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Evaluation of the efficacy and duration of immunity of a

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Article in Veterinary Therapeutics: Research in Applied Veterinary Medicine · February 2004

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 O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz

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

aSchering-PloughAnimal Health bDepartment of Pathobiological Sciences

Research and Development School of Veterinary Medicine
21401 West Center Road University of Wisconsin-Madison
Elkhorn, NE 68022 2015 Linden Drive West
Madison, WI 53706

C L INI CAL R E L E VANCE

The results of this study confirmed that dogs vaccinated subcutaneously with a
commercially available multivalent vaccine containing modified-live canine dis-
temper 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 follow-
ing parvovirus and infectious canine hepatitis experimental infections. All vacci-
nates were protected against mortality and 90% against clinical disease follow-
ing distemper challenge. These data support at least a 4-year duration of
immunity for these three “core” fractions in the combination vaccine.

■ INTRODUCTION ic agent for CPV infections is a nonenveloped

Canine parvovirus (CPV), infectious canine DNA-containing virus, CPV type 2 (CPV-2).
hepatitis (ICH), and canine distemper virus The virus is very stable in the environment and
(CDV) infections are potentially fatal diseases highly contagious.1 In the United States, the
of dogs and other related species. The etiolog- CPV-2b genotype, through mutations,2 has
*This research was funded by Schering-Plough Ani-
largely replaced previously isolated genotypes
mal Health, Elkhorn, Nebraska. (CPV-2 and CPV-2a). In the Far East3,4 and

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Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004

Europe,5,6 both CPV-2a and CPV-2b may be each of these three antigens,21–24 the potential
present, but current published information is for environmental exposure to these viruses un-
not available. Canine distemper infection is der field conditions compromises the conclu-
caused by CDV, a contagious Morbillivirus be- sion that protection is solely due to vaccine-
longing to the Paramyxoviridae family. CDV induced antibodies.
affects Canidae and other carnivores such as This article presents vaccine efficacy and du-
pandas, raccoons, and large felids.7,8 ICH is ration of immunity data obtained from direct
caused by canine adenovirus type 1 (CAV-1). animal vaccination and experimental challenge
The virus is stable in the environment and af- studies. The vaccinated animals were housed
fects Canidae and Ursidae (bears).9 under high levels of biosecurity throughout the
Immunization of dogs through the use of holding period to prevent extraneous virus ex-
vaccination during the past decades has greatly posure. Control nonvaccinated dogs were
reduced the incidence of these infectious dis- maintained in the same facilities throughout

Safe and efficacious vaccines against CDV, CPV, and ICH

infections were recently designated as “core vaccines.”
eases in dog populations.10–12 Modified-live the period and were shown to remain antibody
vaccines were shown to induce superior and negative to the three viruses of interest.
longer-lasting immunity compared with inacti-
vated vaccines.9–11 Safe and efficacious vaccines ■ MATERIALS AND METHODS
against CDV, CPV, and ICH infections are Animals
considered essential vaccines that every dog Ten beagle puppies seronegative to CPV-2b,
should receive and were recently designated as CAV-1, canine adenovirus type 2 (CAV-2),
“core vaccines” by Schultz,13 the American Vet- and CDV were vaccinated at 7 to 8 weeks of
erinary Medical Association Council on Bio- age with two doses of vaccine given 3 weeks
logical and Therapeutic Agents (COBTA),14 apart; the puppies were held in isolation until
and the American Animal Hospital Association challenge: CPV-2b challenge was administered
(AAHA) Canine Vaccine Task Force.15 55.1 months after vaccination, CAV-1 chal-
Determination of vaccination frequency for lenge at 55.8 months, and CDV challenge at
the core vaccines was a subject of debate and 56.5 months. Seven age-matched unvaccinated
differing views among experts in the field.14,16–18 puppies were held separately at the same facil-
Veterinarians have to make decisions regarding ity as controls. Three of the seven controls were
the frequency of vaccination based on safety, ef- entered into the study at the time the other
ficacy, and duration of immunity as well as such dogs were vaccinated, and the remaining four
factors as animal susceptibility, animal environ- were introduced 5 months after vaccination.
ment, risk of exposure, breed involved, and These controls are referred to as “adult control
other considerations.14,16,17 To determine the dogs” in this article. An additional three sets of
duration of immunity of “core” vaccines, some five young beagle puppies were used as young
authors relied on serologic data obtained from susceptible controls to validate the virulence of
field studies.19,20 Although serologic response is each challenge (five per challenge) and are re-
a good indicator of the level of protection for ferred to as “young control puppies” in this ar-

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 O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz

ticle. All dogs in this study were treated ac- ditional sets of five young control puppies were
cording to animal welfare regulations outlined used to validate CAV-1 and CDV challenges
in 9 CFR, Subchapter A, Part 3, Subpart A. (five puppies per challenge).
Animal care and use committee (IACUC) ap-
proval was obtained at each site before the Experimental Challenge
study was conducted. Canine Parvovirus Type 2b Challenge
The 10 vaccinates, four adult controls, and
Vaccine five young control puppies were all challenged
The test vaccine is a commercially available oronasally (day 0) with virulent CPV-2b (ob-
multivalent freeze-dried vaccine (DA2PPv) tained from the USDA Center for Veterinary
containing modified-live CDV, CAV-2, canine Biologics). Each dog received 104.1 TCID50 of
parainfluenza virus (CPI), and CPV-2b anti- challenge virus. Inoculated dogs were observed
gens. The vaccine is marketed by Schering- for clinical signs, temperature, and mortality
Plough Animal Health in the United States, for 14 days after challenge. Whole blood (in
Canada, and South Africa under the trade EDTA tubes) and fecal swabs were collected
name Galaxy and in Europe and other markets daily to evaluate leukocyte counts and virus
under the trade names Procyon Dog or Quan- shedding, respectively.
tum Dog. The test vaccine was rehydrated with
liquid vaccine containing killed Leptospira cani- Canine Adenovirus Type 1 Challenge
cola, Leptospira icterohaemorrhagiae, and coro- After the vaccinates were rested for 1 week,
navirus antigens at the time of vaccination and they and three adult controls were inoculated
administered subcutaneously in 1-ml doses. intravenously (day 21) with CAV-1 (obtained
from the USDA Center for Veterinary Biolog-
Animal Trial Design ics). Five young control puppies were chal-
Before vaccination, puppies were blocked by lenged in a similar manner to validate the chal-
litter and gender and were randomly assigned lenge dose. Each dog received at least 103.3
as vaccinates or controls. Vaccinates (n = 10) TCID50 of challenge virus. The inoculated
received two doses of vaccine given 3 weeks dogs were observed daily for clinical signs for
apart and were held with the controls (n = 7) 21 days following challenge.
in an isolation facility in pens at Liberty Re-
search (Waverly, NY) for 55 months. During Canine Distemper Virus Challenge
this period, they were cared for and blood sam- Three weeks following CAV-1 challenge
ples were collected regularly to monitor their (day 42), the 10 vaccinates and four adult con-
antibody status. At the end of the holding pe- trols (the same adult controls used in the par-
riod, the designated vaccinates and adult con- vovirus challenge) were physically examined
trol dogs were transferred to the University of and determined to be fit before they were in-
Wisconsin animal facility in Madison, Wiscon- oculated intranasally and intravenously with
sin, for experimental challenge. The dogs were virulent CDV Snyder Hill challenge virus
acclimated for 8 days and determined to be fit (provided by R. S.). Five young (12-week-old)
before challenge inoculation. Five 12-week- control puppies were challenged separately in
old, CPV-2b-seronegative puppies (young con- advance to validate the challenge virus viru-
trol puppies) arrived at the facility at the same lence and were observed for 15 days following
time for use in the CPV-2b challenge. Two ad- challenge. Inoculated adult vaccinates and con-

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Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004

trols were observed daily for clinical signs typ- pressed as the reciprocal of the dilution, which
ical of distemper disease for 21 days after chal- neutralized 50% of the virus, as calculated by
lenge. the Spearman–Karber method.25 For purpose
of analysis, SN titers below 2 were given a val-
Serum Neutralization ue of 1. Antibody titers that were not end-
Antibody responses to CPV, CAV-1, CAV-2, pointed at dilution 1:4,096 were given the val-
and CDV were measured by serum-neutraliza- ue of 4,096. Geometric mean titers (GMTs)
tion (SN) assays performed by a microtitration obtained in these cases were indicated as
method using flat-bottomed 96-well microtiter >GMT value.
plates. Twofold serial dilutions of serum sam-
ples were prepared in Dulbecco’s modified Ea- Detection of Canine Parvovirus in Fecal
gle’s medium (DMEM) supplemented with 2 Samples by Hemagglutination Test
mm L-glutamine and gentamicin (50 µg/ml). CPV in rectal swabs was quantified by the
Four wells of a microtiter plate were inoculated hemagglutination assay (HA) using 96-well
with 50 µl of neat or diluted serum from each plates. Briefly, the swabs were stored in tubes
dilution. An equal volume (50 µl) of the specif- containing 1 ml of phosphate-buffered saline
ic SN challenge virus (CPV, CAV-2, CAV-1, or (PBS) and frozen until thawed for extraction.

Following vaccination, the vaccinates developed high

levels of SN antibodies to all fractions.

CDV [distemperoid strain]) containing 50 to At extraction, tubes were vortexed, swabs were
300 log10 TCID50 was added to each well. After removed, and 500 µl chloroform was added.
30 to 60 minutes of incubation at 36 ± 2˚C, Tubes were vortexed intermittently for 15 min-
cell substrate (dog kidney cell in the case of utes and centrifuged, and the supernatant was
CPV, CAV-1, and CAV-2; Vero cells in the case removed and stored frozen until tested. The
of CDV) was added to each well (seeded at ap- sample was twofold serially diluted (in PBS
proximately 1 to 2 × 104 cells/0.1 ml/well in with bovine serum albumin) in duplicates of
DMEM supplemented with 2 mm L-gluta- wells of a round-bottomed 96-well plate. After
mine, gentamicin [50 µg/ml], and 5% fetal sample dilutions were made, 1% of washed
bovine serum). Plates were incubated in a hu- porcine erythrocytes was added to each well
midified carbon dioxide (4% to 6 %) incubator and plates were refrigerated for 4 to 8 hours.
for 3 days (CPV) or 5 to 7 days (CAV-1, CAV- Titers were calculated as the reciprocal of the
2, and CDV) at 36 ± 2˚C. highest dilution of sample that produced com-
At the end of the incubation period, SN an- plete agglutination. Positive (sample with
tibody response was determined by examina- known titer) and negative (sample with no
tion of wells for typical CDV or CAV cyto- virus) controls were used to monitor the assay.
pathic effect. For CPV, plates were fixed with
80% acetone (100 µl/well) and stained with Leukocyte Counts
CPV-specific fluorescein-labeled antibody con- Leukocyte counts were determined using an
jugate (75 µl/well). SN antibody titers were ex- ADVIA 120 Hematology System (Bayer

176
 O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz

HealthCare, Tarrytown, NY). The EDTA Canine Distemper Virus Response

blood sample was mixed with ADVIA 120 Nine of the 10 vaccinates (90%) responded
BASO solution containing acid and surfactant. with high SN titers following the second vacci-
After erythrocytes were hemolyzed, the leuko- nation. One dog did not develop neutralizing
cyte counts were analyzed using two-angle laser antibodies after vaccination. The highest SN
light scatter signals. GMT was obtained 14 days after the second
vaccination (GMT >846). CDV-specific anti-
■ RESULTS bodies were maintained for 56.5 months after
Antibody Response vaccination and until challenge (GMT = 24).
All puppies were confirmed to be seronega-
tive to CPV, CAV-1, CAV-2, and CDV before Canine Parvovirus Type 2b Challenge
vaccination (Table 1, Figure 1). Following vac- Clinical Evaluation
cination, the vaccinates developed high levels Starting 5 days after CPV-2b challenge, the
of SN antibodies to all fractions. The control young unvaccinated controls developed severe
dogs remained seronegative throughout the clinical signs typical of parvovirus, including di-
holding period, indicating lack of extraneous arrhea, bloody diarrhea, vomiting, dehydration,
exposure to these agents. and depression. One of the puppies died on day
7 after challenge, and the remaining four were
Canine Parvovirus Type 2b Response euthanized on the same day for humane reasons,
All 10 vaccinates (100%) responded with resulting in 100% mortality (Table 2, Figure 2).
very high SN titers, with the highest GMT Two of the adult controls showed sporadic clini-
obtained 1 month after vaccination (GMT cal signs, including inappetance, diarrhea, de-
>3,956). The GMT was maintained above pression, and vomiting, between days 5 and 8 af-
1,078 during the entire postvaccination period. ter challenge. Total average clinical score was
GMT was above 2,656 before challenge (55.1 37.4 for the young controls and 1.8 for the adult
months after vaccination). The hemagglutina- controls. By contrast, the vaccinates remained in
tion inhibition (HI) antibody GMT for the vac- good health and none showed any clinical signs
cinates at this point was 844 (data not shown). (clinical score = 0).

Canine Adenovirus Type 1 Response Fecal Virus Shedding

All 10 vaccinates (100%) responded with Results of fecal virus shedding are shown in
high SN titers after the first vaccination (GMT Table 3 and Figure 3. All dogs in the two control
= 388). The SN GMTs were maintained at high groups (100%) shed challenge virus between
levels during the entire postvaccination period days 4 and 8 after challenge. The highest shed-
and until challenge (55.8 months after vacci- ding was scored on day 6 (GMT = 4,096) for the
nation), when the GMT was 274. young controls (one day before death/euthana-
sia) and on day 7 (GMT = 512) for the adult
Canine Adenovirus Type 2 Response controls (Table 3, Figure 3). None of the vacci-
All 10 vaccinates (100%) responded with nates shed virus in feces as determined by HA.
very high SN titers within 1 month of vaccina-
tion (GMT > 3,385). The high SN titers were Leukopenia
maintained through the entire postvaccination Measurement of total leukocytes (data not
period and until challenge (GMT = 152). shown) has shown that leukopenia was ob-

177
Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004

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
AC f <2 <2 <2 <2 <2 <2 <2 <2 <2
a Titerswere 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.
b 22.6-month point for CPV.
c Challenge point = 55.1 months after vaccination for CPV, 55.8 months after vaccination for ICH, and 56.5 months af-

ter vaccination for CDV.

served in three of the five young controls control developed severe clinical signs fol-
(60%) on day 7 after challenge. While no clin- lowed by death on day 6 after challenge. All
ical leukopenia (i.e., greater than 50% diminu- three adult controls (100%) developed clinical
tion of circulating leukocytes from baseline signs following challenge, and two of three
values) occurred in the adult controls, one dog (66%) showed severe clinical signs, including
experienced significant reduction in leukocyte depression, dehydration, conjunctivitis, oral
count (37% below baseline value) on day 7 af- hemorrhages, and/or petechiae. One of the
ter challenge. None of the vaccinates devel- adult controls was euthanized on day 6 after
oped leukopenia following challenge. challenge. The total average daily score was 25
for adult controls and 17 for young controls.
Rectal Temperature By contrast, vaccinates remained in good
One of the adult controls and two of the health and showed no clinical signs (clinical
young controls developed rectal temperatures score = 0).
above 103.1˚F. None of the vaccinates showed
temperatures above 103.1˚F (data not shown). Rectal Temperature
None of the dogs developed clinical fever af-
Canine Adenovirus Type 1 Challenge ter challenge (data not shown).
Clinical Evaluation
Average daily clinical scores are shown in Canine Distemper Virus Challenge
Table 4 and Figure 4. Following CAV-1 chal- Clinical Evaluation
lenge, four of the five young unvaccinated Average daily clinical score results are shown
control dogs developed moderate clinical signs in Table 5 and Figure 5. Following CDV chal-
typical of canine hepatitis. The fifth young lenge, all five young unvaccinated control dogs

178
 O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz

Months After Vaccination

Challenge Postchallenge
16.5 19.6 b 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

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.

Vaccinates Developed High Levels of Serum-Neutralizing Antibodies

Geometric Mean Serum-Neutralization Titer

10,000

1,000

100

10
CPV CAV-1 CAV-2 CDV AC

1
C
.6
.4

.5

.7

.7

.7

.4

.0
7

PC
0

0.

1.

1.

1.

3.

6.

9.

19
13

16

25

31

36

50

53

Months Postvaccination
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.

(100%) developed severe clinical signs typical rhea, dehydration, depression, and vomiting.
of distemper. Clinical signs displayed included One dog developed nervous signs. Four of the
ocular discharge, conjunctivitis, bloody diar- five young controls (80%) were either eutha-

179
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
Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004

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
aFecalsamples 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

Unvaccinated Controls Developed Severe Clinical Signs of Parvovirus

28
24 AC SC YC
Average Daily Score

20
16

12
8
4
0
–2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days Postchallenge

Figure 2. Average daily clinical score following CPV-2b challenge. No clinical score recorded for young unvaccinat-
ed controls (YC) beyond day 7 after challenge because of 100% mortality. AC = adult unvaccinated controls; SC =
subcutaneously vaccinated dogs.

Control Dogs Shed Parvovirus After Challenge

4,400
4,000
Hemoagglutination Virus Titer

AC SC YC
3,600
3,200
2,800
2,400
2,000
1,600
1,200
800
400
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days Postchallenge

Figure 3. Geometric mean fecal virus shedding by hemagglutination assay (HA) following CPV-2b challenge. Fe-
cal samples were evaluated for parvovirus challenge virus by HA. Titers were obtained daily for each group after chal-
lenge. Young controls (YC) had 100% mortality on day 7 after challenge. AC = adult unvaccinated controls; SC =
subcutaneously vaccinated dogs.

nized or died between days 11 and 14 after By contrast, only one of the 10 vaccinates
challenge. All four adult controls developed showed significant distemper clinical signs,
typical distemper. Two of four (50%) died (one and it recovered. Except for a mild and tran-
on day 13 and one on day 16 after challenge). sient ocular/nasal discharge for 1 day in one

181
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
aClinicalsigns 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; con-
junctivitis; 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

Unvaccinated Control Dogs Developed Severe Clinical Signs of Canine Hepatitis Disease
14

12 AC SC YC
Average Daily Score

10

0
–2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days Postchallenge

Figure 4. Average daily clinical score following CAV-1 challenge; also see Table 4. AC = adult unvaccinated con-
trols; SC = subcutaneously vaccinated dogs; YC = young unvaccinated controls.

Unvaccinated Control Dogs Developed Severe Clinical Signs of Distemper Disease

16

14 AC SC YC

12
Average Daily Score

10

0
–2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Days Postchallenge

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 con-
trols; SC = subcutaneously vaccinated dogs.

dog, the remaining nine vaccinates (90%) were Rectal Temperature

normal throughout the postchallenge period. One young control puppy developed a rectal
The total average daily score was 45 for the temperature of 103.4˚F after challenge. None
adult unvaccinated controls and 2.1 for the of the vaccinates developed a temperature
vaccinates (Table 5). above 102.5˚F (data not shown).

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Veterinary Therapeutics • Vol. 5, No. 3, Fall 2004

■ DISCUSSION contrast to unvaccinated controls that shed sig-

Results obtained from this study provided nificant amounts of virus and developed
evidence that vaccination of young (7- to 8- leukopenia. These findings collectively indicate
week-old) susceptible puppies with a commer- that the vaccine was able to induce immunity
cially available combination vaccine containing to CPV in dogs that lasted for at least 55
modified-live CPV-2b, CDV, and CAV-2 anti- months following vaccination.
gens provided and maintained adequate im- The subsequent CAV-1 challenge was con-
munity for at least 4 years following vaccina- firmed to have sufficient virulence to judge the
tion. Except for one dog’s response to CDV, efficacy of the vaccine because it caused 100%
the three antigens induced high levels of SN morbidity and 33% mortality in the adult un-
antibodies following vaccination, and those vaccinated controls (Table 4, Figure 4). Vacci-
levels persisted at protective levels21–24 for the nates remained in good health and showed no
entire postvaccination period until challenge clinical signs. The postchallenge antibody re-
(Table 1, Figure 1). HI antibody titers for CPV sponse (GMT = 3,327 to CAV-1 and 3,158 to
were also high (GMT = 844) at 55 months af- CAV-2 [Table 1, Figure 1]) suggests the pres-
ter vaccination (data not shown). The adult ence of effective immunologic memory.
unvaccinated control dogs housed in the same The experimental distemper challenge result-
facility remained seronegative, indicating lack ed in 100% morbidity in the combined adult

Vaccination of young susceptible puppies with a

commercially available combination vaccine
provided and maintained adequate immunity
for at least 4 years following vaccination.
of extraneous exposure to these agents and and young controls, 80% mortality in the
confirming that the immunity induced in the young controls, and 50% mortality in the adult
vaccinates was from the vaccine. The CPV controls, validating the severity of the challenge
challenge was confirmed adequate to measure and its adequacy to judge vaccine efficacy. Nine
the efficacy of the vaccine because it resulted in of the 10 vaccinates (90%) were protected
100% morbidity and mortality in the young against clinical signs associated with distemper
unvaccinated controls. except for mild transient conjunctivitis and oc-
Adult controls experienced transient clinical ular discharge in one dog for 1 day. One of the
signs, including diarrhea, vomiting, and depres- vaccinates developed clinical signs of distemper,
sion. The difference in the severity of disease including bloody diarrhea, depression, and
between young and adult controls is expected nervous signs, but fully recovered by day 14 af-
because of the difference in age susceptibility to ter challenge. This dog was a nonresponder and
parvovirus infection.26 All vaccinates (100%) did not mount an antibody response following
were protected against clinical disease and mor- vaccination. Nonresponse to CDV vaccination
tality. Furthermore, vaccinates were completely is not uncommon in the field, and nonrespon-
protected against fecal virus shedding (Table 3, ders constitute approximately 1% of cases (R.
Figure 3) and leukopenia (data not shown) in S., personal observation).

184
 O. Y. Abdelmagid, L. Larson, L. Payne, A. Tubbs, T. Wasmoen, and R. Schultz

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