-CDV antibody
titres in dogs at different time after a routine distemper
vaccination and to analyse the immunity of these animals in
comparison with non-vaccinated dogs.
Materials and Methods
Two hundred and forty-eight healthy and sick dogs brought to
the Clinic of Small Animals of the Warsaw Agricultural
University were examined serologically to determine the
antibody titre against CDV. Seventy-two animals had been
vaccinated previously at least once against distemper, 20 dogs
had never been immunized against this disease and in 156 dogs
the vaccination history was unknown. In the group of
vaccinated animals, the time of the last immunization against
distemper was recorded.
Seven dogs were less than 3 months old, 14 animals were
between 3 and 6 months old, 15 were between 7 and 12 months
old, 21 were between 1 and 3 years old, 59 were between 3 and
6 years old and 132 dogs were older than 6 years.
At the time of blood sampling 22 dogs revealed clinical signs
Results
The geometric mean titres in the groups of dogs with different
vaccination history are shown in Fig 1.
The anti-CDV antibody titres depending on the time of the
most recent distemper vaccination are shown in Fig. 2.
Among 156 dogs with unknown vaccination history only 26
(19%) had antibody titre indicating immunity (‡100) against
distemper, whereas most (127) animals appeared to be
susceptible to this disease (Fig. 3).
Among 22 dogs suspected of having distemper in nine
animals the CDV infection was confirmed by the immunoflu-
orescence test or nested RT-PCR. Only in five of these nine
infected patients high titres of anti-CDV antibody were
detected. Among these five dogs with strong serologic response
(‡100), three recovered completely and in two animals only
localized twitching of head and limb muscles were seen till the
end of the observation period (>12 weeks). On the contrary,
all four patients with low antibody response (<100) to CDV
died. Data regarding the age, breed, sex, vaccination status,
history, clinical signs and the results of serological and
virological examination of the nine dogs with distemper are
presented in Table 1.
120 114 ± 8.2
Geometric mean

100
suggestive of distemper. Beside serological testing these
animals were also examined by the direct immunofluorescence 80
test or by nested RT-PCR to confirm the CDV infection as
titre

described earlier (Jóźwik and Frymus, 2002; Jóźwik and 60

Frymus, 2004). 40 29 ± 8.8 29 ± 5.0

Blood samples were collected from the cephalic vein and
were centrifuged to obtain sera. Collected sera were frozen at 20

)20C until further analysis. 0

Serum antibody titres were determined by the immunoper-
a b c
oxidase monolayer assay (IPMA) as previously described
Vaccinated Unvaccinated Unknown
(Mizak and Borowski, 1998; Borowski and Kimak, 2001).
n =72 n =20 n =156
Briefly, each serum was serially diluted and added into
microwells, which had been previously overlaid with Vero Fig. 1. Mean anti-canine distemper virus serum antibody titres in dogs
cells infected with the Lederle strain of CDV. Rabbit anti-dog vaccinated at least once against distemper, in unvaccinated and in
IgG (Sigma-Aldrich, Poland) labelled with horseradish per- animals with unknown vaccination history (by immunoperoxidase
monolayer assay).ab: P < 0.05; ac: P < 0.001; bc: P > 0.05.
oxidase was used as a conjugate and 3-amino-9-ethylcarbazole
(Sigma) as a substrate. The plates were read by the use of the
light microscope Jenaval FA (Carl Zeiss Jena, Jena, Germany)
at magnification 100·. For the purpose of determining whether 600
dogs were immune against distemper, the titre ‡100 was 490 ± 4.5
Geometric mean titre

500
considered to indicate immunity ( Borowski and Kimak, 2001;
A. Borowski, B. Mizak and I. Kimak, unpublished data). 400 359 ± 3.8
The animals were divided into groups depending on the 300 269 ± 1.9
vaccination status (vaccinated, unvaccinated, unknown his- 204 ± 4.6
200
tory) and time since the most recent immunization against
distemper (group A: up to 1 month; group B: 1–2 months; C: 100
21 ± 8.3 39 ± 7.6
2–12 months; D: 12–24 months; E: 25–36 months and F: 36 0
or more months). Antibody titres in each group were A B C D E F
up – 1 1–2 2 – 12 12 – 24 25 – 36 more than
expressed in log2 and geometric mean as well as standard month months months months months 36 months
deviations were obtained. The significance of the differences (n =13) (n =6) (n =20) (n =4) (n =9) (n =18)
between the groups were determined by the Student’s t-test Time since the last distemper vaccination

using the computer program Statistica (StatSoft, Kraków,

Fig. 2. Mean anti-canine distemper virus serum antibody titres in dogs
Poland) and Microsoft Excel (Microsoft, Troy, NY, USA). at different time since the most recent distemper vaccination (by
For statistical analysis, a value of P < 0.05 was considered immunoperoxidase monolayer assay). AB: P > 0.05; AC: P > 0.05;
significant. AD: P > 0.05; AE: P < 0.01; AF: P < 0.001.
Antibody Titres Against Canine Distemper Virus in Vaccinated and Unvaccinated Dogs 101

of muscles

of muscles
19%

twitching

twitching
Outcome

Localized

Localized
Recovery

Recovery
Recovery
disease
of the

Death

Death

Death

Death
Titre indicating
immunity
Titre <100

Anti-CDV
Ab titre
(IPMA)

1280

1280
160

640

320

40

40

10

5
81%

CDV detected
by RT-PCR
Fig. 3. Percentage of dogs with serum antibody titres indicating

Not done

Not done
(blood)
immunity to distemper (‡ 100 in the immunoperoxidase monolayer

+
+

+
assay) among 156 animals with unknown vaccination history.

Discussion

CDV detected
by IF assay
(imprints)
The standard for assessment the level of immunity to

+
)
)

)
distemper is to challenge the vaccinated animals with a
virulent strain, but as an alternative method, measurement
of serum antibody titres has been widely used (McCaw
et al., 1998; Carmichael, 1999; Schultz, 1999). The import-

Purulent nasal discharge, localized twitching

signs, hyperkeratosis of nose and footpads
Localized twitching of the muscles of limbs

Laryngotracheitis, bronchitis, neurological

ance of the humoural immune response of a dog in
determining the outcome of infection by CDV has been

Localized twitching of muscles, ataxia,

Purulent ocular and nasal discharge
well documented (Appel, 1969; Krakowka et al., 1975; Rima

neurological signs, hyperkeratosis

Laryngotracheitis, bronchitis and

et al., 1991; Greene and Appel, 1998). However, titres

Laryngotracheitis, bronchitis,
considered to indicate immunity vary among laboratories

Clinical signs

of the nose and footpads

and methodologies. Krakowka et al. (1975) reported that

Purulent nasal discharge

At present not observed
vaccinated dogs with antibody titres 16 in the serum
neutralization (SN) test survived challenge with virulent
CDV. Rima et al. (1991) reported that dogs with neutral-

gastroenteritis
izing antibody titres higher than 50 in the SN test recovered

of muscles
from distemper. Carmichael (1999) showed in vaccinated

paresis
beagle dogs kept in strict isolation that neutralizing antibody
titre ‡80 indicated immunity to CDV. McCaw et al. (1998)
Table 1. Epidemiological, serological and virological data of nine dogs with clinical distemper

considered in vaccinated dogs SN antibody titre ‡96 as

protective. Gillespie (1966) showed that pups with maternal

Neurological signs since 1 month

An episode of laryngotracheitis

antibody SN titres above 100 were definitely immune and

Recently visited a dog show,

Derived from a dog shelter

that this is a serologic assurance that the dog is resistant to

Lives in a dog shelter, sick

Derived from a rural area

Pneumonia 2 weeks ago

distemper.
Besides the SN assay other methods have been used to
History

Contacts with dogs

sick since 9 days
determine the antibody level to CDV in dogs. Twark and
Sick since 3 days

since 1 month

Dodds (2000) studied the post-vaccinal immunity to CDV by

10 days ago

the indirect immunofluorescent antibody test. We used a

similar method, the immunoperoxidase monolayer assay
(IPMA). The comparative studies of Borowski and Kimak
(2001) of the SN test and IPMA revealed a high correlation
coefficient (r ¼ 0.94) between both methods and a high
Vaccinated

distemper
against

repeatability of titres using IPMA. Within distemper vaccines

Yes

Yes
Yes

Yes
No

No

No

No

No

control system they demonstrated that only these dogs

survived the challenge, which developed IPMA titre at least
100 (A. Borowski, B. Mizak and I. Kimak, unpublished data).
Sex

#
$
$

Thus we considered in our study this antibody level as

indicating immunity. Also our findings in dogs suffering from
(m-ths)

distemper confirm that only patients with IPMA titre ‡100 are
Age

2,5

2,5
12

12
5

able to recover (Table 1).

St Bernardshund

Our studies were performed on dogs routinely vaccinated

Siberian husky

with commercially available live virus products. There is one

napoletano
Dachshund
Breed

Polish distemper vaccine in the market, but to the best of our

Mastino

knowledge the most frequently used products in Warsaw in

Mixed
Mixed

Mixed

Mixed

Mixed

the last decade were manufactured by Fort Dodge, Pfizer,

Intervet and Merial. We found that in dogs vaccinated at
752A

726A
759A

818A

738A

815A

731A

least once against distemper, the mean serum antibody titre

33A

32A
No.

to CDV was 114, whereas animals never immunized against

102
this disease as well as dogs with unknown vaccination history
had significantly lower mean antibody titre of 29 (Fig. 1).
However, there were no differences between the titres in
unvaccinated dogs when compared with animals with
unknown vaccination history. It means that antibody levels
in vaccinated dogs indicated their immunity (‡100), in
contrast with unvaccinated animals or dogs with unknown
vaccination history. Therefore, lack of data regarding dis-
temper immunization should raise suspicion, that an animal is
not protected against CDV.
As can be seen from Fig. 2 vaccinated animals had antibody
titres indicating immunity within the first as well as the second
year after immunization and later the antibody level decreased
significantly to fall below 100. These data may not correspond
with post-vaccinal immunity as the dogs could have contacts
with wild CDV, so a natural booster was not excluded. The
animals used in this study lived under natural conditions, as
exist in a big town where distemper cases are occasionally
confirmed (Jóźwik and Frymus, 2002). Besides, they were
vaccinated with different products. Nevertheless, from this
data it is apparent that dogs from an urban population
vaccinated with commercial distemper vaccines have for
2 years antibody titres indicating immunity, and then antibody
levels significantly decrease. Therefore, it seems reasonable to
revaccinate urban adult dogs against distemper every second
year.
It has been postulated that the course of canine distemper
strongly depends on the ability of an infected dog to
quickly develop an immunological response to CDV (Appel,
1969; Krakowka et al., 1975; Rima et al., 1991; Greene and
Appel, 1998). If the serum antibody titre reaches high level
within 8–9 days of infection, the virus disappears from the
lymphatic and other tissues and the infection remains
subclinical or mild. However, if the immunological response
is poor or delayed, the CDV disseminates through many
tissues causing an acute or chronic disease with high
mortality. Our results confirm the role of humoural
immunity in recovering from this disease. Among nine dogs
with distemper confirmed virologically five patients had high
titres of anti-CDV serum antibody (Table 1). All these dogs
with strong serologic response recovered, three completely
and in two animals only twitching of some muscles
remained till the end of the observation period
(>12 weeks). On the contrary, all four patients with low
antibody response to CDV died. They suffered from
distemper for longer than 9 days but in none of them
antibody titre achieved 100 despite administration of anti-
CDV immunoglobulin therapy to two of them. The results
shown in Table 1 confirm that serological examination of a
distemper patient may have a prognostic value.
It can be concluded that dogs from an urban population
vaccinated with commercial modified live virus distemper
vaccines have for 2 years antibody titres indicating immu-
nity, and then the antibody level significantly decreases.
Unknown vaccination history should raise suspicion, that
the dog is not protected against CDV and prompt for basic
vaccination.
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