Vaccine 19 (2001) 4307– 4317

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Clinical and virological evaluation of the efficacy of an inactivated

EHV1 and EHV4 whole virus vaccine (Duvaxyn EHV1,4).
Vaccination/challenge experiments in foals and pregnant mares
Jacobus G.M. Heldens a,*,1, Duncan Hannant d, Ann A. Cullinane b,
Michael J. Prendergast c, Jennifer A. Mumford d, Maura Nelly b, Julia H. Kydd d,
Marien W. Weststrate a, Rene van den Hoven a,2
a
Fort Dodge Animal Health Holland, Department of Bio R&D, C.J. 6an Houtenlaan 36, 1381 CP Weesp, The Netherlands
b
Irish Equine Centre, Johnstown, Naas, Co. Kildare, Ireland
c
Ash Stream Ltd, Hollymount Claremorris, Co. Mayo, Ireland
d
Animal Health Trust, P.O.B. 5, Newmarket, Suffolk CB8 7DW, UK
Received 4 September 2000; received in revised form 9 March 2001; accepted 22 March 2001

Abstract

Pregnant mares and young foals were vaccinated with Duvaxyn EHV1,4, an inactivated and adjuvanted vaccine containing both
the EHV-1 and 4 antigens. SN and CF antibody titres were induced two weeks after first vaccination. Antibody levels were
boosted after second vaccination, however they never reached the levels induced after virus challenge. Young foals were
challenged with virulent EHV-1 and EHV-4 field viruses. Pregnant mares were challenged with the highly abortigenic EHV-1
strain Ab4. Vaccinated animals showed a clear reduction in clinical signs and virus excretion compared to unvaccinated control
animals. Log transformed antibody levels could be correlated to duration of virus excretion. The incidence of EHV-1 induced
abortions was drastically reduced in vaccinated mares. Therefore, although vaccinated animals are not fully protected against
disease, Duvaxyn EHV1,4 clearly reduces clinical symptoms, the duration of virus shedding and the quantity of virus shed. It can
be concluded that vaccination of foals and pregnant mares with Duvaxyn EHV1,4 significantly reduces the risk of abortions and
outbreaks of respiratory disease caused by circulating field viruses. © 2001 Elsevier Science Ltd. All rights reserved.

1. Introduction were distinct virus species [1–3]. Nevertheless, the de-

Equine herpesvirus type 1 (EHV-1) and equine her-
pesvirus type 4 (EHV-4) are alpha herpesviruses and
are genetically related to bovine herpesvirus 1 (BHV-1),
herpes simplex viruses 1 and 2 (HSV-1 & HSV-2) and
pseudo rabies virus (PRV). Originally EHV-1 and
EHV-4 were considered variants of the same virus
species, but restriction endonuclease and nucleotide se-
quence analysis of their genomes confirmed that they
* Corresponding author. Tel.: + 31-485-587600; fax: +31-485-
577333.
E-mail address: jacco.heldens@intervet.com (J.G.M. Heldens).
1
Present address: Intervet International BV, P.O. Box 31, 5830 AA
Boxmeer, The Netherlands.
2 nodes and limb oedema [4]. Infected horses shed large
Present address: 1st Medical Clinic for Ungulates and Small
Animals, Veterinärmedizinische Universität Wien, Veterinärplatz 1,
A-1210 Wien, Austria.
duced amino acid sequences of many EHV genes show
high homology between EHV-1 and EHV-4 and less
but significant relatedness to BHV-1 and HSV-1 and 2.
EHV-1 and EHV-4 are endemic in horse populations
worldwide and cause significant economic loss due to
respiratory disease, abortion and more rarely paralysis.
EHV-1 and EHV-4 enter the host via the mucosa of the
respiratory tract. The first cycle of replication takes
place in the tissues of upper respiratory tract especially
in cells from the nasopharynx. Infection of young im-
munologically naı̈ve horses with either EHV-1 or EHV-
4 results in an acute respiratory disease characterised by
fever, anorexia, ocular and nasal discharge, coughing,
enlarged intermandibular and parapharyngeal lymph
amounts of virus, which can be recovered from the
nasopharynx for up to 12 days post infection. The viral

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PII: S0264-410X(01)00131-1
4308 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317
titres in the nasal fluid may vary considerably depend-
ing on the strain of challenge virus [5– 7].
Equine herpesvirus abortion occurs annually in
horse populations worldwide. Most outbreaks consists
of only one or two abortions but abortion storms
sometimes occur resulting in the loss of many foals [8].
Abortion usually occurs in the last trimester of preg-
nancy and may be due to the reactivation of latent
virus.
Both EHV-1 and EHV-4 can induce abortion. How-
ever the majority of abortions are caused by EHV-1.
EHV-4 is primarily associated with respiratory disease.
It is occasionally associated with sporadic abortion and
rarely associated with neurological disorders [4]. EHV-
1 is more endotheliothropic than EHV-4 and the cell
associated viraemia which characterises EHV-1 infec-
tions permits systemic spread of virus to additional
sites of replication including the endothelium of blood
vessels in the endometrium and the central nervous
system [9– 11].
EHV-1 neurological disease is less common than
abortion but has been recorded all over the world. The
symptoms vary from mild ataxia to paresis and are
caused by damage to the endothelium of vessels in the
spinal cord and brain resulting in anoxia [12–16].
These clinical signs have been reproduced experimen-
tally in horses by virus challenge [17].
Both EHV-1 and EHV-4 induce Serum Neutralisa-
tion (SN) and Complement Fixation (CF) antibodies in
infected horses. High SN titres are detected in the
serum of naturally infected animals for several months.
In contrast CF titres generally decline within weeks of
infection. Traditionally, SN antibody levels were con-
sidered as an indicator of protection against EHV
disease [18,19]. Later however it became clear that
horses with high vaccine– induced SN antibody titres
were not protected against the disease on re-infection
[20]. Furthermore it was clearly demonstrated that un-
der field conditions there was no correlation between
SN and CF antibody levels and the clinical signs or
virological findings post challenge [21]. The current
knowledge of the cell-associated basis of the pathogen-
esis of these viruses clearly indicates that other compo-
nents of the immune systems such as local immunity
and cell mediated immunity play a major role in the
protection of the horse against EHV induced diseases
[21,22, review, 9,5].
The inability of classical inactivated or modified life
vaccines to provide complete protection against equine
herpesvirus associated diseases has stimulated research
into alternative vaccination approaches ([22], review).
Recombinant DNA techniques enabled the develop-
ment of DNA and subunit vaccines ([12] for review)
and vaccines based on EHV-deletion mutant strains
[23,24]. Some of these experimental vaccines appeared
to be safe, were capable of inducing high antibody
titres in horses and were protective against virulent
EHV challenges in a mouse model [12,25–27]. It
should be noted however that the real value of these
vaccines can only be assessed in virus challenge experi-
ments in horses [21]. Until the efficacy of these novel
vaccines, and in case of recombinant live vaccines their
safety in relation to animals and the environment, have
been thoroughly investigated, classical approaches us-
ing inactivated virus and new adjuvants have to be
pursued.
The main objective of vaccination is to prevent an
outbreak of EHV associated disease. The ability of a
vaccine not only to minimise clinical symptoms but
also to reduce the amount of virus excreted and the
risk of viral spread are pivotal in achieving this goal.
This paper describes the assessment of the efficacy of
Duvaxyn EHV1,4, a whole virus, carbomer adjuvanted,
inactivated, equine herpesvirus vaccine containing both
EHV-1 and 4 virus strains in this perspective.
2. Materials and methods
2.1. Vaccines and 6accination schemes
All horses were vaccinated with Duvaxyn EHV1,4
by deep intramuscular injection in accordance with the
datasheet. The foals received two vaccinations with an
interval of 4 weeks. Pregnant mares were vaccinated in
months 5, 7–9 of gestation.
2.2. Viruses
The highly pathogenic Ab4 EHV-1 strain, isolated
by [28] from the nasal turbinate of a quadriplegic mare
was used to challenge the pregnant mares. The virus
was propagated on primary equine embryonic lung
(EEL) cells for 8 passages and stored at − 70°C.
Another pathogenic EHV-1 strain 121412 isolated
from the lung of an aborted fetus in 1997 (A.A. Culli-
nane, unpublished data) was used to challenge 15 foals.
The virus was passaged for 6 passages in EEL cells and
stored at − 70°C.
An EHV-4 strain 122324 isolated from a nasopha-
ryngeal swab collected from a horse suffering from
respiratory disease in 1998 (A. A. Cullinane, unpub-
lished data) was used to challenge 15 foals. The virus
was propagated on EEL cells for 11 passages and
stored at − 70°C.
2.3. Animals and animal management
Thirty recently weaned animals both fillies and colts
were used in the foals trial. One foal in a control group
had to be withdrawn due to colic shortly before chal-
 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317
lenge. At the time of challenge all foals were between 5
and 8 months of age. The foals were of common
Irish breeds, such as pure and crossbred Connemara
ponies, draft horses and hunters. At the commence-
ment of the trial, care was taken to include only foals
that were free of VN or CF antibodies against EHV-1
and EHV-4. At the start of the trial the 30 foals were
randomly allocated to one of 4 groups. Two groups of
5 were left unvaccinated and 2 groups of 10 were
vaccinated. One day before challenge a vaccinated
group and a control group were transferred to a barn
approximately 2 miles away from the remaining ponies
to prevent cross contamination with the other chal-
lenge virus. The ponies in one location were challenged
with EHV-1 and those in the other location were
challenged with EHV-4. The controls were kept in a
separate box to the vaccinates to prevent nose to nose
contact.
Nine pregnant Welsh mountain ponies were used in
the abortion trial. The mares were approximately 36
months of age at the start of the study. They had no
evidence of previous experimental infection or vaccina-
tion histories for EHV1/4. However, they were all
previously primed by natural exposure(s) to these ubiq-
uitous viruses as evidenced by the detection of serum
CF and VN antibody titres [29] before the vaccination
programme began. Blood samples were collected from
the mares 6 times before their first vaccination for
serological screening to ensure that they had not been
exposed to either EHV-1 or EHV-4 within the four
months prior to the start of the study. All mares had
been vaccinated before the start of the trial against
tetanus. Prior to challenge the 5 vaccinated mares were
kept together with the 4 control animals. 3 days before
challenge the mares were separated and housed indi-
vidually.
In both trials the diet consisted of pasture grass, hay
or grass silage ad libitum. Drinking water from public
water works was also supplied ad libitum by automatic
drinkers.
2.4. Challenge
All foals were challenged two weeks after the second
vaccination. Each animal received 2 ml of an aerosol
containing105.0 TCID50 EHV-1 or 106 TICD50 EHV-4
intranasally. The aerosol was produced by inserting a
plastic nozzle attached to a syringe in the nostrils and
subsequently emptying the syringe contents under high
pressure. After challenge ponies were kept in close
confinement for 1 h to facilitate circulation of virus.
The pregnant mares were challenged 4 weeks after
the third vaccination. Each mare received 2 ml of an
aerosol containing 106.0 TCID50 per ml of the EHV-1
Ab4 isolate intranasally.
4309
2.5. Serological tests
Blood samples were collected from the foals for
serological examination on the day of first vaccination,
7 and 14 days after first vaccination and on the day of
second vaccination. Further samples were collected on
the day of challenge and on days 3, 7, 10, 14 and 21
post challenge.
Blood samples were collected from the mares for
serological examination on the day of first vaccination,
2 and 4 weeks after first vaccination, on the day of
second vaccination, 2 weeks after second vaccination,
on the day of third vaccination and two weeks after
third vaccination. Further samples were collected on
the day of challenge and 2 and 4 weeks post challenge.
Complement Fixation (CF) and Virus Neutralisation
(VN) tests were carried out in principle as described by
[29]. The appropriate homologous antigens were used
to establish the antibody responses induced by the
different viruses. CF titres were expressed as the recip-
rocal of the highest dilution of serum displaying 50%
haemolysis, whereas VN titres were expressed as the
reciprocal of the serum dilution displaying cytopathic
effect in 50% of the wells of a particular dilution.
2.6. Virus isolation from nasal swabs and white blood
cells
Virus shedding was monitored the day before virus
challenge, the day of virus challenge and for 14 days
post challenge in the foal trial and for 10 days post
challenge in the pregnant mares. Nasopharyngeal
swabs were collected and immersed immediately in
virus transport medium. Swabs were transported to the
laboratories in contact with ice packs or on wet ice.
Swab extracts were titrated on rabbit kidney cells
(RK13) for the growth of EHV-1 or on EEL cells for
the growth of EHV-4.
Viraemia was monitored on alternate days for 14
days post challenge in the foals challenged with EHV-1
and for 21 days post challenge in the mares. The
leucocytes from heparinised blood samples were cocul-
tivated with RK13 cells as described by [21].
Samples of lung, liver, thymus and spleen from
aborted foetuses and stillborn foals were pooled, ho-
mogenised and inoculated onto RK13 cells.
The identity of all viruses isolated was confirmed by
immunofluorescence using type specific monoclonal an-
tibodies [30].
2.7. Clinical monitoring
Following each vaccination all horses were observed
for evidence of adverse local and/or systemic reactions
to vaccination.
4310 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317

On the day before challenge, the day of challenge and
for 21 days post challenge the foals were clinically
examined and scored as follows.
Appetite: Animal immediately eating the concen-
trates offered − 0 points. Poorly interested in feed, but
still coming after delay of 1 minute to the bucket or
trough −1 point. Not interested in feed − 2 points.
Demeanour: Active, alert and bright − 0 points.
Depressed animals − 1 point. Recumbent but still ca-
pable getting to their feet after stimulation − 2 points.
Paralysed or dead animal −10 points.
Respiration: normal respiration (\ 20/min) − 0
points. Mildly increased rate (20 to B 40/min) − 1
point. Severely increased (\40/min) −2 points.
Nasal discharge: Serous nasal discharge: no discharge
− 0 points. Slight − 1 point. Moderate − 2 points.
Severe −3 points. Mucopurulent discharge: no dis-
charge −0 points. Some − 1.5 points. Moderate − 3
points. Severe − 4.5 points.
Ocular discharge: Serous ocular discharge: no dis-
charge − 0 points. Slight −1 point. Moderate − 2
points. Severe −3 points. Mucopurulent discharge: no
discharge −0 points. Slight −1.5 points. Moderate
− 3 points. Severe −4.5 points.
Coughing: no coughing −0 points. Induced by lar-
ynx palpation − 1 point. Infrequent cough just 1–2
times per occasion − 2 points. Severe cough heard
more than 2 times on each occasion − 3 points.
Mandibular lymph node enlargement: hardly palpa-
ble node −0 points. Cherry sized node − 1 point.
Walnut sized node − 2 points. Mandarin sized node
− 3 points.
The daily clinical score was the sum of the daily
symptom scores of each horse. Rectal temperatures
were taken daily from day 3 before challenge through
to day 14-post challenge. Temperatures \38.8°C were
regarded as abnormal.
Following challenge the mares were observed twice
daily to parturition for nasal and ocular discharge,
coughing, sub-mandibular lymph node enlargements,
ataxia and abortions. Rectal temperatures were taken
daily from the day before challenge through day 10-
post challenge. After parturition foals were monitored
for at least 2 weeks for abnormalities. Aborted and
stillborn foals were subjected to gross and histopatho-
logical examination.
2.8. Statistical analysis
Clinical scores, duration of symptoms, viraemia and
virus excretion and non-transformed titres were
analysed by Mann-Whitney U-test. Two-tailed P-tests
were reported. Rectal temperatures were analysed by
ANOVA, contrasts on day 2 and day 6 were subse-
quently analysed by Student t-test. The Fisher’s exact
test was used for analysis of proportions.
3. Results

3.1. Vaccination of foals to pre6ent respiratory disease

Table 1 and general malaise
Summary of clinical and virological observations after EHV-1 and
EHV-4 challenge in vaccinated and unvaccinated control groups
( 9 SD) 3.1.1. Febrile Response post challenge
Rectal temperatures were measured for 14 days post
Parameter/Group V-EHV-1 C-EHV-1 V-EHV-4 C-EHV-4 viral challenge. Mean group temperatures were calcu-
Mean rectal temp 39.0 90.5 39.4 90.7 38.6 9 0.4 38.9 9 0.6 lated (Table 1; Fig. 1). The mean number of days of
Mean Total 51.4 9 17.8 68.9 916.3 36.1 9 14.4 62.6 9 14.2
pyrexia (T] 38.8°C) in the week after EHV-1 challenge
clinical score
Mean group titre 0.70 9 0.92 1.83 90.84 0.45 9 0.43 1.10 9 0.19 in the vaccinated group was 6.3 days versus 7.2 days in
the control group. This difference in duration of fever
Clinical parameter/patient days in group
–Appetite 0.4 0.6 0 0
was not significant (P= 0.13).
0.1 0.2 0 0 Two-way analysis of variance (ANOVA) of the tem-
–Demeanour 4.7 7 0.9 2.25 peratures over the week after EHV-1 challenge showed
–Respiratory signs a significant individual (P= 0.005) and time (PB0.005)
–Nasal discharge 16.5 18 13.5 21.25 effect. Subsequent analysis of the temperatures at day 2
–Ocular discharge 0.4 0.6 0 0.25 and day 6 showed significant differences between the
–Cough 6.4 8 5.5 11.5 vaccinates and the controls (PB0.005 for day 2 and
–Lymph node 13.6 14.2 11.7 17.75
P= 0.011 for day 6).
swelling
The mean number of days with fever (T] 38.7°C)
1
V =Vaccinated animals; C =Unvaccinated control animals. from 1 to 8 days after EHV-4 challenge in the vaccine
Rectal temperatures (°C) during the first week after challenge are group was 2.1 days versus 3 days in the control group.
depicted. The cumulative clinical score is for the entire observation This difference in duration of fever was not significant
period. Mean group titre (10log) represents the mean titre of the virus
isolated from nasal swabs, between the 2nd and 14th day post EHV
(P= 0.23).
challenge. Total patients-days indices were calculated for symptoms Two-way ANOVA of the temperatures over the week
indicated. after EHV-4 challenge showed a significant individual
 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317
Fig. 1. Daily mean rectal temperature. Group V-EHV-1 and V-EHV-
4 were the vaccinated groups, C-EHV-1 and C-EHV-4 were the
unvaccinated (control) groups. Group C-EHV-1 and V-EHV-1 were
challenged with EHV-1, Groups C-EHV-4 and V-EHV-4 were chal-
lenged with EHV-4.
Fig. 2. Daily mean clinical score. Group V-EHV-1 and V-EHV-4
were the vaccinated groups, C-EHV-1 and C-EHV-4 were the unvac-
cinated (control) groups. Group C-EHV-1 and V-EHV-1 were chal-
lenged with EHV-1, Groups C-EHV-4 and V-EHV-4 were challenged
with EHV-4.
(P B 0.001) and time (P B 0.001) effect. Subsequent
analysis of the temperatures at day 2, day 3 and day 5
showed significant differences between the vaccinates
and the controls for day 3 and 5 (P = 0.012 and P=
0.025 resp.).
3.1.2. Clinical symptoms
No clinical signs were observed in the vaccinates or
in the controls for two days post EHV-1 challenge.
Between the 2nd and the 7th day post challenge 1 of the
vaccinates (days 2, 3 and 5) and 1 of the controls (days
2, 4 and 5) exhibited a decrease of appetite, 1 of the
4311
vaccinates (day 3) and 1 of the controls (day 4) were
depressed and 1 of the vaccinates (day 3 and 4), 2
vaccinates (day 5) and 1 of the controls (day 4, 5, 7)
exhibited mild ocular discharge on at least one occa-
sion. Nasal discharge persisted from the 2nd day post
challenge throughout the entire 21-day observation pe-
riod. Between the 5th and the 8th day post challenge
80–100% of horses in both groups had nasal discharge
at each observation period. The controls had a higher
respiration rate than the vaccinates (Table 1). No spon-
taneous coughing was heard in either group but cough-
ing could be induced by deep laryngeal palpation in
40–80% of the horses in both groups each day post
challenge. Lymph node enlargement occurred at day
5-post challenge and persisted throughout the study in
some horses.
The mean clinical scores on each day for all of the
clinical symptoms for both vaccinates and controls are
summarised in Table 1 and are shown graphically in
Fig. 2. The cumulative individual clinical scores calcu-
lated for the entire observation period after challenge in
the vaccine group were not significantly less than for
the control group (P=0.06). The duration of fever
although shorter in the vaccinated group than the
control group was not significantly different.
Appetite and demeanour were not affected in any of
the horses challenged with EHV-4. One of the horses in
the control group suffered mild ocular discharge for 1
day (day 7). Nasal discharge and coughing were
recorded in both vaccinated and unvaccinated horses
throughout the study. The respiratory rate was more
elevated in the control horses than in the vaccinated
horses. In the control group the highest nasal discharge
scores were recorded on days 5–6 post challenge and
the highest coughing scores were recorded on days 4, 9,
12, 14 and 15-post challenge. The cumulative individual
clinical scores for the vaccinates were significantly less
than for the control group (P= 0.013). The duration of
fever was not significantly (P= 0.62) different (Table
1).
The number of horses showing a specific symptom on
each day in each group was summed for the entire
observation period and divided by the number of
horses per group (Table 1). Nasal discharge and en-
largement of lymph nodes were the most prominent
and common clinical signs.
Challenge with EHV-1 affected appetite and de-
meanour more profoundly than challenge with EHV-4
and resulted in a greater increase in respiratory rate.
EHV-4 challenge was characterised more by inflamma-
tion of the mucosal membranes of the eye and upper
airways and spontaneous coughing (Table 1).
3.1.3. Virus excretion and 6iraemia
Both viraemia and virus excretion were monitored in
the EHV-1 challenge study. Viraemia was demonstrated
4312 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317
in 3 of the 10 vaccinated foals while 4 of the 5 non-vac-
cinates were viraemic. The mean duration of EHV-1
viraemia in the vaccinated group was 0.39 0.5 days
which was significantly shorter (P= 0.03) than the
2.2 9 1.5 days of the control group.
All of the horses irrespective of vaccinal status shed
virus after challenge with EHV-1. However, the mean
duration of nasal shedding was significantly (P =0.02)
shorter in the vaccinates than in the controls. Vacci-
nated horses shed virus for 5.291.6 days while the
controls shed virus for 109 3.2 days. The group mean
virus excretion titre of vaccinated and control groups
are depicted graphically in Fig. 3. With the exception of
one horse peak virus titres were recorded on the day
after challenge which suggests that at least some of the
horses may have had residual challenge virus in the
nasopharynx at that time. The maximum virus titre
detected in the nasal secretions of the control horses
and the vaccinates between the 2nd and 14th day post
challenge was 105.0 TCID50 per 0.1 ml and 104.25
TCID50 per 0.1 ml respectively. The mean virus log10
titres per 0.1 ml nasal secretions of the horses that were
excreting virus between the 2nd and 14th day post
challenge were 0.709 0.92 and 1.839 0.84 for the vac-
cinates and the controls respectively (Table 1). The
difference was significant (P =0.014).
Virus excretion was monitored in the EHV-4 chal-
lenge study. All of the non-vaccinates and 6 of the 10
vaccinates shed virus after challenge. The group mean
virus excretion titre of vaccinated and control groups
are depicted graphically in Fig. 3. As for EHV-1 the
mean duration of nasal EHV-4 shedding was signifi-
cantly shorter (P= 0.007) in the vaccinates (2.89 2.6
days) than in the controls (8.392.1 days).
Unlike the horses in the EHV-1 challenge study only
one horse challenged with EHV-4 (one of the control
group), shed virus on the day after challenge. This
horse shed virus for 6 consecutive days and the peak
titre was recorded on day 4-post challenge. Peak titres
Fig. 3. EHV-1 and EHV-4 virus isolation from nasal swabs after viral challenge of the young foals. V =Vaccinated animals C = Unvaccinated
control animals. Virus titer is expressed as 10log TCID50/0.1 ml swab extract.
for the horses challenged with EHV-4 were recorded on
days 5, 6–7 post challenge. The maximum virus titre
detected in the nasal secretions of the control horses
and the vaccinates was 102.75 TCID50 per 0.1 ml and 102
TCID50 per 0.1 ml respectively. Log10 titres of the nasal
secretions of many vaccinated horses rapidly decreased.
The mean 10log virus titres per 0.1 ml of nasal secre-
tions were 0.4590.43 and 1.109 0.19 for the vacci-
nates and the controls respectively (Table 1). The
difference was significant (P= 0. 03).
On the day of challenge and the day after a few
horses in each group were shedding EHV-2. At the end
of the trial it appeared that all horses challenged with
EHV-1 shed EHV-2 for a few days. In contrast, the
EHV-2 infected horses in the EHV-4 challenge group
did not spread this virus through the population.
3.1.4. Antibody response to 6accination
Mean CF and SN antibody titres against EHV-1 are
given in Table 2. CF antibody titres\5 were detected
at 2 weeks after the 1st vaccination in just 4 of 20
vaccinated horses. After the 2nd vaccination however
19 of 20 horses showed anamnestic responses. SN titres
were hardly detectable in 15 of 20 horses at 2 weeks
after the first vaccination, but 2 weeks after the second
vaccination all horses had SN titres against EHV-1.
None of the unvaccinated controls had detectable levels
of antibodies to EHV-1 prior to virus challenge.
Mean CF and SN antibody titres against EHV-4 are
given in Table 2. CF antibody titres\ 5 were detected
at 2 weeks after the 1st vaccination in just 4 of 20
vaccinated horses. After the 2nd vaccination however
19 of 20 horses showed anamnestic responses. The poor
responder was the horse that also failed to mount a CF
antibody response to the EHV-1 component of the
vaccine. SN titres were hardly detectable in 11 of 20
horses at 2 weeks after the first vaccination, but 2 weeks
after the second vaccination 19 of 20 horses had SN
 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317 4313

Table 2
CF and SN antibody titre at various weeks after first vaccination (A) and at various days after EHV-1 and EHV-4 challenge (B)

A Test Antigen/weeks 0 1 2 4 6 6.5

Vacc. Animals CF EHV-1 0 0 4 4 54 33
EHV-4 0 0 3 1 27 16
SN EHV-1 0 0 3 1 283 213
EHV-4 0 0 6 2 565 532
B Challenge virus Antigen Group/days 0 7 10 14 21
EHV-1 CF EHV-1 V 50 570 608 557 320
EHV-1 C 0 23 79 116 52
EHV-4 V 24 538 602 589 312
EHV-4 C 0 32 72 105 22
EHV-1 SN EHV-1 V 244 307 826 1536 1675
EHV-1 C 0 0 42 235 55
EHV-4 V 99 94 955 1298 1066
EHV-4 C 0 0 0 51 61
EHV-4 CF EHV-1 V 58 113 336 192 70
EHV-1 C 0 0 0 0 0
EHV-4 V 29 194 599 392 190
EHV-4 C 0 0 0 4 0
EHV-4 SN EHV-1 V 321 254 2549 1890 1839
EHV-1 C 0 0 3 3 5
EHV-4 V 1032 1096 3125 3445 2814
EHV-4 C 0 0 10 24 53

2
V, Vaccinated animals; C, Unvaccinated control animals

titres against EHV-4. None of the unvaccinated con-
trols had detectable levels of antibodies to EHV-4 prior
to virus challenge.
3.1.5. Antibody response after challenge
By seven days after EHV-1 challenge the CF anti-
body titres against EHV-1 and EHV-4 of all vaccinated
animals had increased significantly (Table 2). Only one
of the 5 unvaccinated controls had seroconverted,
whereas the others had no detectable levels of CF
antibodies in their serum. By 14 days post challenge 3
of the 5 horses in the control group had seroconverted
to both viruses and the other two had seroconverted to
EHV-1 but not EHV-4. The CF titres of the majority of
horses had started to decline by 21 days post challenge.
Seven days after EHV-1 challenge SN antibody titres
against EHV-1 had not increased in 6 vaccinated ani-
mals, while none of the 5 unvaccinated horses had
detectable SN antibodies. By 10 days post challenge all
unvaccinated horses had low SN antibody titres against
EHV-1 antigen and all vaccinated horses had increased
titres against EHV-1 and EHV-4 antigen. The highest
titres for both groups were recorded at 14 days post
challenge but peak titres were recorded for a minority
of horses at 21 days post challenge (Table 2).
By seven days after EHV-4 challenge 9 vaccinated
horses had mounted a CF antibody response to EHV-4
and 6 vaccinated horses had seroconverted to EHV-1.
None of the 4 unvaccinated horses had seroconverted
(Table 2).
By 10 days post challenge all vaccinates had high CF
antibody titres against both EHV-1 and EHV-4, while
none of the control horses had significant CF titres
against either virus (Table 2). By 14 days post challenge
just 2 unvaccinated horses had low CF antibody titres
against EHV-4. None of the non-vaccinates had titres
against EHV-1.
By 7 days post challenge 3 of the vaccinated horses
had seroconverted to EHV-4 as measured by SN. By 10
days post challenge only one vaccinate had failed to
seroconvert to EHV-4 and 6 had seroconverted to
EHV-1. One horse in the control group had serocon-
verted to EHV-4 but not to EHV-1. By 21 days post
challenge this horse had seroconverted to EHV-1 and
the other horses in the group had mounted an SN
response to EHV-4 but not to EHV-1 (Table 2).
4. Vaccination as an aid in the prevention of abortion
4.1. Serological response to 6accination and challenge
Geometric group mean CF antibody responses to
EHV-1 after vaccination and challenge are shown in
Table 3. At the start of the trial no significant differ-
ences were found in mean CF antibody titres between
vaccinated and control ponies. For the control group a
test for linear trend was performed from week 0
through 20 (ANOVA, F-test). No significant rises in
CF antibody titres occurred during this period in the
controls (P\ 0.10). Within the vaccinated group differ-
ences in CF antibody titres between time points were
analysed using the ANOVA, F-test. Significant rises in
CF antibody titres occurred 2 weeks after both the first
4314 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317

(P B 0.001) and second vaccination (P =0.014). No
significant increases occurred after the third vaccination
(P = 0.34) or virus challenge (P =0.34).
Geometric group mean SN antibody responses to
EHV-1 after vaccination and challenge are shown in
Table 3. Mean SN titres of vaccinated and control
ponies were compared by means of the t-test. At no
time point, was the GM SN of vaccinates significantly
different from the GM SN of the controls. None of the
vaccinates showed a significant rise in SN antibody titre
following vaccination (Table 3).
4.2. Clinical symptoms
Two control animals developed bi-phasic fever after
challenge, typical of EHV-1 infection [21]. In the vacci-
nated group none of the ponies showed this bi-phasic
response although increase in rectal temperature (\
38.8°C) was observed between day 0 and 2 post chal-
lenge in the vaccinated group. The mean significant
temperature for the vaccinates was 39.4°C and for the
controls 39.3°C. The mean duration of this significant
temperature increase was in the controls slightly longer
than in the vaccinates and also the range of days in
which this occurred was longer (Table 4). Both vacci-
nated and control mares developed respiratory symp-
toms (Table 4) but the mean duration of these
symptoms was less in the vaccinated mares than in the
controls. One control mare developed transient ataxia
on day 9-post infection. After 7 days the symptoms had
ameliorated (Table 4).
4.3. Virus isolation from nasopharynx and 6iraemia
All control mares shed virus from the respiratory
tract. The mean duration of shedding was 3.5 days
(range 2– 5 days). Peak titres of 105.5 TCID50/ml were
recorded on day 3-post challenge. Similarly, all vacci-
nates shed virus in respiratory secretions for an average
of 2.2 days (range 1–4 days). Maximum virus titres of
103.75 TCID50/ml were detected in the nasopharyngeal
extracts of individual mares, with peak titres recorded
on day 2-post challenge (Table 5; Fig. 4). At each time
point post infection, the geometric mean virus titres
excreted by the controls were higher than those of the
vaccinates (Table 5). At day 3 p.i. this difference was
statistically significant (Wilcoxon test, P two-sided=
0.02).
All mares, irrespective of vaccination status, became
viraemic. Virus could be isolated from buffy coats up to
day 14 post infection in the vaccinates and up to 21
days post infection in the controls. The mean duration
of viraemia in the vaccinates was 4.0 days which was
not significantly different from the 4.3 days for the
controls.
4.4. Outcome of pregnancy
All controls became infected as indicated by the
serological and virological responses and all aborted
between day 15 and 65 post infection (Table 6). Post
mortem examination of the foetuses showed character-
istic necrotic herpes viral lesions in the spleen, thymus,

Table 3
CF and SN antibody titres following vaccination and challenge to EHV-1 on indicated weeks

CF SN

0 (V1) 2 4 8 (V2) 10 16 (V3) 18 20 (Ch) 22 24 0 (V1) 8 (V2) 16 (V3) 20 (Ch) 22

V 1.7 2.5 2.4 2.2 2.7 2.4 2.6 2.6 2.7 2.8 1.6 1.9 1.8 1.8 2.9
C 1.3 1.3a 1.2a 1.2a 1.3a 1.3a 1.2a 1.5a 2.4 2.8 1.1 1.2 1.1 1.2 2.7

a
Significant difference between vaccinated and control group (t-test with level of significance set at P two-sidedB0.05). V = vaccinated animals,
C =Unvaccinated control animals. Vaccination 1, 2, 3 are represented by Vx. Ch = EHV-1 challenge.

Table 4
Summary of clinical responses to challenge with EHV-1. V = Vaccinated animals, C =Unvaccinated control animals

Nasal discharge Lymph nodes Pyrexia Ataxia

No. Daysa No. Days No. Days

V 5/5 4.4 (1–8) 4/5 1.8 (0–4) 4/5 1.2 (0–2) 0/5
C 4/4 6.0 (3–8) 3/4 2.0 (0–3) 2/4 1.5 (0–4) 1/4b

a
Days and range (brackets).
b
Lasting from day 9 through 16 post infection.
 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317
Table 5
Isolation of EHV-1 from nasopharyngeal swabs. V =Vaccinated animals, C =Unvaccinated control animals
V/C GMT (TCID50/ml) in nasopharyngeal swabs on indicated day p.i.
1 2 3 4
V 1.70 2.05 0.95 0.0
C 2.75 3.42 3.63a 1.00
a
Significant difference between vaccinated and control group (Wilcoxon test, level of significance set at P two-sidedB0.05).
liver and regional lymph nodes and EHV-1 was recov-
ered from all 4 fetuses.
In contrast, only 1 of 5 vaccinated mares aborted on
day 16 p.i. This mare was pyrexic and viraemic for 10
d.p.i. EHV-1 was isolated from her nasopharynx for
just one-day post challenge. Histopathological exami-
nation of the fetus showed characteristic necrotic viral
lesions in the liver, spleen, thymus, small intestines and
associated lymph nodes, with intra nuclear inclusion
bodies in degenerating cells. EHV-1 was isolated from a
pooled sample of fetal liver, lung, thymus and spleen.
The remaining 4 mares delivered healthy foals after a
normal gestational period of 347 and 362 days. No
pathological condition was observed in any of the foals
during a 3– 6 weeks postnatal period.
The incidence of abortion in the vaccinated group
was significantly lower than in the control group (P
two-sided =0.048, Fisher’s exact test).
5. Discussion
This paper describes two vaccination challenge exper-
iments to assess the efficacy of Duvaxyn EHV1,4, an
adjuvanted liquid vaccine containing inactivated EHV-
1 and EHV-4.The vaccine can be considered as classical
vaccine. It was administered according to the recom-
mendations of the manufacturer in pregnant mares
under field conditions, and in recently weaned foals.
The latter were seronegative for both EHV-1 and 4 and
the absence of an anamnestic response confirmed their
naive immune status at the start of the experiment. The
immune status of the mares suggested previous expo-
sure to EHV-1 as they all had low CF and SN antibody
titres to EHV-1 and EHV-4 in the weeks prior to
vaccination. During the time before vaccination there
was no evidence of field infection with EHV-1, al-
though CF antibody titres in individual animals fluctu-
ated considerably.
The clinical and virological efficacy of the vaccine
was assessed in both trials. In the foal trial two primary
doses of vaccine induced CF and SN antibody titres
against EHV-1 and EHV-4. After challenge with either
EHV-1 or EHV-4 the levels of CF and SN antibodies
4315
5 6 7 8 9 10
0.30 0.0 0.0 0.0 0.0 0.0
1.67 0.0 0.0 0.0 0.0 0.0
increased rapidly in the vaccinated animals whereas
antibody titres increased more slowly in the control
animals. Furthermore, vaccination was associated with
a reduction of clinical signs and virus excretion in foals.
It has been suggested by numerous authors that anti-
body levels after EHV vaccination are not a reliable
parameter for the prediction of clinical protection and
that cellular and/or local immunity may play a more
important role in providing protection against clinical
disease [21,22, review].
However, in this study when Spearman rank analysis
(n =10) of log transformed data was performed to test
this generally accepted hypothesis that clinical protec-
tion is not related to antibody levels a correlation
between the SN titres and the duration of virus excre-
tion (z= − 0.73) and between the CF titres and the
total clinical score (z= − 0.76) in the vaccinated group
after challenge with EHV-1 was found. The duration of
fever correlated strongly to the total clinical score (z=
0.82). In contrast, no correlation between CF titres and
virus titre of the nasal secretions or duration of EHV-1
virus excretion could be demonstrated (z= −0.47;
z= − 0.43). Similar analysis revealed that log trans-
formed SN and CF titres correlated with the virus titre
of the nasal secretions (z= − 0.87; z= − 0.90) and
with the duration of virus excretion (z= − 0.76; z=
− 0.74) after EHV-4 challenge. These results clearly
Fig. 4. EHV-1 Virus isolation from nasal swabs taken on various
days post challenge. Virus titer is expressed as geometric group mean
10
logTCID50/ml swab extract taken from vaccinated pregnant mares
(V) and unvaccinated control pregnant mares (C).
4316 J.G.M. Heldens et al. / Vaccine 19 (2001) 4307–4317
Table 6
Outcome of pregnancy. V =vaccinated animals, C = Unvaccinated control animals
V/C Mare No. Outcome
35 Foaled
V 37 Foaled
39 Foaled
40 Aborted
42 Foaled
36 Aborted
C 38 Aborted
41 Aborted
43 Aborted
suggest that CF and VN antibodies may limit the
duration of virus excretion and serve as a useful man-
agement tool. The reduction of virus excretion by
horses that are seropositive as a result of vaccination
decreases the risk they pose to their cohorts and re-
duces the duration and severity of disease outbreaks.
Vaccinated animals therefore reduce the level of virus
challenge spread into the environment.
The pony mares used in the abortion trial were
seropositive prior to the commencement of the trial.
EHV-1 and EHV-4 are endemic in the horse popula-
tion. Therefore the serological status of the mares was
a true reflection of the field situation and the challenge
of such mares an evaluation of vaccine efficacy under
natural conditions. There was no significant difference
in the mean antibody titres of the two groups of mares
at the start of the trial. The vaccinates mounted a
significant CF but not SN antibody response to their
first two vaccinations. They did not seroconvert after
the third vaccination. Both vaccinated and control
mares developed respiratory symptoms, became vi-
raemic and shed virus from the respiratory tract. How-
ever the vaccinates shed less virus than the controls and
the vaccine was clearly efficacious in the prevention of
abortion. All four controls aborted but only one of the
five vaccinated mares aborted. Apparently, vaccine effi-
cacy was independent of pre-vaccinal antibody titre in
this case. It is possible that cellular immunity plays the
dominant role in preventing EHV-1 infected leukocytes
from crossing the placenta.
The challenge virus used in this study EHV-1 Ab4/8,
was originally isolated from a quadriplegic horse. Ex-
perimental infection with this virus is associated with a
high rate of abortion and with neurological disease.
Other viruses isolated from cases of abortion in the
field have proven to be less virulent in experimental
challenge studies for example, EHV-1 V592 isolated
from a major abortion storm [8,21] and EHV-1 121412
used in the foal trial reported here. Furthermore, in-
tranasal inoculation of Ab4/8 has been shown to induce
higher rates of abortion than exposure by aerosol [21]
Days p.i. Day of gestation Abortion incidence
61 349
72 352 1/5
88 362
16 305
87 347
15 294
20 305 4/4
45 315
65 324
P= 0.048
which bears a closer resemblance to the mechanism of
virus exposure in the field. Thus, the vaccine was sub-
jected to a most demanding evaluation of its efficacy
and it would not be unreasonable to expect that based
on the results, horses vaccinated with Duvaxyn EHV1,4
would be protected against abortion in the majority of
situations that arise in the field.
Although the majority of EHV associated abortions
are caused by EHV-1, EHV-4 is not an uncommon
cause of sporadic abortion. EHV-1 and EHV-4 are
antigenically closely related and several vaccines mar-
keted as an aid in the prevention of abortion contain
only EHV-1. Our results demonstrate that that horses
vaccinated with a bi-valent vaccine are susceptible to
infection with both EHV-1 and EHV-4. Thus it is
unlikely that a monovalent vaccine would offer ade-
quate protection against heterologous challenge. The
genetic differences identified by the determination of
the nucleotide sequence of the viral genomes [2,3] also
suggest that the relatedness of the two viruses may have
been overestimated historically and that it would be
unwise to rely on cross protection.
In summary Duvaxyn EHV-1,4 is a bivalent vaccine
which protects against EHV-1 abortion, reduces the
severity of respiratory disease associated with EHV-1
and 4 and minimises the risk to an infected animals
cohorts by decreasing the titre of virus shed and the
duration of virus excretion.
Acknowledgements
The authors would like to thank Drs P.H. Flore and
J.M. Minke for excellent support during the set-up of
the pregnant mare study and the interpretation of the
data.
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