4yr 7mon distemper parvo hepatitis vaccination DOI

[malernee]

O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz
173
■ INTRODUCTION
Canine parvovirus (CPV), infectious canine
hepatitis (ICH), and canine distemper virus
(CDV) infections are potentially fatal diseases
of dogs and other related species. The etiologic
agent for CPV infections is a nonenveloped
DNA-containing virus, CPV type 2 (CPV-2).
The virus is very stable in the environment and
highly contagious.1 In the United States, the
CPV-2b genotype, through mutations,2 has
largely replaced previously isolated genotypes
(CPV-2 and CPV-2a). In the Far East3,4 and
Evaluation of the Efficacy and Duration of Immunity
of a Canine Combination Vaccine Against Virulent
Parvovirus, Infectious Canine Hepatitis Virus, and
Distemper Virus Experimental Challenges*
Omar Y. Abdelmagid, BVSc, MS, PhDa
Laurie Larson, DVMb
Laurie Payne, BAa
Anna Tubbs, BSa
Terri Wasmoen, PhDa
Ronald Schultz, MS, PhD, DACVMb
*This research was funded by Schering-Plough Animal
Health, Elkhorn, Nebraska.
CLINICAL RELEVANCE
The results of this study confirmed that dogs vaccinated subcutaneously with a
commercially available multivalent vaccine containing modified-live canine distemper
virus, canine adenovirus type 2, canine parvovirus type 2b, and canine
parainfluenza virus antigens were protected against sequential experimental
challenge 55 to 57 months after initial vaccination given at 7 to 8 weeks of age.
All 10 vaccinates were protected against clinical diseases and mortality following
parvovirus and infectious canine hepatitis experimental infections. All vaccinates
were protected against mortality and 90% against clinical disease following
distemper challenge. These data support at least a 4-year duration of
immunity for these three “core” fractions in the combination vaccine.
aSchering-Plough Animal Health
Research and Development
21401 West Center Road
Elkhorn, NE 68022
bDepartment of Pathobiological Sciences
School of Veterinary Medicine
University of Wisconsin-Madison
2015 Linden Drive West
Madison, WI 53706
Europe,5,6 both CPV-2a and CPV-2b may be
present, but current published information is
not available. Canine distemper infection is
caused by CDV, a contagious Morbillivirus belonging
to the Paramyxoviridae family. CDV
affects Canidae and other carnivores such as
pandas, raccoons, and large felids.7,8 ICH is
caused by canine adenovirus type 1 (CAV-1).
The virus is stable in the environment and affects
Canidae and Ursidae (bears).9
Immunization of dogs through the use of
vaccination during the past decades has greatly
reduced the incidence of these infectious diseases
in dog populations.10–12 Modified-live
vaccines were shown to induce superior and
longer-lasting immunity compared with inactivated
vaccines.9–11 Safe and efficacious vaccines
against CDV, CPV, and ICH infections are
considered essential vaccines that every dog
should receive and were recently designated as
“core vaccines” by Schultz,13 the American Veterinary
Medical Association Council on Biological
and Therapeutic Agents (COBTA),14
and the American Animal Hospital Association
(AAHA) Canine Vaccine Task Force.15
Determination of vaccination frequency for
the core vaccines was a subject of debate and
differing views among experts in the field.14,16–18
Veterinarians have to make decisions regarding
the frequency of vaccination based on safety, efficacy,
and duration of immunity as well as such
factors as animal susceptibility, animal environment,
risk of exposure, breed involved, and
other considerations.14,16,17 To determine the
duration of immunity of “core” vaccines, some
authors relied on serologic data obtained from
field studies.19,20 Although serologic response is
a good indicator of the level of protection for
each of these three antigens,21–24 the potential
for environmental exposure to these viruses under
field conditions compromises the conclusion
that protection is solely due to vaccineinduced
antibodies.
This article presents vaccine efficacy and duration
of immunity data obtained from direct
animal vaccination and experimental challenge
studies. The vaccinated animals were housed
under high levels of biosecurity throughout the
holding period to prevent extraneous virus exposure.
Control nonvaccinated dogs were
maintained in the same facilities throughout
the period and were shown to remain antibody
negative to the three viruses of interest.
■ MATERIALS AND METHODS
Animals
Ten beagle puppies seronegative to CPV-2b,
CAV-1, canine adenovirus type 2 (CAV-2),
and CDV were vaccinated at 7 to 8 weeks of
age with two doses of vaccine given 3 weeks
apart; the puppies were held in isolation until
challenge: CPV-2b challenge was administered
55.1 months after vaccination, CAV-1 challenge
at 55.8 months, and CDV challenge at
56.5 months. Seven age-matched unvaccinated
puppies were held separately at the same facility
as controls. Three of the seven controls were
entered into the study at the time the other
dogs were vaccinated, and the remaining four
were introduced 5 months after vaccination.
These controls are referred to as “adult control
dogs” in this article. An additional three sets of
five young beagle puppies were used as young
susceptible controls to validate the virulence of
each challenge (five per challenge) and are referred
to as “young control puppies” in this ar-
Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004
174
Safe and efficacious vaccines against CDV, CPV, and ICH
infections were recently designated as “core vaccines.”
O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz
175
ticle. All dogs in this study were treated according
to animal welfare regulations outlined
in 9 CFR, Subchapter A, Part 3, Subpart A.
Animal care and use committee (IACUC) approval
was obtained at each site before the
study was conducted.
Vaccine
The test vaccine is a commercially available
multivalent freeze-dried vaccine (DA2PPv)
containing modified-live CDV, CAV-2, canine
parainfluenza virus (CPI), and CPV-2b antigens.
The vaccine is marketed by Schering-
Plough Animal Health in the United States,
Canada, and South Africa under the trade
name Galaxy and in Europe and other markets
under the trade names Procyon Dog or Quantum
Dog. The test vaccine was rehydrated with
liquid vaccine containing killed Leptospira canicola,
Leptospira icterohaemorrhagiae, and coronavirus
antigens at the time of vaccination and
administered subcutaneously in 1-ml doses.
Animal Trial Design
Before vaccination, puppies were blocked by
litter and gender and were randomly assigned
as vaccinates or controls. Vaccinates (n = 10)
received two doses of vaccine given 3 weeks
apart and were held with the controls (n = 7)
in an isolation facility in pens at Liberty Research
(Waverly, NY) for 55 months. During
this period, they were cared for and blood samples
were collected regularly to monitor their
antibody status. At the end of the holding period,
the designated vaccinates and adult control
dogs were transferred to the University of
Wisconsin animal facility in Madison, Wisconsin,
for experimental challenge. The dogs were
acclimated for 8 days and determined to be fit
before challenge inoculation. Five 12-weekold,
CPV-2b-seronegative puppies (young control
puppies) arrived at the facility at the same
time for use in the CPV-2b challenge. Two additional
sets of five young control puppies were
used to validate CAV-1 and CDV challenges
(five puppies per challenge).
Experimental Challenge
Canine Parvovirus Type 2b Challenge
The 10 vaccinates, four adult controls, and
five young control puppies were all challenged
oronasally (day 0) with virulent CPV-2b (obtained
from the USDA Center for Veterinary
Biologics). Each dog received 104.1 TCID50 of
challenge virus. Inoculated dogs were observed
for clinical signs, temperature, and mortality
for 14 days after challenge. Whole blood (in
EDTA tubes) and fecal swabs were collected
daily to evaluate leukocyte counts and virus
shedding, respectively.
Canine Adenovirus Type 1 Challenge
After the vaccinates were rested for 1 week,
they and three adult controls were inoculated
intravenously (day 21) with CAV-1 (obtained
from the USDA Center for Veterinary Biologics).
Five young control puppies were challenged
in a similar manner to validate the challenge
dose. Each dog received at least 103.3
TCID50 of challenge virus. The inoculated
dogs were observed daily for clinical signs for
21 days following challenge.
Canine Distemper Virus Challenge
Three weeks following CAV-1 challenge
(day 42), the 10 vaccinates and four adult controls
(the same adult controls used in the parvovirus
challenge) were physically examined
and determined to be fit before they were inoculated
intranasally and intravenously with
virulent CDV Snyder Hill challenge virus
(provided by R. S.). Five young (12-week-old)
control puppies were challenged separately in
advance to validate the challenge virus virulence
and were observed for 15 days following
challenge. Inoculated adult vaccinates and con-
pressed as the reciprocal of the dilution, which
neutralized 50% of the virus, as calculated by
the Spearman–Karber method.25 For purpose
of analysis, SN titers below 2 were given a value
of 1. Antibody titers that were not endpointed
at dilution 1:4,096 were given the value
of 4,096. Geometric mean titers (GMTs)
obtained in these cases were indicated as
>GMT value.
Detection of Canine Parvovirus in Fecal
Samples by Hemagglutination Test
CPV in rectal swabs was quantified by the
hemagglutination assay (HA) using 96-well
plates. Briefly, the swabs were stored in tubes
containing 1 ml of phosphate-buffered saline
(PBS) and frozen until thawed for extraction.
At extraction, tubes were vortexed, swabs were
removed, and 500 µl chloroform was added.
Tubes were vortexed intermittently for 15 minutes
and centrifuged, and the supernatant was
removed and stored frozen until tested. The
sample was twofold serially diluted (in PBS
with bovine serum albumin) in duplicates of
wells of a round-bottomed 96-well plate. After
sample dilutions were made, 1% of washed
porcine erythrocytes was added to each well
and plates were refrigerated for 4 to 8 hours.
Titers were calculated as the reciprocal of the
highest dilution of sample that produced complete
agglutination. Positive (sample with
known titer) and negative (sample with no
virus) controls were used to monitor the assay.
Leukocyte Counts
Leukocyte counts were determined using an
ADVIA 120 Hematology System (Bayer
Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004
176
trols were observed daily for clinical signs typical
of distemper disease for 21 days after challenge.
Serum Neutralization
Antibody responses to CPV, CAV-1, CAV-2,
and CDV were measured by serum-neutralization
(SN) assays performed by a microtitration
method using flat-bottomed 96-well microtiter
plates. Twofold serial dilutions of serum samples
were prepared in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 2
mm L-glutamine and gentamicin (50 µg/ml).
Four wells of a microtiter plate were inoculated
with 50 µl of neat or diluted serum from each
dilution. An equal volume (50 µl) of the specific
SN challenge virus (CPV, CAV-2, CAV-1, or
CDV [distemperoid strain]) containing 50 to
300 log10 TCID50 was added to each well. After
30 to 60 minutes of incubation at 36 ± 2°C,
cell substrate (dog kidney cell in the case of
CPV, CAV-1, and CAV-2; Vero cells in the case
of CDV) was added to each well (seeded at approximately
1 to 2 × 104 cells/0.1 ml/well in
DMEM supplemented with 2 mm L-glutamine,
gentamicin [50 µg/ml], and 5% fetal
bovine serum). Plates were incubated in a humidified
carbon dioxide (4% to 6 %) incubator
for 3 days (CPV) or 5 to 7 days (CAV-1, CAV-
2, and CDV) at 36 ± 2°C.
At the end of the incubation period, SN antibody
response was determined by examination
of wells for typical CDV or CAV cytopathic
effect. For CPV, plates were fixed with
80% acetone (100 µl/well) and stained with
CPV-specific fluorescein-labeled antibody conjugate
(75 µl/well). SN antibody titers were ex-
Following vaccination, the vaccinates developed high
levels of SN antibodies to all fractions.
O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz
177
HealthCare, Tarrytown, NY). The EDTA
blood sample was mixed with ADVIA 120
BASO solution containing acid and surfactant.
After erythrocytes were hemolyzed, the leukocyte
counts were analyzed using two-angle laser
light scatter signals.
■ RESULTS
Antibody Response
All puppies were confirmed to be seronegative
to CPV, CAV-1, CAV-2, and CDV before
vaccination (Table 1, Figure 1). Following vaccination,
the vaccinates developed high levels
of SN antibodies to all fractions. The control
dogs remained seronegative throughout the
holding period, indicating lack of extraneous
exposure to these agents.
Canine Parvovirus Type 2b Response
All 10 vaccinates (100%) responded with
very high SN titers, with the highest GMT
obtained 1 month after vaccination (GMT
>3,956). The GMT was maintained above
1,078 during the entire postvaccination period.
GMT was above 2,656 before challenge (55.1
months after vaccination). The hemagglutination
inhibition (HI) antibody GMT for the vaccinates
at this point was 844 (data not shown).
Canine Adenovirus Type 1 Response
All 10 vaccinates (100%) responded with
high SN titers after the first vaccination (GMT
= 388). The SN GMTs were maintained at high
levels during the entire postvaccination period
and until challenge (55.8 months after vaccination),
when the GMT was 274.
Canine Adenovirus Type 2 Response
All 10 vaccinates (100%) responded with
very high SN titers within 1 month of vaccination
(GMT > 3,385). The high SN titers were
maintained through the entire postvaccination
period and until challenge (GMT = 152).
Canine Distemper Virus Response
Nine of the 10 vaccinates (90%) responded
with high SN titers following the second vaccination.
One dog did not develop neutralizing
antibodies after vaccination. The highest SN
GMT was obtained 14 days after the second
vaccination (GMT >846). CDV-specific antibodies
were maintained for 56.5 months after
vaccination and until challenge (GMT = 24).
Canine Parvovirus Type 2b Challenge
Clinical Evaluation
Starting 5 days after CPV-2b challenge, the
young unvaccinated controls developed severe
clinical signs typical of parvovirus, including diarrhea,
bloody diarrhea, vomiting, dehydration,
and depression. One of the puppies died on day
7 after challenge, and the remaining four were
euthanized on the same day for humane reasons,
resulting in 100% mortality (Table 2, Figure 2).
Two of the adult controls showed sporadic clinical
signs, including inappetance, diarrhea, depression,
and vomiting, between days 5 and 8 after
challenge. Total average clinical score was
37.4 for the young controls and 1.8 for the adult
controls. By contrast, the vaccinates remained in
good health and none showed any clinical signs
(clinical score = 0).
Fecal Virus Shedding
Results of fecal virus shedding are shown in
Table 3 and Figure 3. All dogs in the two control
groups (100%) shed challenge virus between
days 4 and 8 after challenge. The highest shedding
was scored on day 6 (GMT = 4,096) for the
young controls (one day before death/euthanasia)
and on day 7 (GMT = 512) for the adult
controls (Table 3, Figure 3). None of the vaccinates
shed virus in feces as determined by HA.
Leukopenia
Measurement of total leukocytes (data not
shown) has shown that leukopenia was ob-
control developed severe clinical signs followed
by death on day 6 after challenge. All
three adult controls (100%) developed clinical
signs following challenge, and two of three
(66%) showed severe clinical signs, including
depression, dehydration, conjunctivitis, oral
hemorrhages, and/or petechiae. One of the
adult controls was euthanized on day 6 after
challenge. The total average daily score was 25
for adult controls and 17 for young controls.
By contrast, vaccinates remained in good
health and showed no clinical signs (clinical
score = 0).
Rectal Temperature
None of the dogs developed clinical fever after
challenge (data not shown).
Canine Distemper Virus Challenge
Clinical Evaluation
Average daily clinical score results are shown
in Table 5 and Figure 5. Following CDV challenge,
all five young unvaccinated control dogs
Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004
178
served in three of the five young controls
(60%) on day 7 after challenge. While no clinical
leukopenia (i.e., greater than 50% diminution
of circulating leukocytes from baseline
values) occurred in the adult controls, one dog
experienced significant reduction in leukocyte
count (37% below baseline value) on day 7 after
challenge. None of the vaccinates developed
leukopenia following challenge.
Rectal Temperature
One of the adult controls and two of the
young controls developed rectal temperatures
above 103.1°F. None of the vaccinates showed
temperatures above 103.1°F (data not shown).
Canine Adenovirus Type 1 Challenge
Clinical Evaluation
Average daily clinical scores are shown in
Table 4 and Figure 4. Following CAV-1 challenge,
four of the five young unvaccinated
control dogs developed moderate clinical signs
typical of canine hepatitis. The fifth young
TABLE 1. Serum-Neutralizing Antibody Response (Geometric Mean Serum-Neutralization
Titers) to CPV, CAV-1, CAV-2, and CDV After Vaccinationa
Months After Vaccination
Antigen 0 0.7 1.2 1.4 1.9 3.0 6.1 9.0 13.4
CPV <2 >3,956 >2,749 1,878 1,078 1,499 >1,814 >2,013 >2,352
CAV-1 <2 388 >712 664 431 >1,097 >2,352 >1,783 1,878
CAV-2 <2 >3,385 1,473 >2,312 558 >3,628 2,120 >3,051 >2,565
CDV <2 >68 >846 >494 68 126 243 104 99
ACf <2 <2 <2 <2 <2 <2 <2 <2 <2
aTiters were obtained for the vaccinate group (10 animals) against each antigen (CPV, CAV-1, CAV-2, and CDV [rows
1–4]) at the indicated months after first vaccination. Titers preceded by > sign were not end-pointed.
b22.6-month point for CPV.
cChallenge point = 55.1 months after vaccination for CPV, 55.8 months after vaccination for ICH, and 56.5 months after
vaccination for CDV.
O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz
179
(100%) developed severe clinical signs typical
of distemper. Clinical signs displayed included
ocular discharge, conjunctivitis, bloody diarrhea,
dehydration, depression, and vomiting.
One dog developed nervous signs. Four of the
five young controls (80%) were either eutha-
Months After Vaccination
Challenge Postchallenge
16.5 19.6b 25.7 31.7 36.7 50.4 53.0 Point c Time Point d
>3,866 >3,327 >3,888 >3,504 >2,947 >1,944 >3,214 >2,656 >2,272
737 609 630 568 344 469 356 274 3,327
>2,048 >1,399 955 342 299 274 289 152 3,158
122 124 85 99 64 23 ND 24 891
<2 <2 <2 <2 <2 <2 <2 <2 f
Figure 1. Serum-neutralizing antibody response to CPV, CAV-1, CAV-2, and CDV after vaccination. Refer to Table
1 for detailed description and antibody responses to the three antigens for the postchallenge (PC) time point. AC =
adult unvaccinated controls; C = challenge time point.
Vaccinates Developed High Levels of Serum-Neutralizing Antibodies
1
10
100
1,000
10,000
0
0.7
1.2
1.4
1.9
3.0
6.1
9.0
13.4
16.5
19.6
25.7
31.7
36.7
50.4
53.0
C
PC
Months Postvaccination
Geometric Mean Serum-Neutralization Titer
CPV CAV-1 CAV-2 CDV AC
dPostchallenge time point = 14 days for CPV-2b and 21 days for ICH and CDV.
eUnvaccinated adult controls (AC) used for CPV-2b challenge (four animals), ICH challenge (three animals), and CDV
challenge (four animals) remained seronegative (SN < 2) until challenge.
f Postchallenge antibody response of AC was >3,922 against CPV-2b, >4,096 against CAV-1, and 181 against CDV.
ND = no data.
Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004
180
TABLE 2. Average Daily (±SD) Clinical Score Following CPV-2b Challengea
Days After Challenge
Treatment Total
Group –2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Score
Adult 0 0 0 0 0 0 0 0.5 ± 1 0 0.5 ± 1 0.8 ± 1.5 0 0 0 0 0 0 1.8 ± 2.4
controls
Vaccinates 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Young 0 0 0 0 0 0 0 4.8 ± 0.8 7.6 ± 1.3 25 ± 0 All puppies died or were euthanized 37.4 ± 2.1
controls
aClinical signs observed following CPV-2b challenge in unvaccinated controls included vomiting, bloody diarrhea, dehydration, depression, and inappetance. No
clinical signs were observed in the vaccinates. Animals were observed for 14 days after challenge.
TABLE 3. Geometric Mean (±SD) Fecal Virus Shedding Titers Determined by Hemagglutination Assay Following
CPV-2b Challengea
Days After Challenge
Treatment
Group 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Adult 0 0 0 0 0 2 ± 16 23 ± 2,037 512 ± 2,328 45 ± 1,008 0 0 0 0 0 0
controls
Vaccinates 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Young 0 0 0 0 256 ± 194 891 ± 1,984 4,096 ± 0 All puppies died or were euthanizedb
controls
aFecal samples were evaluated for parvovirus challenge virus by hemagglutination assay. Titers were obtained for each group for each day after challenge.
bYoung controls had 100% mortality on day 7 after challenge.
0 = no virus detected at the lowest sample dilution (1:10).
O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz
181
nized or died between days 11 and 14 after
challenge. All four adult controls developed
typical distemper. Two of four (50%) died (one
on day 13 and one on day 16 after challenge).
By contrast, only one of the 10 vaccinates
showed significant distemper clinical signs,
and it recovered. Except for a mild and transient
ocular/nasal discharge for 1 day in one
Figure 2. Average daily clinical score following CPV-2b challenge. No clinical score recorded for young unvaccinated
controls (YC) beyond day 7 after challenge because of 100% mortality. AC = adult unvaccinated controls; SC =
subcutaneously vaccinated dogs.
Unvaccinated Controls Developed Severe Clinical Signs of Parvovirus
28
Days Postchallenge
Average Daily Score
0
4
8
12
16
20
24
–2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
YC SC AC
Figure 3. Geometric mean fecal virus shedding by hemagglutination assay (HA) following CPV-2b challenge. Fecal
samples were evaluated for parvovirus challenge virus by HA. Titers were obtained daily for each group after challenge.
Young controls (YC) had 100% mortality on day 7 after challenge. AC = adult unvaccinated controls; SC =
subcutaneously vaccinated dogs.
Control Dogs Shed Parvovirus After Challenge
4,400
4,000
3,600
3,200
2,800
2,400
2,000
1,600
1,200
800
400
0
Days Postchallenge
Hemoagglutination Virus Titer
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
YC SC AC
TABLE 4. Average (±SD) Daily Clinical Score Following CAV-1 Challengea
Days After Challenge
Treatment Total
Group –2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Score
Adult 0 0 0 0 0 0 0.7 ± 1.2 2.3 ± 2.5 13 ± 21.7 5.5 ± 4.9 3 ± 4.2 3 ± 4.2 2 ± 2.8 0.5 0 0 0 25.3 ± 21.7
controls
Vaccinates 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ±0
Young 0 0 0 0 0 0 0 2.6 ± 5.8 6.2 ± 10.6 5.1 ± 5.1 2.5 ± 1.9 1 ± 2 0.8 ± 1.5 0.5 ± 1 0.3 ± 0.5 0.3 ± 0.5 0.3 ± 0.5 17.3 ± 13
controls
aClinical signs observed following CAV-1 challenge in unvaccinated controls included nasal/ocular discharges, vomiting, oral hemorrhages, dehydration, depression, edema, conjunctivitis,
corneal opacity, and icterus signs. One of the three adult controls was euthanized on day 6 after challenge. One of the five young unvaccinated controls was found dead on day 6 after challenge.
No clinical signs were observed in the vaccinates. Animals were observed for 21 days after challenge.
TABLE 5. Average (±SD) Daily Clinical Score Following CDV Challengea
Days After Challenge
Treatment Total
Group –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Score
Adult 0 0 0 0 0 0 0 0 0.5 ± 1 0.5 ± 1 2.3 ± 2.9 3.3 ± 3.8 3.8 ± 2.5 5.8 ± 4.5 10.8 ± 9.9 6.7 ± 4.9 6.3 ± 5.5 10.3 ± 13.1 1.5 ± 2.1 45 ± 28.7
controls
Vaccinates 0 0 0 0.2 ± 0.6 0 0 0 0 0 0.2 ± 0.6 0.4 ± 1.3 0.6 ± 1.9 0.3 ± 0.9 0.1 ± 0.3 0.2 ± 0.6 0.1 ± 0.3 0 0 0 2.1 ± 6.0
Young 0 0 0 0 0 0 0 0 0.2 ± 0.4 3 ± 2.4 6 ± 0.7 7.4 ± 0.5 14.2 ± 9.9 13 ± 10.4 6 ± 4.2 12.5 ± 17.7 0 ND ND 46 ± 11.9
controls
aClinical signs observed following CDV challenge in unvaccinated controls included ocular discharges; vomiting; bloody diarrhea; dehydration; depression; central nervous system signs; conjunctivitis;
and emaciation. Two of the four adult controls (50%) died after the challenge (one on day 13 and one on day 16). Four of the five (80%) young unvaccinated controls died between
days 11 and 14 after challenge. One of the 10 vaccinates developed clinical signs for 7 days after challenge. A second vaccinate showed mild ocular discharge for 1 day.
ND = no observations were made for the surviving young control beyond day 15 after challenge.
O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz
183
dog, the remaining nine vaccinates (90%) were
normal throughout the postchallenge period.
The total average daily score was 45 for the
adult unvaccinated controls and 2.1 for the
vaccinates (Table 5).
Rectal Temperature
One young control puppy developed a rectal
temperature of 103.4°F after challenge. None
of the vaccinates developed a temperature
above 102.5°F (data not shown).
Figure 4. Average daily clinical score following CAV-1 challenge; also see Table 4. AC = adult unvaccinated controls;
SC = subcutaneously vaccinated dogs; YC = young unvaccinated controls.
Unvaccinated Control Dogs Developed Severe Clinical Signs of Canine Hepatitis Disease
Days Postchallenge
Average Daily Score
0
2
4
6
8
10
12
14
–2 –1 0 1 2 3 4 5 6 7 8 9
YC SC AC
10 11 12 13 14
Figure 5. Average daily clinical score following CDV challenge; also see Table 5. No further observations were made
for the surviving young unvaccinated controls (YC) beyond day 15 after challenge. AC = adult unvaccinated controls;
SC = subcutaneously vaccinated dogs.
Unvaccinated Control Dogs Developed Severe Clinical Signs of Distemper Disease
Days Postchallenge
Average Daily Score
0
2
4
6
8
10
12
14
16
–2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
YC SC AC
Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004
184
■ DISCUSSION
Results obtained from this study provided
evidence that vaccination of young (7- to 8-
week-old) susceptible puppies with a commercially
available combination vaccine containing
modified-live CPV-2b, CDV, and CAV-2 antigens
provided and maintained adequate immunity
for at least 4 years following vaccination.
Except for one dog’s response to CDV,
the three antigens induced high levels of SN
antibodies following vaccination, and those
levels persisted at protective levels21–24 for the
entire postvaccination period until challenge
(Table 1, Figure 1). HI antibody titers for CPV
were also high (GMT = 844) at 55 months after
vaccination (data not shown). The adult
unvaccinated control dogs housed in the same
facility remained seronegative, indicating lack
of extraneous exposure to these agents and
confirming that the immunity induced in the
vaccinates was from the vaccine. The CPV
challenge was confirmed adequate to measure
the efficacy of the vaccine because it resulted in
100% morbidity and mortality in the young
unvaccinated controls.
Adult controls experienced transient clinical
signs, including diarrhea, vomiting, and depression.
The difference in the severity of disease
between young and adult controls is expected
because of the difference in age susceptibility to
parvovirus infection.26 All vaccinates (100%)
were protected against clinical disease and mortality.
Furthermore, vaccinates were completely
protected against fecal virus shedding (Table 3,
Figure 3) and leukopenia (data not shown) in
contrast to unvaccinated controls that shed significant
amounts of virus and developed
leukopenia. These findings collectively indicate
that the vaccine was able to induce immunity
to CPV in dogs that lasted for at least 55
months following vaccination.
The subsequent CAV-1 challenge was confirmed
to have sufficient virulence to judge the
efficacy of the vaccine because it caused 100%
morbidity and 33% mortality in the adult unvaccinated
controls (Table 4, Figure 4). Vaccinates
remained in good health and showed no
clinical signs. The postchallenge antibody response
(GMT = 3,327 to CAV-1 and 3,158 to
CAV-2 [Table 1, Figure 1]) suggests the presence
of effective immunologic memory.
The experimental distemper challenge resulted
in 100% morbidity in the combined adult
and young controls, 80% mortality in the
young controls, and 50% mortality in the adult
controls, validating the severity of the challenge
and its adequacy to judge vaccine efficacy. Nine
of the 10 vaccinates (90%) were protected
against clinical signs associated with distemper
except for mild transient conjunctivitis and ocular
discharge in one dog for 1 day. One of the
vaccinates developed clinical signs of distemper,
including bloody diarrhea, depression, and
nervous signs, but fully recovered by day 14 after
challenge. This dog was a nonresponder and
did not mount an antibody response following
vaccination. Nonresponse to CDV vaccination
is not uncommon in the field, and nonresponders
constitute approximately 1% of cases (R.
S., personal observation).
Vaccination of young susceptible puppies with a
commercially available combination vaccine
provided and maintained adequate immunity
for at least 4 years following vaccination.
O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz
185
In summary, this report provided evidence
for the efficacy and duration of immunity of
this combination vaccine based on validated
vaccination and experimental challenge studies.
It is the first published report in which
dogs were maintained in a known virus-free
environment that supports more than 4 years
of immunity based on challenge evaluation under
controlled conditions. Data obtained from
challenge evaluation are far more reliable than
serologic response evaluation from field studies
previously described.19,20 Although serologic response
is a good correlate of protection for
each of these three antigens,21–24 extraneous
virus exposure to these agents is likely to occur
in dogs in the field, thereby compromising
findings. This report provides veterinarians
with critically needed information regarding
duration of immunity of commercial “core”
vaccines to help in assessing and designing appropriate
vaccination programs according to
the recently issued recommendations by
AAHA,15 COBTA,14 and published literature.16
■ CONCLUSION
The modified-live CPV-2b, CDV, and CAV-
2 fractions in the multivalent vaccine evaluated
in this study induced immunity in 7- to 8-
week-old susceptible puppies that persisted for
more than 4 years after vaccination. The immunity
in vaccinated dogs was evaluated
against virulent experimental sequential parvovirus,
infectious hepatitis, and distemper
challenges. All 10 vaccinates (100%) were protected
against clinical diseases and mortality
following parvovirus and infectious hepatitis
experimental infections. All vaccinates (100%)
were protected against mortality, and 90%
were protected against clinical disease following
distemper challenge. The data obtained
support at least 55 months’ duration of immunity
to the CPV-2b, CDV, and CAV-2 fractions
in the combination vaccine.
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