D6188.PDF

Rev. sci. tech. Off. int. Epiz., 2009, 28 (1), 275-291
Efficacy of a commercial inactivated
H5 influenza vaccine against highly pathogenic
avian influenza H5N1 in waterfowl evaluated
under field conditions
M. Rudolf (1), M. Pöppel (2), A. Fröhlich (3), T. Mettenleiter (4),
M. Beer (1) & T. Harder (1)*
(1) World Organisation for Animal Health (OIE) and National Reference Laboratory for Avian Influenza,
Institute for Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, D-17493 Greifswald-Insel Riems,
Germany
(2) Veterinary Practice, Poultry Specialisation, Delbrück, Germany
(3) Institute of Epidemiology, Friedrich-Loeffler-Institut, Wusterhausen, Germany
(4) Institute of Molecular Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany
*Corresponding author: [email protected]
Summary
Highly pathogenic avian influenza virus (HPAIV) can cause devastating losses in
the poultry industry. In addition, several HPAIV exhibit a zooanthroponotic
potential and can cause fatal infections in humans. These attributes particularly
apply to HPAIV H5N1 of Asian origin. Due to the absence of overt clinical
symptoms, introduction and subsequent spread of HPAIV H5N1 in domestic
waterfowl (especially ducks) may occur undetected, which increases the risk of
transspecies transmissions to highly vulnerable gallinaceous poultry and
mammals, including humans. Humans may also become infected with HPAIV
H5N1 by food products from slaughtered, silently infected ducks. Vaccination
against HPAIV can raise a protective barrier against an incursion of HPAIV since,
at least under experimental conditions, the reproduction factor R0 is lowered to
<1, which ensures eradication of the virus. The objective of this study was to
analyse whether these results can also be obtained under free-ranging field
conditions in commercially reared flocks of goose parents and fattening ducks
injected with a licensed, adjuvanted inactivated H5N2 vaccine. The time and
labour required for the vaccination of these geese and duck flocks exceeded
expected values, mainly due to animal sorting according to foot ring labels. No
adverse effects directly associated with vaccination were observed.
Serologically, a homogenous H5-specific antibody response was induced. Titres
varied with temporal distance from the last application of vaccine. Geese
parents were clinically protected against challenge with HPAIV A/Cygnus
cygnus/Germany/R65/06 (H5N1), but still could be infected and spread HPAIV
H5N1, albeit at lower levels and for shorter periods compared to unvaccinated
controls. Fattening Pekin ducks proved to be clinically resistant against
challenge virus infection and shed very little virus.
Keywords
Avian influenza – Field validation – Protective efficacy – Vaccination – Waterfowl.
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Introduction
Highly pathogenic avian influenza virus (HPAIV) can cause
devastating losses in poultry. In several developing
countries where small-scale, so-called ‘sector four’ or
backyard poultry production is a significant source of food
for the population, continuing HPAIV outbreaks may even
lead to protein deprivation and malnutrition (2, 23). In
addition, several HPAI viruses (HPAIV) exhibit a
zooanthroponotic potential and may cause fatal infections
in humans (5). All of these attributes particularly apply to
HPAIV H5N1 of Asian origin. This virus, whose precursor
surfaced in 1996 in the Chinese province Guangdong,
spread over large parts of Asia and Europe and, in 2005,
also reached the African continent (6). In several Southeast
Asian countries, as well as in parts of Africa and the Middle
East, representatives of different lineages of this virus have
established endemic infections with year-round viral
activity detectable in poultry (11).
There are several ways in which highly pathogenic
infectious agents can establish endemicity. The clinical
picture of an infection with HPAIV depends, among other
factors, on the bird species affected (1). In gallinaceous
poultry, after a short time of incubation, a severely
depressed general condition becomes apparent, and the
birds die rapidly within days. In domestic waterfowl,
however, substantial differences in clinical characteristics
following an HPAIV infection have been noticed. This has
been analysed in greater detail for HPAIV H5N1 of Asian
origin (21, 29, 30). Factors influencing the clinical course
relate to species, age of animals and the virus strain (19,
18). Introduction and subsequent spread of HPAIV H5N1
in duck flocks may, therefore, be clinically silent, as
observed in live bird markets in Southeast Asia, where
HPAIV H5N1 can be isolated year-round, mainly from
clinically inconspicuous domestic water fowl, particularly
ducks (11, 26). Recent strains isolated from such
outbreaks induce no clinical symptoms in ducks, but
remain highly pathogenic for chickens and turkeys. Thus,
during syndrome surveillance, infection will only be
detected after spread into highly susceptible gallinaceous
species (11). This situation is not necessarily confined to
backyard and rice-paddy rearing systems: industrial duck
holdings with high biosecurity standards may also be
affected, as demonstrated by clinically silent infections in
two German holdings of fattening ducks in 2007 (12).
Furthermore, silently infected ducks living in close contact
with humans increase the risk that HPAIV will cross the
species barrier (17). Human contacts with HPAIV are also
conceivable via food products from silently infected ducks.
Therefore,
outbreaks
legislation
based on
measures to prevent, control or eradicate
of these viruses form an integral part of
worldwide (4, 22). Prevention is principally
biosecurity to prohibit virus incursion into
poultry holdings. However, the level of success depends on
the structure of the poultry industry and on HPAIV
epidemiology. Biosecurity measures are easier to
implement in regions with a high percentage of wellcontrolled industrial poultry holdings. Sector four
holdings, spatially highly clustered and inhomogeneous
concerning age and species of poultry, pose a more severe
problem in this respect.
Vaccination against HPAIV can increase barriers against
an incursion of HPAIV. Successful vaccination is
determined by:
– absence of symptoms after infection
– an increased ability of vaccinees to resist infection
– an effective reduction of virus shedding after infection.
Transmission of field virus will be interrupted when the
reproduction factor R0 drops below 1, thus ensuring
eradication of the virus (32). However, in a modelling
approach, Savill et al. (24) demonstrated that even 90%
vaccination coverage with currently available vaccines does
not prevent clinically ‘silent’ field virus infections and
further virus spread. A field vaccination study of chickens
in Hong Kong resulted in 81.7% successfully vaccinated
animals (7). Today, in the European Union (EU), three
inactivated oil emulsion vaccines are licensed and
commercially available. Two of them are specific for
subtype H5 (8). Inactivated vaccines have to be
administered individually via injection, causing significant
logistical challenges. While it has been experimentally
shown that R0 can drop below 1 by using these vaccines
(3, 31, 32), it remains to be elucidated whether these
results, obtained in a laboratory setting with very limited
numbers of animals, can be extrapolated to conditions in
commercial poultry production. Besides questions about
the protective efficacy of the available vaccine in a field
situation, there are also apprehensions that widespread and
sustained, but uncontrolled, vaccination may facilitate the
development by antigenic drift of variants which escape
immunity induced by the vaccines (16, 26).
This study evaluated the time and labour required for the
vaccination of geese and duck flocks against HPAIV H5
under commercial conditions. Vaccine efficacy was
investigated by the induction of antibodies, and by
challenge experiments. The authors show that vaccinated,
commercially reared geese parents are protected from
disease but still can be infected and shed HPAIV H5N1
albeit at reduced levels and for shorter periods compared
to non-vaccinated controls. Pekin ducks in this study
proved to be clinically resistant against challenge virus
infection and shed only very little virus, if any at all.
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Material and methods
Study design and supervision of flocks
Two commercial, professionally reared free-range flocks
were selected: one of goose parents (n = 1,200) and one of
fattening Pekin ducks (n = 1,500). The holdings were
operated under fully commercial conditions in
Northwestern Germany; however, neither products nor
animals from these flocks were allowed to be marketed
during the trial. The geese flock was kept from November
2006 onwards on 7.5 ha pastures but had an open stable
available. During times of reduced meadow regeneration
(November 2006 to January 2007 and September 2007 to
January 2008) the birds received supplements of carrots,
corn and wheat. During laying periods special breeding
forage (loose forage, supplied by Goldott-Entenzucht p,
Germany) was provided.
Six-week-old fattening ducks were kept under free-range
conditions with sheds available. The geese and duck flocks
were monitored for 20 and 5 months, respectively, for
productivity determinants (daily feed consumption, loss
rate, production rate), clinical status and avian influenza
virus (AIV)-specific virological and serological parameters.
The study received full legal approval as an animal
experiment (authorisation no. LALLF M-V/TSD/7221.31.1-040/05). Since AIV vaccination is legally prohibited in
Germany and in the EU, derogation was granted by the
European Commission (EC) as laid down in decision
2006/705/EC.
Vaccine and immunisation schemes
An inactivated and adjuvanted vaccine based on low
pathogenic avian influenza virus (LPAIV) strain
A/duck/Potsdam/1402/86 (H5N2) was used to immunise a
subpopulation of the flocks. A total of 800 of 1,200 geese,
and 1,000 of 1,500 ducks were vaccinated according to the
manufacturer’s protocols (1.0 ml vaccine per animal).
Primary AIV H5 vaccination of geese was performed at
17 weeks of age (Fig. 1a, squares). Ducks received the
primary AIV H5 vaccination at seven weeks of age (Fig. 1b,
squares). Standard vaccinations against parvovirosis and
a) Geese
Vaccination (V)
Sampling (S)
Challenge
experiment (C)
16 18 20 22 24 26 … 46 48 50 … 74 76 78 80 82
b) Ducks
Vaccination (V)
Sampling (S)
Challenge
experiment (C)
Fig. 1
Vaccination and sampling scheme of geese (a) and duck flocks (b)
The geese were vaccinated (□) at the age of 17 weeks (V1), 21 weeks (V2), 48 weeks (V3) and 77 weeks (V4). Sampling (○, swabs and blood) was
done at week 17 (S1), 21 (S2), 25 (S3), 47 (S4), 50 (S5), 76 (S6) and 81 (S7). Challenge experiments (∆) were conducted in week 21 (C1), 25 (C2),
47 (C3) and 77 (C4)
The ducks were vaccinated (□) at the age of 7 weeks (V1) and 11 weeks (V2). Sampling (○, swabs and blood) was done in week 7 (S1), 10 (S2),
14 (S3) and 25 (S4). Challenge experiments (∆) were conducted in week 10 (C1), 14 (C2) and 26 (C3)
278
salmonellosis were carried out twice before the laying
period in geese, and Pasteurella anatipestifer vaccine, based
on a strain isolated from this herd, was applied at 12 days
of age in ducks. All vaccines were given by intramuscular
injection. The second AIV H5 vaccination (‘booster’) was
performed four weeks after primary vaccination. The geese
were revaccinated twice, after six and 12 months,
following the booster vaccination. Avian influenza virusvaccinated animals were identified by a coloured plastic
foot-ring imprinted ‘AIV’.
Flock sampling schedules
Combined oropharyngeal and cloacal swabs (Virocult®
MW 950, Medical Wire & Equipment Co. Ltd, Corsham,
Wiltshire, England) were taken from 60 animals of each
group (vaccinated, non-vaccinated) in each flock at time
points indicated by the circular symbols (S) in Figures 1a
and 1b. Heparinised blood samples (lithium-heparin
tubes, Kabe Labortechnik, Germany) were taken from the
same birds in parallel to swabbing. Swabs and blood
samples were kept cooled until arrival in the laboratory
within 48 h.
Challenge experiments
Vaccine efficacy as defined by protection against disease,
infection, and excretion of HPAIV, was evaluated by
challenge experiments using HPAIV A/Cygnus
cygnus/Germany/R65/06 (H5N1). This strain had been
isolated from a naturally infected whooper swan dying
from the infection in Germany in spring 2006 (34). At the
time points (C) indicated by triangles in Figures 1a and 1b,
eight vaccinated and eight non-vaccinated birds were
withdrawn from each of the flocks and transported to the
high containment facilities of the Friedrich-LoefflerInstitut, Insel Riems, Germany. Ducks and geese were
transported separately and had no other contact. For
challenge infection, each vaccinated animal was inoculated
by the oculo-oronasal route with 106 50% egg infective
dose (EID50) of HPAIV H5N1 diluted in cell culture
medium containing 10% foetal calf serum. Three nonvaccinated animals were inoculated with the same amount
as controls. An aliquot of each inoculum was re-titrated to
ensure proper dosage of the challenge virus. After 24 h,
five non-vaccinated birds were added to the respective
groups to check for contact infection. For the last challenge
experiment (geese: C4), vaccinated birds were added
instead. Animals were observed for ten days and clinical
symptoms were recorded. Oropharyngeal and cloacal
swabs were taken at days one, two, three, four, seven, nine
and ten post inoculation (PI). Animals that died or had to
be euthanised in a moribund state were referred for
pathological investigations. Blood samples were obtained
from all animals before inoculation and from all surviving
birds at the end of the observation period.
Rev. sci. tech. Off. int. Epiz., 28 (1)
Virus detection by real-time reverse
transcriptase polymerase chain reaction
Ribonucleic acid was isolated from swabs either manually
using the Viral RNA kit of Qiagen (Hilden, Germany) or
automatically with the Nucleospin 96 Virus Extraction Kit
(Macherey & Nagel, Germany) on a Tecan Freedom Evo®
pipetting platform. One-step real-time reverse
transcriptase polymerase chain reaction (rRT-PCR) for
detection of an M-gene fragment (27, modified by 33) was
carried out for swabs taken from the flocks. Five of these
swabs were pooled for rRT-PCR analysis. Single swabs from
positive pools were re-tested. Single positive swab samples
were then tested by rRT-PCR for presence of H5- (25) and
H7-genome fragments (27, modified by 33).
Swabs taken from birds during the challenge experiments
were processed individually and examined by the
H5-specific rRT-PCR only.
Molecular characterisation
of non-H5/H7 avian influenza virus
Swab samples taken from the flocks which apparently
harboured AIV RNA were further subjected to
conventional reverse transcriptase polymerase chain
reaction (RT-PCR) for an HA2-gene fragment (21).
Resulting amplification products were sequenced directly
as described (28). Assembled sequences were used for
BLASTN2 database searches to identify the corresponding
haemagluttinin (HA) subtype. The neuraminidase (NA)
subtype was identified following the method of Fereidouni
et al. (10). These samples were also subjected to virus
isolation.
Virus isolation in embryonated chicken eggs
Swabs were processed and supernatants inoculated into
embryonated chicken eggs according to the EU diagnostic
manual (9). Two passages each of five days were carried out
after inoculation of nine- to 11 day old embryonated eggs.
Detection of haemagglutinating activity in amnio-allantoic
fluids prompted further molecular investigation, as
described above, to verify the presence of AIV.
Virus titration in cell culture
Serial tenfold dilutions were made from swab fluids or
challenge virus inocula in cell culture medium containing
10% foetal calf-serum, and used to infect MV1Lu cells
(ATCC CCL 64) during seeding into microtitre plates
(100 µl per well; four replicas per dilution). MV1Lu
cultures were grown in Dulbecco’s modified Eagle’s
medium containing 10% foetal calf serum. The cultures
were incubated at 37°C in 5% CO2 for three days, after
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which infected cells were identified by an immune
peroxidase monolayer assay (IPMA) targeting the viral
nucleocapsid protein (NP). To this end, cell monolayers in
microtitre plates were fixed using a 1/1 (vol/vol) mixture of
methanol and acetone at –20°C after removing the
medium and rinsing the monolayer once with phosphatebuffered saline (PBS). Fixed cells were rehydrated with
PBS, and then overlaid with appropriately diluted anti-NP
monoclonal antibody (ATCC HB 65) for one hour at room
temperature. After two washes with PBS containing 0.05%
(vol/vol) Tween 20, the cells were overlaid with anti-Mouse
IgG peroxidase conjugate (Sigma, Saint Louis, Missouri,
United States of America) for 1 h. Following another wash,
cells were stained with 3-aminoethylcarbazole and
H2O2. Coarse intracellular granular reddish precipitates
were used to identify antigen-positive cells by light
microscopy at a magnification of 200×. Infectivity titres
were estimated according to Karber (13).
Detection of avian influenza virus
nucleocapsid protein-specific
antibodies by competitive enzyme-linked
immunosorbent assay
Heparinised blood samples were screened for AIV-specific
antibodies using the AI A blocking enzyme-linked
immunosorbent assay (ELISA) (Institut Pourquier,
Montpellier, France), or the ID Screen® Influenza A
NP Antibody Competition ELISA (ID VET, Montpellier,
France). These tests enable a qualitative detection of
antibodies directed against the influenza A virus
NP protein of all subtypes. Both assays appeared to have
comparable performance according to the authors’
validation data (not shown) and were therefore
used interchangeably. The instructions of the suppliers
were followed exactly. Positive samples were further tested
for subtype-specific reactivity by haemagglutination
inhibition (HI).
Statistical analysis
Statistical analyses were performed using the open-source
software package R 2.1.1 (The R Foundation for Statistical
Computing [2005]; www.r-project.org) and STATISTICA
für Windows (Software-System für Datenanalyse) Version
6.0 (StatSoft, Inc. [2001]; www.statsoft.com) to assess
statistical differences of clinical signs or serological titres.
Depending on the small sample size of different groups,
Fisher’s exact test was used to verify differences in the
distributions of data measured. Survival analysis
was performed by the log rank test. The sign-test was
performed to compare connected samples of values on an
ordinal scale, e.g. titres. For result interpretation a strictly
defined significance level at p-value 0.05 was used.
Multiple tests on the same data according to the critical
significance levels were not corrected.
Results
Monitoring trial flocks reveals
undisturbed general health
but suboptimal production parameters
During the whole study period the health status of the
geese and duck flocks was very good. Gross-pathological
investigations of dead or randomly selected diseased birds
revealed that these cases were largely the result of injuries
accidentally caused by manipulation during sampling and
vaccination (particularly manipulation to obtain blood
samples). After blood sampling, a few (4 to 6) birds
per flock died, due to uncontrolled bleeding from the
injection site, or had to be euthanised because of broken
wings or leg injuries incurred when the birds panicked
during capture.
Geese
Detection of avian influenza
virus subtype-specific antibodies
by haemagglutination inhibition
Hemagglutination inhibition antigen was prepared from
inactivated allantoic fluids of embryonated chicken
eggs inoculated with either the vaccine (A/duck/Potsdam/
1402/86 [H5N2]) or the challenge virus (A/Cygnus
cygnus/Germany/R65/06 [H5N1]). In addition, duck
sera were also tested against a recent H10 field virus
isolate from Germany (A/mallard/NVP/Wv1015/04,
H10N7). Haemagglutination inhibition was performed as
outlined in the World Organisation for Animal Health
(OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial
Animals (35).
During the first and second laying periods, 82 and
61 animals, respectively, died or were selected for
pathological examination for various reasons. These figures
include 13 losses by predators (foxes). A more frequent
cause of loss was oviduct-peritonitis in laying geese,
probably also provoked or aggravated by capture. Sixtyfour geese were removed for challenge infections. The
birds produced on average 32 eggs each. This value is at
the lower range of normal production. It could well be that
frequent handling for sampling and vaccination had a
negative influence on egg production. Likewise, forage
consumption of 3.37 kg/egg and 1.97 kg/egg, in the first
and second laying periods, respectively, is not
representative, due to extra rations used for baiting the
animals during manipulations.
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Ducks
In the duck flock 52 birds died during the 126 day
observation period due to accidents during handling and a
further 48 birds were used for challenge experiments. The
average animal weight at the end of the fattening period
was 3,680 g (a gain of 880 g). Usually, a fattening period
for ducks does not exceed 49 days; however, in this
experiment other conditions prevailed according to the
objectives of the study. Consequently, no values for forage
consumption are indicated.
Avian influenza virus vaccination
by injection was logistically
demanding but prompted no significant
vaccine-induced side-effects in the birds
Special care had to be taken when marking vaccinated
birds by ringing. Open rings have proven inapt, so since
only closed rings can be used there is only a short time
span during which the rings can be fitted onto the leg
without immediate loss of the ring. It took three people
5 hours to ring 800 geese, and four people 4 hours to ring
1,000 ducks.
Avian influenza virus-specific
antibody titres in flocks decreased with
temporal distance from revaccination
All blood samples taken prior to vaccination tested
negative for AIV NP-specific antibodies. Considering the
performance of the ELISAs used, the AIV seroprevalence in
the herds prior to vaccination was estimated to be <4.87%,
and the herds were classified to be seronegative and fully
susceptible for AIV infections.
Geese
a) Geese
b) Ducks
Log2
Only one AIV NP and H5 seropositive sample was detected
throughout the observation period (n = 420 samples)
among the non-vaccinated birds. Most likely this sample
was from a vaccinated bird which had lost its foot ring.
Generally, 89.54% ELISA-positive samples turned out to
be positive (≥ 4 log2) in HI-test. In vaccinated geese, there
was a gradual increase of H5-specific mean titres over the
course of the observation period (Fig. 2a). After the second
vaccination (S3), 92% of the vaccinated geese showed
H5 vaccine-specific antibody titres ≥ 5 log2. In the
Log2
No adverse reactions during and after application of the
vaccine were observed in geese or ducks. During capture
and manipulation of the animals for vaccination
considerable care had to be taken to prevent injuries,
overheating and suffocation of the birds, particularly in
layers (see above). Precautionary measures demanded up
to two additional technical assistants than usually
considered for vaccination of gallinaceous poultry. For
vaccination of 800 geese within three hours five persons
were required. This included additional work to separate
vaccinated geese from non-vaccinated controls during
revaccination runs. Two thirds of the ducks
(1,000 animals) were vaccinated within three hours by a
total of five people.
Rev. sci. tech. Off. int. Epiz., 28 (1)
Fig. 2
Average HI-titres against the vaccine antigen (A/duck/Potsdam/1402/86 [H5N2], ●) and the challenge virus antigen (A/Cygnus
cygnus/Germany/R65/06 [H5N1], □) in vaccinated geese (a) and ducks (b) at various time points after vaccination (S, see Figure 1)
For the duck flock average HI titres are also shown against antigen of an AIV H10N7 isolate (A/Mallard/NVP/Wv 1015/04 [H10N7], ▲). The mean
titre and 95% confidence intervals are depicted. The dotted line represents a titre of 5 log2 indicative of a solid vaccine-induced immunity which was
shown to correlate with protection against disease in chickens (15)
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observation period that followed titres declined with
temporal distance from the last vaccination to below
4 log2 and were boosted after each revaccination
(Fig. 2a). Titres against the challenge virus were 3 to
7 log2 steps lower.
Immunologically naïve geese and ducks
were susceptible to challenge virus infection
but only geese developed significant central
nervous symptoms and mortality
Geese
Ducks
Development of AIV H5-specific antibodies in vaccinated
ducks (Fig. 2b) took a similar course and all tested birds
showed AIV-specific antibody titres ≥5 log2 after booster
vaccination (S3), which only marginally decreased after
11 weeks (S4). Surprisingly, and in contrast to the geese
flock, AIV NP-specific antibodies were also detected in
non-vaccinated ducks (Fig. 2b) from week 10 onwards
(S2). According to virological investigations (see below),
this was most likely due to a natural infection with an
AIV field isolate of subtype H10. Using a recent
H10N7 isolate originating from wild birds in Germany,
development and rapid decline of H10-specific mean
HI antibody-titres was evident in non-vaccinated ducks
(Fig. 2b, triangles). Also, some vaccinated ducks
developed H10-specific antibodies, albeit at lower
frequency and titres compared to non-vaccinated animals.
Virological monitoring revealed a natural
avian influenza virus infection in the
free-range duck herd but not in the geese flock
Geese
By generic AIV rRT-PCR specific for an M gene fragment no
evidence for naturally occurring influenza A virus
infections was obtained during the study period, in either
the vaccinated or non-vaccinated subpopulation.
Ducks
Prior to vaccination (S1) all swabs tested negative for
AIV RNA. However, at three weeks after the first
vaccination (S2), 40 of 120 swabs (20/60 from nonvaccinated and 20/60 from vaccinated birds) were positive
in M-gene specific rRT-PCR. Cycle threshold values
(Ct-values) ranged between 39 and 30. No viruses were
isolated from any of the PCR positive swabs. By
conventional RT-PCR for the HA2 encoding part of the
HA gene, specific amplification products were obtained
from two animals and sequenced (sequences available in
GenBank under accession number EU883664).
The AIV A/duck/Shimane/45/1997 (H10N7) proved to be
the closest relative of these sequences in a BlastN2 database
search. At the monitoring time points that followed
(S3, S4) no influenza A virus RNA was detectable in
the swabs.
In the first two challenge experiments (C1, C2), three nonvaccinated animals were each inoculated with 106 EID50 of
HPAIV H5N1 as controls. All of these animals fell sick, and
five out of six died. Clinically, central nervous symptoms
such as altered behaviour, phases of circular motion and
severe torticollis were observed from 4 days post
inoculation (dpi). The geese died or were euthanised in
moribund status between dpi 6 and 10. Pathological
alterations found in dead animals were non-specific,
consisting primarily of mucosal haemorrhages in
proventriculus or pancreatitis. For animal welfare reasons
no non-vaccinated controls were used in challenge
experiments C3 and C4 since the pathogenicity of the
challenge strain for susceptible geese had been
demonstrated adequately.
Ducks
During each of the three challenge experiments three nonvaccinated ducks were inoculated with 106 EID50 of HPAIV
H5N1 as controls. All of these animals had low titres of
H10-specific antibodies (mean titre C1: 4 log2; C2: 4 log2;
C3: 2.7 log2). None of these ducks developed any
clinical signs.
Absence of clinical signs in challenged
vaccinated geese and ducks
With the single exception of one animal which died during
challenge at C4, vaccinated geese fully resisted an
inoculation of 106 EID50 of HPAIV H5N1 with respect to
clinical symptoms, even after a single vaccination (Fig. 3).
The one dead animal shed virus up to a Ct-value of 25 and
showed a mild rhinitis and pancreatitis. As expected,
vaccinated animals did not show clinical symptoms upon
exposure to 106 EID50 of HPAIV H5N1 (not shown).
Like the non-vaccinated ducks, the vaccinated ducks
displayed no clinical signs.
Vaccinated geese and ducks transmitted
challenge virus to non-vaccinated
and vaccinated sentinels, although
reduced challenge virus excretion in level
and duration was evident
Geese
During challenge trial C1, three weeks after the first
vaccination, all five non-vaccinated in-contact animals
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c) Challenge experiment C3
1.2
1.2
1.0
1.0
Ratio of healthy animals
Ratio of healthy animals
a) Challenge experiment C1
0.8
0.6
0.4
0.8
0.6
0.4
0.2
0.2
0.0
0.0
Time (days)
Time (days)
d) Challenge experiment C4
1.2
1.2
1.0
1.0
Ratio of healthy animals
Ratio of healthy animals
b) Challenge experiment C2
0.8
0.6
0.8
0.6
0.4
0.4
0.2
0.2
0.0
0.0
Time (days)
Time (days)
Fig. 3
Kaplan-Meier curves of the geese challenge experiments C1 to C4
Vaccinated animals are plotted as grey squares, controls as black circles and associated in-contact birds as white lozenges. For animal welfare
reasons, no controls were included in C3 and C4. In-contact birds of C1 and C2 were taken from the non-vaccinated subpopulation, in C3 and C4
vaccinated in-contact birds were chosen
died on dpi 10 due to HPAIV H5N1 infection, while all
vaccinated animals survived without developing clinical
symptoms. At C2, three weeks after the second
vaccination, two of five non-vaccinated in-contact animals
died on dpi 10. At C3, one of five non-vaccinated animals
died on dpi 8. C4 was conducted six months after the first
revaccination. In contrast to previous challenge
experiments, the in-contact birds had also been vaccinated.
While none of the vaccinated contact animals died, one of
eight vaccinated and inoculated geese fell severely ill and
died due to challenge virus infection.
Intermittent virus shedding started at dpi 1 in inoculated
geese (Table I). Until dpi 10, episodic excretion from the
oropharynx and/or the cloaca was evident for vaccinated
geese in all challenge experiments. Non-vaccinated in-
contact animals started virus shedding after two days of
contact. Ct-values of up to 24 were reached between days
7 and 9 post challenge. Shedding from the oropharynx was
significantly more pronounced than shedding from the
cloaca (geese: 127 rRT-PCR-positive oropharyngeal swabs
out of 383 [127/383] versus 34/381 positive cloacal swabs;
ducks: 36/329 positive oropharyngeal swabs versus 3/329
positive cloacal swabs; p < 0.0001, Fisher’s exact test). In
cell culture, HPAIV H5N1 was re-isolated only from swabs
obtained from non-vaccinated in-contact birds. The 50%
tissue culture infectious dose (TCID50) values ranged
between 101.6 and 105.1 per ml swab fluid. It was not
possible to re-isolate challenge virus from swabs taken
from vaccinated in-contact animals (C4), except for two
vaccinated and inoculated geese of C4, one of which
succumbed to the infection. These birds excreted
283
Rev. sci. tech. Off. int. Epiz., 28 (1)
Table I
Results of molecular (a) and virological (b) examination in challenge trials – geese
a) RNA detection
DPI
Day 3
Day 7
Day 10
Animal status
Parameters
Vaccinated, inoculated Combined swabs
In-contact,
non-vaccinated
Combined swabs
Control, inoculated
Combined swabs
Vaccinated, inoculated Combined swabs
In-contact,
non-vaccinated
Combined swabs
Control, inoculated
Combined swabs
Vaccinated, inoculated Combined swabs
In-contact,
non-vaccinated
Combined swabs
Control, inoculated
Combined swabs
C1
Challenge experiments
C2
C3
n/m
Ct
n/m
C4
n/m
Ct
3/8
32.58
0/8
>40
0/5
2/3
>40
35.85
0/5
2/3
>40
31.11
3/8
33.22
0/8
>40
3/5
0/2
30.57
>40
2/5
0/0
30.66
–
4/5
32.03
No controls
1/5
38.75
No controls
0/8
>40
6/8
37.95
1/8
1/7
5(b)/5
0/1
30.95
>40
5/5
0/0
30.03
>40
4/4
31.50
No controls
2/8
Ct
n/m
Ct
36.34
5/8
34.43
1/5
32.20
No controls
0/5(a)
>40
No controls
4/8
2/7
36.09
35.86
34.49
36.59
0/5
>40
No controls
DPI: days post inoculation
n/m: number of swabs tested positive in real-time reverse transcription polymerase chain reaction (rRT-PCR) / total number of swabs tested (for the purposes of the experiment no distinction
was made between oropharyngeal and cloacal swabs)
Ct: average cycle threshold value in H5-specific rRT-PCR <40
(a) vaccinated contact animals were used in the in fourth trial
(b) all birds dead on day 10
b) Excretion of infectivity
Gaps in this table indicate that by previous investigation using real-time reverse transcription polymerase chain reaction (see Table Ia) no evidence for
virus infection was obtained and, therefore, no virus isolation attempts were initiated
DPI
Animal status
Parameters
C1
n/m
Day 3
Vaccinated, inoculated
In-contact,
non-vaccinated
Control, inoculated
Combined swabs
0/3
Combined swabs
Combined swabs
0/2
Vaccinated, inoculated
In-contact,
non-vaccinated
Control, inoculated
Combined swabs
0/3
Combined swabs
Combined swabs
0/3
Day 10 Vaccinated, inoculated
In-contact,
non-vaccinated
Control, inoculated
Combined swabs
Day 7
Combined swabs
Combined swabs
VI
0(b)/5
Challenge experiments
C2
C3
n/m
VI
n/m
C4
VI
n/m
VI
0/2
2/5
2.3 102
0/4
1/2
1.3 103
0/2
0/2
2/4
0/6
0/1
0/5
1/4
2.6 102
0/1(a)
0/1
2.1 102
DPI: days post inoculation
n/m: number of swabs tested positive in cell culture / total number of real-time reverse transcription polymerase chain reaction positive swabs (for the purposes of the experiment no
distinction was made between oropharyngeal and cloacal swabs)
VI: average virus isolation and titration in MV1Lu-cells
(a) vaccinated contact animals were used in the fourth trial
(b) all birds dead on day 10
infectious HPAIV H5N1 detectable in cell culture with a
TCID50 of 102.1 to 103.6 per ml. Two of five vaccinated
contact birds seroconverted against the challenge virus,
indicating that they had become infected.
Ducks
In Table II the results of the duck challenge trials are
summarised. Only low loads (Ct-values >30) of H5specific AIV RNA were detected in a few cases scattered
across the observation period. The HPAIV H5N1 was re-
isolated from only two cases: a vaccinated duck (C1, dpi
1), and one non-vaccinated in-contact animal (C3, dpi 4).
Vaccinated birds revealed increased avian influenzaspecific haemagglutination inhibition titres after challenge
experiments
As shown in Figure 4, in each challenge experiment with
HPAIV H5N1 a booster effect on the vaccine-induced
H5-specific antibody titres (>4 log2 steps) was evident in
the vaccinated birds. The increase in antibody titre was
most significant for antibodies against the challenge virus,
284
Rev. sci. tech. Off. int. Epiz., 28 (1)
Table II
Results of molecular (a) and virological (b) examination in challenge trials – ducks
a) RNA detection
DPI
Day 3
Day 7
Day 10
Animal status
Parameters
C1
Challenge experiments
C2
n/m
Ct
n/m
Ct
Vaccinated, inoculated Combined swabs
In-contact,
non vaccinated
Combined swabs
Control, inoculated
Combined swabs
0/8
>40
1/8
0/5
2/3
>40
27.22
Vaccinated, inoculated Combined swabs
In-contact,
non vaccinated
Combined swabs
Control, inoculated
Combined swabs
1/8
Vaccinated, inoculated Combined swabs
In-contact,
non vaccinated
Combined swabs
Control, inoculated
Combined swabs
C3
n/m
Ct
36.37
0/8
>40
0/5
3/3
>40
34.44
0/4(a)
2/3
>40
34.75
36.83
0/8
>40
0/8
>40
0/5
0/3
>40
>40
0/5
0/3
>40
>40
0/4
1/3
>40
37.62
0/8
>40
0/8
>40
0/8
>40
0/5
1/3
>40
37.52
0/5
0/3
>40
>40
0/4
0/3
>40
>40
DPI: days post inoculation
n/m: number of swabs tested positive in real-time reverse transcription polymerase chain reaction (rRT-PCR) / total number of swabs tested (for the purposes of the experiment no distinction
was made between oropharyngeal and cloacal swabs)
Ct: average cycle threshold value in H5-specific rRT-PCR <40
(a) only four animals remaining after the death of one goose
b) Excretion of infectivity
Gaps in this table indicate that by previous investigation using real-time reverse transcription polymerase chain reaction (see Table IIa) no evidence
for virus infection was obtained and, therefore, no virus isolation attempts were initiated)
DPI
Animal status
Parameters
C1
n/m
Day 3
Day 7
Day 10
Vaccinated, inoculated
In-contact,
non vaccinated
Control, inoculated
Vaccinated, inoculated
In-contact,
non vaccinated
Control, inoculated
Vaccinated, inoculated
In-contact,
non vaccinated
Control, inoculated
VI
Combined swabs
Challenge experiments
C2
n/m
VI
C3
n/m
VI
0/1
Combined swabs
Combined swabs
2/2
Combined swabs
0/1
Combined swabs
Combined swabs
6.31 102
0/3
0/2
0/1
Combined swabs
Combined swabs
Combined swabs
0/1
DPI: days post inoculation
n/m: number of swabs tested positive in cell culture / total number of real-time reverse transcription polymerase chain reaction positive swabs (for the purposes of the experiment no
distinction was made between oropharyngeal and cloacal swabs)
VI: average virus isolation and titration in MV1Lu-cells
but an increase against vaccine antigen was also evident in
most cases.
Discussion
An efficacy assessment of an inactivated adjuvanted
vaccine against HPAIV H5N1 infection in commercially
reared flocks of geese parents and fattening ducks was
carried out. Feasibility of application, protection against
disease, and protection against infection with, and
excretion of, HPAIV field virus were investigated.
Feasibility of vaccine application
Intramuscular application of the vaccine posed no
particular problems in 16-week-old geese or in 7-week-old
fattening ducks. About 200 birds were injected per hour by
one person with the help of three or four assistants. This
included time required to identify the bird’s vaccination
status by reading the leg ring. It should be mentioned that
remnants of oil from the absorbed vaccine were detected in
breast muscles of fattening ducks for up to 50 days after
vaccination. This may cause problems in marketing meat
products from these animals; a subcutaneous route of
application would be preferable.
285
Rev. sci. tech. Off. int. Epiz., 28 (1)
Protection against disease
A considerable host- and virus-strain-related variability in
the clinical outcome of HPAIV infections in waterfowl has
been documented (18, 19, 21, 30). The challenge strain
chosen for this study originated from an outbreak in wild
birds in Northern Germany in 2006. It exhibited an
intravenous pathogenicity index of 2.97 in chickens (34)
and caused significant mortality, though not a 100%, in
susceptible adult geese within ten days. This strain, like
others, shows an age-dependent pathogenicity in Pekin
ducks, causing 100% mortality in four week-old animals
(Rudolf et al., in preparation), but did not induce any
detectable clinical signs in adult Pekin ducks. Moreover,
virus replication seemed to be limited in adult ducks in
line with the restricted virus excretion. Nevertheless,
successful challenge virus infection of all animals was
indicated by seroconversion at dpi 10 (Fig. 4). Thus,
concerning protection from disease, no effect of
vaccination could be assessed.
A possible co-protective role of heterosubtypic immunity
induced by the accidental, natural infection with an AIV of
subtype H10 in the free ranging duck flock is unclear. Even
if, at C3, H10-specific antibodies were no longer
detectable, cellular immunological memory functions may
have been still active.
Meier Curve (Fig. 3) shows that there is a 100% probability
that vaccinated animals will stay healthy, only in C4 did
one vaccinated animal die. Analysis by Fisher’s exact test
shows significant (p<0.05) differences between healthy
and diseased/dead animals in vaccinated and nonvaccinated groups.
Protection against infection with, and excretion
of, highly pathogenic avian influenza field virus
One of the gravest concerns about vaccination against
HPAIV is the risk of clinically silent HPAIV infections
Table III
Efficacy of protection of vaccinated geese
Challenge
Disease * (a)
trial
Death ** (b)
Clinically apparent
transmission to
non-vaccinated animals *** (c)
C1
0/8 (0.00606)
0/8 (0.05455)
5/5 (0.00077)
C2
0/8 (0.00606)
0/8 (0.00606)
4/5 (0.00699)
C3
0/8 (no controls)
0/8 (no controls)
2/5 (0.1282)
C4
0/8 (no controls)
1/8 (no controls)
0/5 (1) (d)
a) Geese
b) Ducks
Log2
Log2
In contrast, the protective effects of the vaccination of geese
were statistically verified (Table III): vaccination protected
successfully against illness and death. (In C1 protection
could not be formally verified, as one out of three nonvaccinated control animals survived as well.) A Kaplan-
* number of diseased vaccinated animals after challenge
(a) figures in parentheses indicate the efficacy of protection of vaccinated geese against
disease (Fisher’s exact test, p-values under null-hypothesises)
** number of dead vaccinated animals after challenge
(b) figures in parentheses indicate efficacy of protection of vaccinated geese against death
(Fisher’s exact test, p-values under null-hypothesises)
*** number of diseased non-vaccinated animals after nine days of contact
(c) figures in parentheses indicate efficacy of protection of vaccinated geese against virus
transmission leading to clinically apparent disease in non-vaccinated control birds (Fisher’s
exact test, p-values under null-hypothesises)
(d) vaccinated contact animals were used in the fourth trial
Experiments
Experiments
Fig. 4
Average HI-titres against the vaccine antigen (A/duck/Potsdam/1402/86 [H5N2], light grey) and the challenge virus antigen (A/Cygnus
cygnus/Germany/R65/06 [H5N1], dark grey) in vaccinated geese (a) and ducks (b) at dpi 0 and dpi 10 of the challenge experiments
The median titre and 25% and 75% quartiles are depicted in box plots. Outliers are marked by black dots. The dotted line represents a titre of 5 log2
indicative of a solid vaccine-induced immunity which was shown to correlate with protection against disease in chickens (15)
286
under immune coverage. According to mathematical
modelling (24) very high levels of population immunity
are required to completely suppress both infection and
spread. However, there are no accurate data available on
immunisation rates of different poultry species under field
conditions, since all data were obtained from trials run
under experimental conditions. The HI-specific antibody
titres correlate with clinical protection and, in chickens, an
HI-titre of ≥ 5 log2 was associated with good protection
(15). However, a similar correlation has not been made in
other avian species.
In the two waterfowl species examined here the
development of specific humoral immunity was evaluated
by NP-specific antibody ELISA and by strain-specific HI.
In line with previous experiences, ducks and geese in our
study reached, on average, lower antibody titres compared
with chickens. In addition, in geese, these titres declined
more rapidly (14), so that only 40% of vaccinees had titres
of 5 log2 six months after the basic immunisation.
Therefore, bi-annual revaccinations of geese were required
to keep the average HI titre against the homologous
(vaccine virus) antigen at ≥5 log2 in at least 90% of
vaccinees. In contrast, basic immunisation of ducks was
sufficient to maintain this level during the observation
period. Antibody titres against the heterologous HPAIV
H5N1 challenge virus were significantly lower, which was
expected due to the comparatively large differences
between the two viruses (homology of HA sequences
92%).
However, in the author’s study no vaccinated geese showed
overt signs of disease upon challenge, not even animals
with an average HI titre of 2.4 log2 (±1.4) against the
homologous vaccine virus antigen at C3, which is
considerably lower than anticipated for solid clinical
protection in chickens (15). Similar findings were seen in
the other challenge experiments. The one goose which
died in C4 had no detectable antibodies against the vaccine
antigen prior to challenge. Interestingly, in the same
experiment, two other animals without detectable
antibodies survived the challenge infection. Therefore, the
predictive value of specific HI antibody titres as an
indicator of the protective efficacy of vaccination in geese
seems to be questionable. This may also point towards a
substantial contribution to protection of the cellular
immune response.
Rev. sci. tech. Off. int. Epiz., 28 (1)
immune status [32]), but it was chosen since it more likely
resembles a realistic situation in the field. However, for the
final challenge experiment (C4) vaccinated in-contact
geese were used.
In the geese experiments, challenged vaccinated birds
became infected and excreted challenge virus albeit at
lower titres and intermittently compared to non-vaccinated
control geese which received the same inoculation dose.
Non-vaccinated in-contact geese acquired the infection
after challenge virus transmission from vaccinated birds
and developed clinical disease in experiments C1 and
C2. Only one of five non-vaccinated in-contact geese
succumbed to the infection in the third trial (C3), although
all in-contact birds became infected (TCID50 ranged
between 101.6 and 103.1). The authors were not able to
clearly demonstrate transmission of challenge virus
to vaccinated in-contact animals (C4), as only one out of
five contact animals revealed an rRT-PCR positive swab and
on only one occasion. Nevertheless, seroconversion against
the challenge virus indicates infection of at least two of the
vaccinated in-contact geese of C4 (Table IV).
Successful re-isolation of virus from comparatively few of
the PCR-positive animals (Tables I and II) indicates that the
cell system used is not as sensitive as live sentinel birds.
Thus, despite the inability to re-isolate challenge virus,
transmission of virus from vaccinated animals to nonvaccinated sentinels did occur.
For reasons detailed above, no proper assessment of
the protective efficacy of the vaccination was possible for
the ducks. We only detected low levels of virus shedding,
and virus re-isolation was only possible in isolated cases
even in non-vaccinated animals. Nevertheless, we found
significantly higher numbers of rRT-PCR positive swabs in
the non-vaccinated animals than in the vaccinated birds
(7 out of 384 swabs from vaccinated ducks versus 28 out
of 144 swabs from non-vaccinated ducks; p< 0.0001,
Fisher’s exact test). This indicates that vaccination led to a
detectable reduction of virus shedding, even if none of the
Table IV
Serological response (log2) of vaccinated in-contact geese in
challenge experiment C4
Animal number
In order to evaluate protection from infection with, and
excretion of, HPAIV, vaccinated birds were challenged with
a recent H5N1 HPAIV of Asian origin. In addition to
evaluation of challenge virus excretion by molecular and
virological methods, non-vaccinated in-contact birds
(sentinels) were chosen to closely mimic a field situation in
a flock with a heterogenous H5-specific immune status.
This approach formally interferes with a calculation of
R0 levels (since this would require animals of similar
H5N2 (a)
0 dpc
10 dpc
H5N1 (b)
0 dpc
10 dpc
1
6
7
0
7
2
7
7
0
6
3
3
6
0
0
4
3
5
0
0
5
7
5
0
0
(a) vaccine antigen
(b) challenge virus
dpc: days post challenge
287
Rev. sci. tech. Off. int. Epiz., 28 (1)
animals showed any sign of illness. Beato et al. (3) provided
data on the efficacy of a two-dose vaccination programme
in the first 30 days of life of Pekin ducks. The same vaccine
was used as in the authors’ experiments, but the challenge
virus was HPAIV A/duck/Vietnam/12/05 (H5N1). The
animals were challenged at four weeks of age. Only nonvaccinated animals fell sick and died. Furthermore, viral
shedding by vaccinated animals was marginal. Apparently,
the older age of the ducks used in our experiment
prevented the development of serious clinical signs.
Conclusions
Vaccination of geese under field conditions was successful
in protection against illness and death. However, our
experimental evidence demonstrates that geese vaccinated
under field conditions can become infected with
and transmit challenge virus to non-vaccinated geese.
These contact animals fell ill, shed virus at higher titres
than the vaccinated animals, and served as amplification
hosts for the virus. However, when vaccinated in-contact
geese were used (C4), transmission was greatly reduced.
This indicates that even under field conditions vaccination
is a means to limit and, ideally, abolish virus transmission
if a high level immunity can be achieved within the
population (flock).
Both vaccinated and non-vaccinated ducks were able to
resist infection with HPAIV H5N1 and virus excretion was
found to be intermittent and with low titres. Susceptible
four-week-old ducklings, in contrast, died within a few
days after challenge with the same dose of challenge virus
HPAIV A/Cygnus cygnus/Germany/R65/06 (H5N1)
(Rudolf et al., in preparation). An age-dependent
susceptibility of ducks for different strains of HPAIV has
been demonstrated in other studies (18). These findings,
along with results of the study by Beato et al. (3), suggest
that vaccination of ducks at as young an age as possible
could avoid losses of animals caused by HPAIV. It might
also help reduce virus shedding in adult vaccinated
animals. Ducks have been identified as a major reservoir of
endemic HPAIV H5N1 infections in Southeast Asia, since
syndrome surveillance fails as an early warning system in
ducks. Vaccination of ducks against HPAIV H5N1 may
help reduce infection rates and excretion levels of HPAIVexposed vaccinated birds. More importantly, vaccination in
this species would not interfere with syndrome-based early
warning systems since even without vaccination, older
animals do not exhibit clinical symptoms in contrast to, for
example, chickens or geese. Since clinical observation will
often not detect infection of ducks by HPAIV, other means
of surveillance, including targeted sampling and laboratory
diagnosis, are required to eliminate this important
virus reservoir.
Acknowledgements
This study was funded by a grant from the German Federal
Ministry for Food, Agriculture and Consumer Protection
(FSI1-2.2). We thank Markus Durban for operating
and controlling the automated extraction of RNA. We are
grateful to Julia Feldmann, Diana Wessler, Georg Bauer
and Thorsten Arnold for excellent technical help. We also
thank Bruno Fecke and Cornelia Rehage for their excellent
work in tending the flocks, and Dr Pöppel and his team for
vaccinating the birds and supervising their health status.
Maria Pittman, from the European Union DirectorateGeneral for Health and Consumers, and colleagues from
other authoritative bodies were of great help with issuing
permits and providing support in the planning stages
of this project.
288
Rev. sci. tech. Off. int. Epiz., 28 (1)
Évaluation de l’efficacité sur le terrain d’un vaccin commercial
à virus H5 inactivé contre l’influenza aviaire hautement pathogène
H5N1 destiné aux oiseaux aquatiques
M. Rudolf, M. Pöppel, A. Fröhlich, T. Mettenleiter, M. Beer & T. Harder
Résumé
Le virus de l’influenza aviaire hautement pathogène (IAHP) est responsable
de pertes économiques colossales dans le secteur de l’aviculture. En outre,
plusieurs virus de l’IAHP ont un potentiel anthropozoonotique et peuvent
provoquer une infection létale chez l’homme. Ces caractéristiques s’appliquent
en particulier au sous-type H5N1 du virus de l’IAHP, d’origine asiatique. Parce
qu’il ne provoque pas de signes cliniques manifestes, le virus de l’IAHP de soustype H5N1 peut s’introduire et se propager parmi les populations d’oiseaux
aquatiques domestiques (en particulier les canards) sans être détecté, ce qui
accroît le risque de transmission trans-spécifique aux espèces extrêmement
vulnérables de gallinacés et de mammifères (dont l’homme). L’homme peut
également contracter l’infection par le virus H5N1 de l’IAHP par l’intermédiaire
de produits alimentaires dérivés de canards porteurs silencieux du virus au
moment de l’abattage. La vaccination contre le virus de l’IAHP permet de dresser
une barrière protectrice contre les nouvelles incursions du virus : en effet, dans
des conditions expérimentales, elle fait passer le facteur de reproduction R0 en
dessous de 1, ce qui suffit pour éradiquer le virus. La présente étude a pour objet
de déterminer si des résultats similaires peuvent être obtenus sur le terrain chez
des troupeaux vivant en liberté d’oies reproductrices commerciales et de
canards gras, après injection d’un vaccin autorisé à virus H5N2 inactivé
comprenant un adjuvant. Le temps de travail et la main d’œuvre nécessaires
pour procéder à la vaccination de ces troupeaux d’oies et de canards ont été
plus importants que prévu, en raison de la nécessité de trier les volailles en
tenant compte de leurs bagues d’identification. Aucun effet indésirable
directement imputable à la vaccination n’a été constaté. La vaccination a induit
une réaction sérologique comportant l’apparition d’anticorps homogènes
spécifiques pour H5. Les variations des titres d’anticorps constatées reflétaient
le temps écoulé depuis la dernière application du vaccin. Le vaccin protège
cliniquement les oies reproductrices contre une inoculation d’épreuve avec
la souche A/Cygnus cygnus/Allemagne/R65/06 du virus H5N1 de l’IAHP, mais ne
les empêche pas de contracter l’infection ni de disséminer ce virus, bien qu’à
des niveaux moindres et sur des périodes plus courtes que chez le groupe
témoin non vacciné. Les canards de Pékin étaient protégés cliniquement contre
l’inoculation d’épreuve et excrétaient le virus en très faibles quantités.
Mots-clés
Efficacité protectrice – Influenza aviaire – Oiseaux aquatiques – Vaccination – Validation
sur le terrain.
289
Rev. sci. tech. Off. int. Epiz., 28 (1)
Evaluación de la eficacia en aves acuáticas y en condiciones
naturales de una vacuna comercial contra la influenza aviar
altamente patógena por H5N1 a base de virus H5 inactivados
M. Rudolf, M. Pöppel, A. Fröhlich, T. Mettenleiter, M. Beer & T. Harder
Resumen
Los virus de la influenza aviar altamente patógena (virus IAAP) pueden causar
pérdidas devastadoras para el sector avícola. Además, varios de ellos presentan
potencial zooantroponótico y pueden provocar infecciones mortales en el ser
humano. Estas características se aplican especialmente a la cepa H5N1 del
virus IAAP, de origen asiático. Debido a la ausencia de síntomas clínicos
ostensibles, la penetración y subsiguiente propagación de dicha cepa en aves
acuáticas domésticas (en especial el pato) pueden pasar desapercibidas, lo que
incrementa el riesgo de transmisión a otras especies muy vulnerables
de gallináceas o mamíferos, comprendido el ser humano. Las personas también
pueden resultar infectadas por la cepa H5N1 al ingerir alimentos procedentes
de patos que portaban una infección silente al ser sacrificados. La vacunación
contra el virus puede conferir protección contra una incursión vírica, por cuanto,
al menos en condiciones experimentales, hace descender el factor
de reproducción (factor R0) por debajo de 1, lo que garantiza la eliminación del
virus. Los autores describen un estudio encaminado a determinar si es posible
obtener resultados similares en condiciones naturales tras administrar una
vacuna patentada (a base de virus H5N2 inactivados con adyuvante) a bandadas
industriales de ocas reproductoras y patos de engorde criadas en libertad.
La vacunación de esas bandadas de ocas y patos fue más larga y laboriosa
de lo previsto, sobre todo por la necesidad de clasificar a los animales según sus
anillas identificativas. No se observó ningún efecto adverso relacionado
directamente con la vacunación. Por lo que respecta a la serología, se indujo
una respuesta homogénea de anticuerpos específicos de las cepas H5.
Los títulos de anticuerpos variaban según el tiempo transcurrido desde la última
administración de vacuna. Las ocas reproductoras quedaron clínicamente
protegidas contra la infección experimental por virus IAAP A/Cygnus
cygnus/Germany/R65/06 (H5N1), pero aún podían resultar infectadas por la cepa
vírica H5N1 y a su vez transmitirla, aunque en niveles menores y por períodos
más cortos que los animales testigo no vacunados. Los patos de Pekín,
en cambio, resultaron ser clínicamente resistentes a la inoculación experimental
y excretar muy pocos virus.
Palabras clave
Aves acuáticas – Eficacia de la protección – Influenza aviar – Vacunación – Validación en
condiciones naturales.
290
Rev. sci. tech. Off. int. Epiz., 28 (1)
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