Comprehensive serological analysis of two successive heterologous vaccines against H5N1 avian influenza virus in exotic birds in zoos.

Abstract

In 2005, European Commission directive 2005/744/EC allowed controlled vaccination against avian influenza (AI) virus of valuable avian species housed in zoos. In 2006, 15 Spanish zoos and wildlife centers began a vaccination program with a commercial inactivated H5N9 vaccine. Between November 2007 and May 2008, birds from 10 of these centers were vaccinated again with a commercial inactivated H5N3 vaccine. During these campaigns, pre- and postvaccination samples from different bird orders were taken to study the response against AI virus H5 vaccines. Sera prior to vaccinations with both vaccines were examined for the presence of total antibodies against influenza A nucleoprotein (NP) by a commercial competitive enzyme-linked immunosorbent assay (cELISA). Humoral responses to vaccination were evaluated using a hemagglutination inhibition (HI) assay. In some taxonomic orders, both vaccines elicited comparatively high titers of HI antibodies against H5. Interestingly, some orders, such as Psittaciformes, which did not develop HI antibodies to either vaccine formulation when used alone, triggered notable HI antibody production, albeit in low HI titers, when primed with H5N9 and during subsequent boosting with the H5N3 vaccine. Vaccination with successive heterologous vaccines may represent the best alternative to widely protect valuable and/or endangered bird species against highly pathogenic AI virus infection.

Full text

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CLINICAL AND VACCINE IMMUNOLOGY, May 2011, p. 697–706
1556-6811/11/$12.00 doi:10.1128/CVI.00013-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 18, No. 5
Comprehensive Serological Analysis of Two Successive Heterologous
Vaccines against H5N1 Avian Influenza Virus in Exotic
Birds in Zoos?
Ju ´lia Vergara-Alert,1†* Hugo Ferna ´ndez-Bellon,2† Nu ´ria Busquets,1Gabriel Alca ´ntara,2
María Delclaux,2Bienvenido Pizarro,2Celia Sa ´nchez,3Azucena Sa ´nchez,4
Nata `lia Majo ´,1,5and Ayub Darji1
Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Auto `noma de Barcelona, 08193 Bellaterra,
Barcelona, Spain1; Grupo Veterinario, Asociacio ´n Ibe ´rica de Zoos y Acuarios, Secretariat, Parc Zoolo `gic de Barcelona, Parc de
la Ciutadella s/n, Barcelona, Spain2; Subdireccio ´n General de Sanidad de la Produccio ´n Primaria, Ministerio de
Medio Ambiente y Medio Rural y Marino, 28071 Madrid, Spain3; Departamento de Enfermedades Emergentes,
Laboratorio Central de Veterinaria, Ctra. Algete, km 8, 28110 Algete, Madrid, Spain4; and Departament de
Sanitat i Anatomia Animals, Universitat Auto `noma de Barcelona, 08193 Bellaterra, Barcelona, Spain5
Received 10 January 2011/Returned for modification 7 February 2011/Accepted 14 March 2011
In 2005, European Commission directive 2005/744/EC allowed controlled vaccination against avian influenza
(AI) virus of valuable avian species housed in zoos. In 2006, 15 Spanish zoos and wildlife centers began a
vaccination program with a commercial inactivated H5N9 vaccine. Between November 2007 and May 2008,
birds from 10 of these centers were vaccinated again with a commercial inactivated H5N3 vaccine. During these
campaigns, pre- and postvaccination samples from different bird orders were taken to study the response
against AI virus H5 vaccines. Sera prior to vaccinations with both vaccines were examined for the presence of
total antibodies against influenza A nucleoprotein (NP) by a commercial competitive enzyme-linked immu-
nosorbent assay (cELISA). Humoral responses to vaccination were evaluated using a hemagglutination inhi-
bition (HI) assay. In some taxonomic orders, both vaccines elicited comparatively high titers of HI antibodies
against H5. Interestingly, some orders, such as Psittaciformes, which did not develop HI antibodies to either
vaccine formulation when used alone, triggered notable HI antibody production, albeit in low HI titers, when
primed with H5N9 and during subsequent boosting with the H5N3 vaccine. Vaccination with successive
heterologous vaccines may represent the best alternative to widely protect valuable and/or endangered bird
species against highly pathogenic AI virus infection.
Avian influenza (AI) is an infectious disease caused by type
A influenza viruses of the Orthomyxoviridae family. AI virus
subtypes are classified according to their surface glycoproteins:
hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) (9).
To date, highly pathogenic avian influenza (HPAI) viruses are
restricted mainly to infections with H5 and H7 subtype viruses,
which have caused unprecedented morbidity and mortality in
birds within the last few years (2). Aquatic wild birds, including
Anatidae (ducks, geese, and swans) and Charadriidae (shore-
birds), are widely considered to be the natural reservoir of AI
virus (13). Although wild birds were not known to be impli-
cated in the initial HPAI outbreaks, in 2002, an outbreak of
H5N1 HPAI virus in Hong Kong caused mortality in a wide
range of avian species, including migratory birds and resident
waterfowls (6). Since then, the H5N1 subtype of HPAI virus
has spread throughout Asia and into Europe and Africa, af-
fecting a large number of species. In 2005, an outbreak killed
over 6,000 water birds (mainly bar-headed geese [Anser indi-
cus], great cormorants [Phalacrocorax carbo], Pallas’s gulls
[Larus ichthyaetus], brown-headed gulls [Larus brunnicepha-
lus], and ruddy shelducks [Tadorna ferruginea]) at the Qinghai
Lake National Nature Reserve in northwest China (3). Fur-
thermore, several reports indicate direct bird-to-human trans-
mission in some Asian countries (11, 18). These zoonotic con-
sequences and the ecologic value of protecting avian species
have emphasized the need for effective control measures.
Due to unprecedented morbidity and mortality caused by
H5N1 HPAI virus and given the value of birds kept in zoos, in
2005 the European Commission directive 2005/744/EC allowed
vaccination against AI virus in such birds in zoos, under strict
surveillance (7). In the following years, different European
countries established preventive vaccination campaigns in
zoological institutions. In 2006, 15 Spanish zoos and wildlife cen-
ters underwent a vaccination program with a commercial in-
activated H5N9 vaccine. Between November 2007 and May
2008, birds from 10 of these centers were vaccinated again with
a commercial inactivated H5N3 vaccine, as decided by the
Spanish government. The decision of changing the vaccine
used in the first AI vaccination program (VP1) was based on
experimental results showing that the H5N3 vaccine, a reverse
genetics monovalent vaccine, was shown to elicit a strong im-
mune response and protected chickens (10) and ducks (12)
from experimental H5N1 infection, with no detection of viral
shedding.
The goal of the present study was to compare the seropro-
* Corresponding author. Mailing address: Centre de Recerca en
Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Au-
to `noma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Phone: 34
93 581 45 27. Fax: 34 93 581 44 90. E-mail: julia.vergara@cresa.uab.cat.
† J. Vergara-Alert and H. Ferna ´ndez-Bellon contributed equally to
the studies presented in this paper.
?Published ahead of print on 23 March 2011.
697

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tection elicited by inactivated H5N9 and H5N3 vaccines and
evaluate the boost effect of H5N3 vaccine in inducing immune
responses after priming a wide selection of avian species with
H5N9 in Spanish zoos.
MATERIALS AND METHODS
Vaccination. An inactivated, commercial, water-in-oil adjuvanted H5N9 (A/
CK/Italy/22A/H5N9/1998) vaccine (Poulvac i-AI H5N9-, Fort Dodge Animal
Health, Weesp, Netherlands), containing at least 128 hemagglutination units
(HAU) according to potency test, was used in zoos during the first AI vaccination
program (VP1) in Spain. Vaccination against AI virus in some of the zoos began
in March 2006, with the remaining zoos vaccinating up to September 2006. More
than 2,600 birds were vaccinated in the 15 zoos participating in this study. The
birds were vaccinated twice within a 3-week interval via the subcutaneous route.
Eighteen months later, between November 2007 and May 2008, a second vacci-
nation program (VP2) was carried out. At that time, an inactivated, commercial,
water-in-oil adjuvanted H5N3 (strain rg-A/ck/VN/C58/04) vaccine (Poulvac i-AI
H5N3-, Fort Dodge Animal Health, Weesp, Netherlands), containing at least
256 HAU, was used. Ten out of the 15 zoos took part in the second vaccination
program. More than 450 birds were vaccinated either once (if they had been
previously vaccinated with the H5N9 vaccine) or twice (those being vaccinated
for the first time). Most of the animals receiving the vaccine for the first time
were born after VP1.
Both vaccines are effective against the virus type in circulation and support the
DIVA (differentiating infected from vaccinated animals) principle, as the N
antigen differs from N1, which makes it possible to distinguish vaccinated birds
from H5N1-infected birds while maintaining acceptable efficacy. Further details
may be obtained from the manufacturer. In the two campaigns, the vaccine dose
administrated was adapted to body weight. Thus, birds with a body weight of ?2
kg were given 0.2 ml, those 2 to 10 kg were given 0.5 ml, and those ?10 kg were
given 1 ml. Published mean body weights of the different species were used
instead of using individual weights (4).
Sampling. Blood was collected from the right jugular, brachial, or ulnar vein
(left or right). In VP1, samples were obtained on the days of both first (n ? 2,672
samples from 17 taxonomic orders) and second (n ? 947 samples from 17
taxonomic orders) vaccinations, as well as 9 (n ? 933 samples from 17 taxonomic
orders) and 18 (n ? 542 samples from 16 taxonomic orders) weeks following the
first vaccination dose. In VP2, blood was collected on the day of vaccination (n ?
469 samples from 16 taxonomic orders) and 6 (n ? 398 samples from 14 taxo-
nomic orders) and 12 (n ? 376 samples from 15 taxonomic orders) weeks after
the first vaccination. In VP2, birds receiving an AI vaccine for the first time (107
out of 469) were revaccinated after 6 weeks (Fig. 1).
The official sampling protocol also included collecting cloacal swabs to detect
the presence of AI virus by reverse transcription-PCR (RT-PCR), as described
previously (13).
Serology. Sera prior to vaccinations with H5N9 (A/CK/Italy/22A/H5N9/1998)
and H5N3 (rg-A/ck/VN/C58/04) were examined for the presence of total anti-
bodies against influenza A nucleoprotein (NP) by a commercial competitive
enzyme-linked immunosorbent assay (cELISA) kit (ID VET, Montpellier,
France). The cELISA is based on recombinant AI virus NP as the antigen and a
conjugated antibody directed against the NP of AI virus. The assay was per-
formed according to manufacturer instructions.
To evaluate the humoral immune response induced after both vaccinations,
homologous H5-specific antibody titers were determined by an HI test by fol-
lowing standard procedures (14). Briefly, chicken erythrocytes and 4 HAU of an
H5 antigen (GD-Animal Health Service Deventer, Netherlands) were used for
the test. Sera from some bird species may cause agglutination of the chicken
erythrocytes used in the HI test, which may mask low levels of HI activity. For
that reason, before doing the test, sera from all animals were pretreated with a
50% suspension of chicken erythrocytes for 1 h. Fifty microliters of pretreated
serum was diluted by 2-fold serial dilution (1:2 to 1:4,096) in phosphate-buffered
saline (PBS) solution in U-bottomed microwell plastic plates (Nunc, Copenha-
gen, Denmark), and 4 HAU of virus was added to each well. Following incuba-
tion at room temperature for 30 min, 50 ?l of 0.6 to 0.75% chicken red blood
cells (RBC) was added to each well, and the plates were incubated at room
temperature for 30 to 45 min to allow RBC to settle. The HI titer was determined
as the value of the highest dilution of serum causing complete inhibition of the
4 HAU. Vaccine-induced titers of ?32 were considered to be a measure of
vaccine efficacy, and titers ?16 were considered negative according to 92/40/EEC
guidelines (8). In poultry, HI titers of ?16 were shown to indicate protection
against infection when animals were challenged with HPAI H7N7 virus after
vaccination with inactivated H7 AI vaccines (17). Since performing challenge
experiments in valuable zoo species is not possible and in accordance with the
European Food Safety Authority (EFSA), we chose an HI titer of 32 as a
threshold of protective vaccine efficacy, as vaccine manufacturers do (5).
To evaluate the specific immune response against an HPAI H5N1 virus strain
and to test the breadth of antibody response, postvaccination serum was tested
against A/Mallard/It/3401/05 (H5N1) and A/Tky/Eng/647/77 (H7N7).
No adverse reactions to vaccination were reported in any of the participating
centers.
Statistical analysis. For each species and for each order, the geometric mean
titer (GMT) and the percentage of animals with titers higher than 32 were
calculated. Differences of GMT values between orders were tested with the
Mann-Whitney test. Statistical analyses were performed using SPSS for Win-
dows, version 17.0.
RESULTS
Humoral response against H5N9 vaccination (VP1). De-
tailed data concerning humoral immune response against an
inactivated H5N9 vaccine from each order and species studied
is provided in Table 1. Before receiving the vaccine, only 33
birds out of 2,672 (1.2%) showed antibodies against AI virus
NP when tested by cELISA. Similarly, less than 1% of the
animals were seropositive for H5 AI virus by an HI test using
the homologous antigen. These 25 birds, presenting HI titers
of 32 or higher, belonged to four orders (Phoenicopteriformes
[n ? 19 birds], Anseriformes [n ? 3 birds], Ciconiiformes [n ?
2 birds], and Pelecaniformes [n ? 1 bird]).
HI antibody titers 3 weeks after the first vaccination (at the
time of the second vaccination) (n ? 947 birds) and 9 (n ? 933
FIG. 1. Vaccination and sampling schedule. In VP1, animals were
vaccinated twice with an inactivated H5N9 vaccine, at day 0 and 3
weeks after the first dose. Eighteen months later, birds were vaccinated
with an inactivated H5N3 vaccine (VP2). In VP2, two groups were
differentiated, those being vaccinated for the first time and those that
were previously vaccinated with H5N9. Serum samples were collected
at all the time points indicated in the figure and tested by cELISA and
HI. The numbers of animals tested are also indicated in the rectangles
next to each time point.
698VERGARA-ALERT ET AL.CLIN. VACCINE IMMUNOL.

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TABLE 1. Humoral immune response of avian species in zoos, vaccinated twice (within a 3-week interval) with
an inactivated H5N9 vaccine (VP1)a
Order
Species
No. of
birds
GMT
% of birds with
HI titers of ?32
Common name Scientific name
AnseriformesTotal
Mandarin duck
Egyptian goose
Northern pintail
Northern shoveler
Baikal teal
Eurasian wigeon
Mallard
Chiloe wigeon
Greylag goose
Swan goose
Bar-headed goose
Magpie goose
Canada goose
Barnacle goose
Red-breasted goose
Hawaiian goose
Muscovy duck
Ringed teal
Cape Barren goose
Southern screamer
Australian wood duck
Andean goose
Black swan
Black-necked swan
Mute swan
Fulvous whistling-duck
Marbled duck
Rosybill
Red-crested pochard
Knob-billed duck
Ruddy shelduck
Raja shelduck
Common shelduck
179 6167.2
0
33.3
100
100
100
100
37.5
0
57.1
50
94.1
100
100
100
100
100
50
100
57.1
80
33.4
100
87.5
100
54.5
100
100
88.9
62.5
66.7
16.7
100
100
Aix galericulata
Alopochen aegyptiacus
Anas acuta
Anas clypeata
Anas formosa
Anas penelope
Anas platyrhynchos
Anas sibilatrix
Anser anser
Anser cygnoides
Anser indicus
Anseranas semipalmata
Branta canadensis
Branta leucopsis
Branta ruficollis
Branta sandvicensis
Cairina moschata
Callonetta leucophrys
Cereopsis novaehollandiae
Chauna torquata
Chenonetta jubata
Chloephaga picta
Cygnus atratus
Cygnus melanocorypha
Cygnus olor
Dendrocygna bicolor
Marmaronetta angustirostris
Netta peposaca
Netta rufina
Sarkidiornis melanotos
Tadorna ferruginea
Tadorna radjah
Tadorna tadorna
1
6
1
1
6
2
8
9
4
13
256
256
228
181
19
4
1424
194
17234
321
1
2
9
1
2,048
256
299
512
1618
81
32
3
7
5
3
3
111
8
323
146
256
53
16
1
11
1
1
9
8
3
6
1
2
1,024
32
299
91
51
7
1,024
362
Charadriiformes Total
Eurasian oystercatcher
Audouin’s gull
Caspian gull
Pied Avocet
Masked lapwing
17
4
1
5
5
2
20
23
4
7
42
64
47.1
50
0
20
60
100
Haematopus ostralegus
Larus audouinii
Larus cachinnans
Recurvirostra avosetta
Vanellus miles
Ciconiiformes Total
Abdim’s stork
White stork
Ibis stork
Scarlet ibis
Northern bald ibis
Marabou stork
Yellow-billed stork
Roseate spoonbill
African spoonbill
African sacred ibis
Straw-necked ibis
82 1433.7
100
30
100
5.6
100
22.2
0
66.6
66.7
37
0
Ciconia abdimii
Ciconia ciconia
Ciconia ibis
Eudocimus ruber
Geronticus eremita
Leptoptilos crumeniferus
Mycteria ibis
Platalea ajaja
Platalea alba
Threskiornis aethiopicus
Threskiornis spinicollis
1 256
13
51
20
3
185
4
9
1
3
3
64
13
4
32
128
15 19
18
ColumbiformesTotal
Nicobar pigeon
Speckled pigeon
Rock pigeon
Common wood pigeon
Victoria crowned pigeon
Barbary dove
79
6
7
56
1
2
7
612.5
66.7
0
7.1
0
0
28.6
Caloenas nicobarica
Columba guinea
Columba livia
Columba palumbus
Goura victoria
Streptopelia risoria
20
4
5
4
4
10
Coraciiformes Total
Knobbed hornbill
275
4
7.4
0 Aceros cassidix2
Continued on following page
VOL. 18, 2011 SUCCESSIVE HETEROLOGOUS H5 VACCINES IN ZOOS699

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TABLE 1—Continued
Order
Species
No. of
birds
GMT
% of birds with
HI titers of ?32
Common nameScientific name
Mindanao wrinkled hornbill
Black hornbill
White-crowned hornbill
Great hornbill
Rhinoceros hornbill
Abyssinian ground hornbill
Southern ground hornbill
Silvery-cheeked hornbill
Trumpeter hornbill
Gray-cheeked hornbill
Laughing kookaburra
Aceros leucocephalus
Anthracoceros malayanus
Berenicornis comatus
Buceros bicornis
Buceros rhinoceros
Bucorvus abyssinicus
Bucorvus leadbeateri
Bycanistes brevis
Bycanistes bucinator
Bycanistes subcylindricus
Dacelo novaeguineae
2
2
2
1
1
1
8
1
1
2
4
4
4
4
4
4
4
6
4
4
4
7
0
0
0
0
0
0
12.5
0
0
0
25
FalconiformesTotal
Cinereous vulture
Steppe eagle
Verreaux’s eagle
Red-tailed hawk
Variable hawk
Royal hawk
Turkey vulture
Short-toed eagle
Black vulture
Lanner falcon
Lesser kestrel
Black-chested buzzard eagle
Palm-nut vulture
White-backed vulture
Griffon vulture
Himalayan vulture
White-tailed eagle
Bald eagle
African fish eagle
Black kite
Red kite
Hooded vulture
Egyptian vulture
Osprey
Harris’s hawk
Honey buzzard
Southern caracara
King vulture
White-headed vulture
Andean condor
75
3
3
1
1
1
4
4
3
1
1
3
2
1
1
3
1
2
4
4
3
5
6
2
3
2
1
4
2
1
3
42
8
51
128
16
128
32
11
20
64
33.3
100
100
0
100
50
25
66.7
0
100
100
0
0
0
100
100
100
50
50
66.7
100
83.3
100
66.7
100
0
75
100
0
0
Aegypius monachus
Aquila nipalensis
Aquila verreauxii
Buteo jamaicensis
Buteo poecilochrous
Buteo regalis
Cathartes aura
Circaetus gallicus
Coragyps atratus
Falco biarmicus
Falco naumanni
Geranoaetus melanoleucus
Gypohierax angolensis
Gyps africanus
Gyps fulvus
Gyps himalayensis
Haliaeetus albicilla
Haliaeetus leucocephalus
Haliaeetus vocifer
Milvus migrans
Milvus milvus
Necrosyrtes monachus
Neophron percnopterus
Pandion haliaetus
Parabuteo unicinctus
Pernis apivorus
Polyborus plancus
Sarcoramphus papa
Trigonoceps occipitalis
Vultur gryphus
4
512
203
4
8
4
102
256
32
54
38
64
194
81
362
40
256
4
54
64
4
4
GalliformesTotal
Vulturine guineafowl
Lady Amherst’s pheasant
Golden pheasant
Great curassow
Red junglefowl
Silver pheasant
Indian peafowl
Common pheasant
69 30
25
59.4
66.7
33.3
0
100
69.2
0
61.3
0
Acryllium vulturinum
Chrysolophus amherstiae
Chrysolophus pictus
Crax rubra
Gallus gallus
Lophura nycthemera
Pavo cristatus
Phasianus colchicus
3
3
1
1
8
4
32
55 26
24
3129
24
Gruiformes Total
Blue crane
Demoiselle crane
Black crowned crane
Gray crowned crane
Seriema
Common crane
3110 25.8
0
20
0
45.5
0
33.3
Anthropoides paradisea
Anthropoides virgo
Balearica pavonina
Balearica regulorum
Cariama cristata
Grus grus
34
8
4
10
1
1118
3
3
6
13
Passeriformes Total
Pied crow
Carrion crow
Corn bunting
9
3
1
1
8
4
4
11.1
0
0
0
Corvus albus
Corvus corone
Emberiza calandra 16
Continued on following page
700 VERGARA-ALERT ET AL.CLIN. VACCINE IMMUNOL.

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TABLE 1—Continued
Order
Species
No. of
birds
GMT
% of birds with
HI titers of ?32
Common nameScientific name
Rosy starling
Red-billed chough
Common blackbird
Pastor roseus
Pyrrhocorax pyrrhocorax
Turdus merula
1
1
2
128
16
100
0
04
Pelecaniformes Total
Great white pelican
Pink-backed pelican
Great cormorant
31
20
15
32
38.7
60
0
0
Pelecanus onocrotalus
Pelecanus rufescens
Phalacrocorax carbo
8
3
4
4
Phoenicopteriformes Total
Lesser flamingo
Chilean flamingo
American flamingo
93
19
5
69
122
143
256
111
86
89
80
85.5
Phoeniconaias minor
Phoenicopterus chilensis
Phoenicopterus ruber
Piciformes Total
Toco toucan
Keel-billed toucan
Black-mandibled toucan
3
1
1
1
1333.3
0
100
0
Ramphastos toco
Ramphastos sulfuratus
Ramphastos ambiguus
4
128
4
PsittaciformesTotal
Blue-fronted amazon
Orange-winged amazon
Yellow-shouldered amazon
Festive amazon
Yellow-crowned amazon
Red-spectacled amazon
Vinaceous amazon
Hyacinth macaw
Great green macaw
Blue-and-yellow macaw
Red-and-green macaw
Scarlet macaw
Military macaw
Red-fronted macaw
Chestnut-fronted macaw
Blue-crowned parakeet
Finsch’s parakeet
White cockatoo
Sulfur-crested cockatoo
Goffins cockatoo
Salmon-crested cockatoo
Western corella
Yellow-crested cockatoo
Eclectus parrot
Golden parakeet
Scaly-headed parrot
Pesquet’s parrot
African gray parrot
177 1542.9
33.3
50
56
40
66.7
0
0
100
100
66.7
23.5
86.7
38.5
53.8
25
0
0
25
40
0
100
0
100
57.1
16.7
0
0
40
Amazona aestiva
Amazona amazonica
Amazona barbadensis
Amazona festiva
Amazona ochrocephala
Amazona pretrei
Amazona vinacea
Anodorhynchus hyacinthinus
Ara ambigua
Ara ararauna
Ara chloroptera
Ara macao
Ara militaris
Ara rubrogenys
Ara severa
Aratinga acuticaudata
Aratinga finschi
Cacatua alba
Cacatua galerita
Cacatua goffini
Cacatua moluccensis
Cacatua pastinator
Cacatua sulphurea
Eclectus roratus
Guarouba guarouba
Pionus maximilianii
Psittrichas fulgidus
Psittacus erithacus
3
2
9
5
3
3
3
1
3
8
16
30
21
32
10
10
128
51
16 27
17
15
13
13
9
37
19
16
4
1
1
8
5
1
1
8
1
7
6
1
1
8
4
4
9
11
8
32
4
64
16
11
4
4
1513
Sphenisciformes Total
Humboldt penguin
African penguin
16
5
11
9 18.8
60
0
Spheniscus humboldti
Spheniscus demersus
21
6
Strigiformes Total
Little owl
Eurasian eagle owl
Snowy owl
Barn owl
127 16.7
50
14.3
0
0
Athene noctua
Bubo bubo
Bubo scandiacus
Tyto alba
2
7
2
1
11
7
4
4
Struthioniformes Total
Emu
Greater rhea
Ostrich
33
9
19
5
11
7
9
37
30.3
22.2
21.1
80
Dromaius novaehollandiae
Rhea americana
Struthio camelus
All 93310348.2
aThe geometric mean titers (GMT) and the percentages of birds with a postvaccination serum hemagglutination inhibition (HI) titer of ?32 shown were measured
6 weeks after the second vaccination.
VOL. 18, 2011 SUCCESSIVE HETEROLOGOUS H5 VACCINES IN ZOOS 701

Page 6

birds) and 18 (n ? 542 birds) weeks after the first dose were
determined. After the first vaccine dose, the geometric mean
titer (GMT) was 81, and 31.8% of birds reached a serum
antibody titer of ?32 against the H5 antigen. On average, after
the booster vaccination, the GMT reached 103, and 51.4% had
a titer of ?32 against the H5 antigen. To evaluate longer-
lasting immunity, titers 15 weeks after the second vaccination
were studied. More than 45% of the birds were considered
positive, and the overall GMT was 59. Of the 7 taxonomic
orders for which more than 45 individuals were subjected to
serological follow-up, 6 reached mean titers greater than 32
(Fig. 2). Falconiformes, Pelecaniformes, Phoenicopteriformes,
and Struthioniformes presented HI titers over 120. In contrast,
Psittaciformes and Galliformes showed the lowest GMT val-
ues. However, only Phoenicopteriformes reached prevalences
over 75% of antibody titers at 32 or higher. Over 50% of birds
belonging to the orders of Galliformes, Falconiformes, and
Anseriformes reached a serum antibody titer of ?32.
Humoral response against H5N3 vaccination (VP2). De-
tailed data concerning humoral immune response against an
inactivated H5N3 vaccine from each order and species studied
are provided in Table 2. Of 469 birds tested prior to VP2, 190
tested positive by the cELISA (40%). Most of the seropositive
birds were from the following orders: Phoenicopteriformes
(n ? 74), Anseriformes (n ? 51), Psittaciformes (n ? 16), and
Ciconiiformes (n ? 15). However, only 26 out of 190 animals
were not vaccinated in the previous vaccination program
(VP1). By HI test, 279 out of 469 (60%) birds were seroneg-
ative for H5 AIV.
In VP2, antibody titers at 6 (n ? 398 samples) and 12 (n ?
376 samples) weeks postvaccination were studied. In both
cases, the number of seropositive animals was around 40%,
and the overall GMTs were different between those animals
vaccinated in the previous vaccination program (VP1 with
H5N9) and those vaccinated for the first time with H5N3 (Fig.
3 and 4). Six weeks after the second dose of the H5N3 vaccine,
Galliformes and Pelecaniformes orders (that were included in
the VP2 with only the H5N3 vaccine) manifested a GMT
higher than 150 (Fig. 3). The Falconiformes order showed a
weaker response, with a GMT of 50. The other birds that had
not been vaccinated previously had a GMT of less than 32.
Among animals vaccinated in VP1, Galliformes showed a very
high response (GMT ? 437) 12 weeks after receiving the
H5N3 vaccine. The Psittaciformes and Struthioniformes orders
reached seropositivity with a GMT of 58 and 128, respectively
(Fig. 4).
After H5N3 vaccination, 338 birds were evaluated for the
presence of serum antibody titers against an HPAI H5N1
strain circulating in Europe (A/Mallard/It/3401/05) and for the
presence of A/Tky/Eng/647/77 (H7N7)-specific antibodies. The
response obtained against H5N1 was compared to those elic-
ited against the H5N3 vaccine component. Moreover, two
groups were differentiated between those being H5N9 and
H5N3 vaccinated and those receiving only the H5N3 vaccine.
The frequencies of birds reaching a seroprotective titer (?32)
are similar when testing antibody titers against H5N1 as well as
for the vaccine compound in both the studied groups (Fig. 5).
No immune response against the H7N7 strain was detected in
any of the studied animals.
Virus detection. No AIV antigen was detected in collected
cloacal swabs in VP1. Prior to VP2, two animals that were
RT-PCR positive were probably exposed to AI virus during
this time interval. Both animals were from the Phoenicopteri-
formes order.
DISCUSSION
In the present work, we demonstrate that carrying out two
vaccination programs with successive heterologous vaccines in
wild animals from Spanish zoos can be the key to widely pro-
tect species from taxonomic orders which did not develop HI
antibody to a unique vaccine. In 2005, when the European
Commission directive 2005/744/EC allowed vaccination against
avian influenza (AI) in zoos (7), other European countries also
embarked on the mass vaccination program in zoo birds.
Lately, results from some of the zoos, judging the efficacy of
different vaccine formulations used, have been reported (1, 15,
16). Comparison of different vaccine formulations in eliciting a
strong humoral response could be instrumental to decide fu-
ture vaccination programs against AI virus.
In 2006, both Spain (data from present study, VP1) and
Denmark (1) used inactivated H5N9 vaccines from different
manufacturers in their vaccination programs in zoo birds. We
observed that 51.4% of the H5N9-vaccinated birds in Spanish
zoos had an HI titer of ?32 after booster vaccination, with an
overall GMT of 103. The present data were comparatively
lower than those previously reported by Bertelsen et al. (1),
also using the H5N9 vaccine, where 76% of the zoo birds
developed a titer of 32 with a GMT of 137. The differences in
seroprotection efficacy between our results and those reported
by Bertelsen et al. (1) may be due to different amounts of
antigen or adjuvants used in the vaccine preparation, since the
inactivated H5N9 vaccine studied by the Danish group was
derived from a different manufacturer. Moreover, it should be
noted that the present work is comprised of a large number of
exotic birds (n ? 933 after booster vaccination) from various
orders, which may influence the amount of the overall GMT.
This fact may also explain the heterogeneity in the antibody
FIG. 2. Humoral immune response following vaccination with an
inactivated H5N9 vaccine (VP1). An inactivated H5N9 vaccine was
used and administered twice within a 3-week interval. Bars represent
the geometric mean titers (GMT) with standard errors (SE) of differ-
ent taxonomic orders at different time points. The statistical signifi-
cance of the difference (Mann-Whitney test) between taxonomic or-
ders for each time point is indicated with a letter (P ? 0.05).
702 VERGARA-ALERT ET AL.CLIN. VACCINE IMMUNOL.

Page 7

TABLE 2. Humoral immune response of avian species in zoos vaccinated twice (within a 6-week interval) with an
inactivated H5N3 vaccine (VP2)a
GroupOrder
Species
No. of
birds
GMT
% of birds with HI
titers of ?32
Common nameScientific name
Nonvaccinated in VP1Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Columbiformes
Columbiformes
Columbiformes
Columbiformes
Coraciiformes
Coraciiformes
Falconiformes
Falconiformes
Falconiformes
Falconiformes
Galliformes
Galliformes
Galliformes
Gruiformes
Gruiformes
Pelecaniformes
Pelecaniformes
Pelecaniformes
Phoenicopteriformes
Phoenicopteriformes
Strigiformes
Strigiformes
Strigiformes
Total
Egyptian goose
Mallard
Greylag goose
Bar-headed goose
Magpie goose
Hawaiian goose
Cape Barren goose
Andean goose
Black swan
Mute swan
Fulvous whistling-duck
Rosybill
Total
Common wood pigeon
Diamond dove
Barbary dove
Total
White-crowned hornbill
Total
Common buzzard
Griffon vulture
Black kite
Total
Red junglefowl
Indian peafowl
Total
Demoiselle crane
Total
Great white pelican
Great cormorant
Total
American flamingo
Total
Barn owl
Spectacled owl
44 1011
Alopochen aegyptiacus
Anas platyrhynchos
Anser anser
Anser indicus
Anseranas semipalmata
Branta sandvicensis
Cereopsis novaehollandiae
Chloephaga picta
Cygnus atratus
Cygnus olor
Dendrocygna bicolor
Netta peposaca
440
0
0
1210
2
2
1
5
1
4
6
1
1
5
5
1
2
2
2
2
4
2
1
1
4
32100
40
1620
40
0
0
0
11
16
16
64100
20
40
7
16
Columba palumbus
Geopelia cuneata
Streptopelia turtur
40
64100
8
4
4
0
0
0 Berenicornis comatus
49
108
75
100 Buteo buteo
Gyps fulvus
Milvus migrans
40
128
187
56
512
100
100
100
100
11
Gallus gallus
Pavo cristatus
5
6
1
1
4
3
1
4
4
2
1
1
4
4
0
0 Anthropoides virgo
152
512
75
100Pelecanus onocrotalus
Phalacrocorax carbo4
8
8
0
0
0Phoenicopterus ruber
1150
Tyto alba
Pulsatrix perspicillata
40
32100
Vaccinated in VP1Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Anseriformes
Charadriiformes
Charadriiformes
Ciconiiformes
Ciconiiformes
Ciconiiformes
Ciconiiformes
Columbiformes
Columbiformes
Coraciiformes
Coraciiformes
Coraciiformes
Total
White-cheeked pintail
Chestnut teal
Mallard
Greylag goose
Emperor goose
Swan goose
Barnacle goose
Red-breasted goose
Hawaiian goose
Cape Barren goose
Andean goose
Ashy-headed goose
Ruddy-headed goose
Black swan
Black-necked swan
Rosybill
Red-crested pochard
Ruddy shelduck
Common shelduck
Total
Caspian gull
Total
White stork
Glossy ibis
African sacred ibis
Total
Common wood pigeon
Total
Knobbed hornbill
Mindanao wrinkled
hornbill
Black hornbill
Total
Turkey vulture
Himalayan vulture
Bald eagle
Hooded vulture
Harris’s hawk
Total
Indian peafowl
912042
Anas bahamensis
Anas castanea
Anas platyrhynchos
Anser anser
Anser canagicus
Anser cygnoides
Branta leucopsis
Branta ruficollis
Branta sandvicensis
Cereopsis novaehollandiae
Chloephaga picta
Chloephaga poliocephala
Chloephaga rubidiceps
Cygnus atratus
Cygnus melancoryphus
Netta peposaca
Netta rufina
Tadorna ferruginea
Tadorna tadorna
1
2
4
4
4
0
0
0 11
3
5
5
1
3
1
1
6
2
7
6
3
6
2
40 33.3
0
20
0
33.3
0
0
0
0
0
83.3
0
100
100
78.6
83.3
50.0
50
44
0
64.7
0
0
0
33.3
0
0
4
9
4
16
16
11
4
4
4
102
6
323
181
61
85
13
13
16
14
12
4
Larus cachinnans4
25
Ciconia ciconia
Plegadis falcinellus
Threskiornis aethiopicus
34
1731
5
9
9
6
2
2
4
4
4
9
4
4
Columba palumbus
Aceros cassidix
Aceros leucocephalus
Coraciiformes
Falconiformes
Falconiformes
Falconiformes
Falconiformes
Falconiformes
Falconiformes
Galliformes
Galliformes
Anthracoceros malayanus2
7
2
1
1
1
2
45
9
64
100
28.6
100
0
0
0
0
95.5
95.5
Cathartes aura
Gyps himalayensis
Haliaeetus leucocephalus
Necrosyrtes monachus
Parabuteo unicinctus
4
4
4
4
22
22
437
437 Pavo cristatus
Continued on following page
VOL. 18, 2011 SUCCESSIVE HETEROLOGOUS H5 VACCINES IN ZOOS703

Page 8

responses that we observed in serological analysis in vaccinated
birds, which varied greatly, not only between taxonomic orders
but also between species of a single order and even within
species. Similar observations with an inactivated H7N1 vaccine
were published by Philippa et al. (15), who described a high
seroprotection rate of 81.5% and an overall GMT of 190, with
variations in HI titers among different bird orders examined. In
general, based on the serological analysis from a huge number
of H5N9-vaccinated Spanish zoo birds, we observed that more
than 75% of birds from Phoenicopteriformes manifested a
GMT of ?32, and from the other 15 orders studied after
booster vaccination, 12 had a protection rate less than 50%.
TABLE 2—Continued
GroupOrder
Species
No. of
birds
GMT
% of birds with HI
titers of ?32
Common nameScientific name
Gruiformes
Gruiformes
Gruiformes
Gruiformes
Passeriformes
Passeriformes
Pelecaniformes
Pelecaniformes
Phoenicopteriformes
Phoenicopteriformes
Phoenicopteriformes
Phoenicopteriformes
Psittaciformes
Psittaciformes
Psittaciformes
Psittaciformes
Sphenisciformes
Sphenisciformes
Sphenisciformes
Strigiformes
Strigiformes
Strigiformes
Struthioniformes
Struthioniformes
Struthioniformes
Total
Blue crane
Demoiselle crane
Gray crowned crane
Total
European greenfinch
Total
Pink-backed pelican
Total
Lesser flamingo
Chilean flamingo
American flamingo
Total
Red-and-green macaw
Military macaw
Eclectus parrot
Total
African penguin
Humboldt penguin
Total
Eurasian eagle owl
Snowy owl
Total
Emu
Greater rhea
6
3
2
1
1
1
8
8
9
4
33.3
4
100
0
0
0
0
0
29.7
0
100
35.3
100
100
100
100
0
0
0
0
0
0
33.3
0
100
Anthropoides paradisea
Anthropoides virgo
Balearica regulorum
45
4
4
4
4
4
Carduelis chloris
Pelecanus rufescens
91
31
18
Phoeniconaias minor
Phoenicopterus chilensis
Phoenicopterus ruber
4
9 276
27
58
32
32
128
10
51
7
Ara chloroptera
Ara militaris
Eclectus roratus
1
3
3
16
Spheniscus demersus
Spheniscus humboldti
44
12
3
14
4
Bubo bubo
Nyctea scandiaca
2
1
3
1
2
4
4
128
16
362
Dromaius novaehollandiae
Rhea americana
Total nonvaccinated
in VP1
Total vaccinated in VP1
77 1923.4
299 1638.5
All37618 33.2
aThe geometric mean titers (GMT) and the percentages of birds with a postvaccination serum hemagglutination inhibition (HI) titer of ?32 shown were measured
6 weeks after the second vaccination. Animals are grouped into two groups: the nonvaccinated in VP1 and the ones that were vaccinated in VP1.
FIG. 3. Humoral immune response following vaccination with an
inactivated H5N9 vaccine (VP1). An inactivated H5N9 vaccine was
used and administered twice within a 3-week interval. Bars represent
the geometric mean titers (GMT) with standard errors (SE) of differ-
ent taxonomic orders. The statistical significance of the difference
(Mann-Whitney test) between taxonomic orders for each time point is
indicated with a letter (P ? 0.05).
FIG. 4. Humoral immune response in birds vaccinated with an
inactivated H5N3 vaccine (VP2) and vaccinated previously with an
inactivated H5N9 vaccine in VP1. An inactivated H5N3 vaccine was
used and administered once. Bars represent the geometric mean titers
(GMT) with standard errors (SE) of different taxonomic orders. The
statistical significance of the difference (Mann-Whitney test) between
taxonomic orders for each time point is indicated with a letter (P ?
0.05).
704VERGARA-ALERT ET AL.CLIN. VACCINE IMMUNOL.

Page 9

For the second vaccination program (VP2), the Spanish
Ministry replaced the H5N9 vaccine with an H5N3 recombi-
nant vaccine. The decision was based on the results given by
the manufacturer, showing that H5N3 (a reverse genetics vac-
cine), besides protecting chickens (10) and ducks (12) from
experimental AI infection, also prevented viral shedding. Mask-
ing disease signs while the bird continues to shed viruses may be
a serious problem both for valuable exotic birds and humans.
Thus, limiting virus shedding and further transmission is of ex-
treme importance.
Vaccination with inactivated recombinant H5N3 vaccine was
equally effective as VP1 in eliciting high titers of HI antibodies
against H5 among most of the bird orders studied, except for
birds belonging to Psittaciformes, which did not develop HI
antibodies to either vaccination protocol. Interestingly, how-
ever, priming with H5N9 and subsequently boosting with the
H5N3 vaccine induced a significant antibody response in Psit-
taciformes birds, albeit at lower titers than the others. Simi-
larly, Galliformes and Struthioniformes birds responded to the
H5N3 vaccine with much higher HI titers after booster vacci-
nation. This strategy (prime-boost) could be used in some of
the orders or species which do not respond to a unique vaccine.
However, we also have to carefully pay attention to the anti-
body titer length. As shown in Fig. 2, GMT after 18 months
decreased drastically. Thus, some of the orders receiving H5N3
vaccine only once, because they were previously vaccinated
with H5N9 (Fig. 4), did not show high titers. Philippa et al.
(16), based on previous reports, have pointed to the need of a
revaccination between 6 to 10 months after vaccination to
maintain seroprotective titers among different wild species in
zoos. This was similar to the results we obtained in VP1 18
months after the single vaccination, where seroprotection titers
started to decrease. The effect of a booster vaccination is seen
clearly in VP2, in those animals nonvaccinated previously in
VP1 (Fig. 3), especially for the orders of Galliformes and
Pelecaniformes, where GMT increased four times. These re-
sults are similar to those obtained by Philippa et al. (16), after
booster vaccination increased the GMT by 30% (from 50.5%
after single vaccination to 80.5% after booster vaccination)
(16).
To design future vaccination strategies in exotic wild birds, it
is important to evaluate both the response against the vaccine
and the durability of HI antibodies. Sera 80 weeks after a single
H5N9 dose were analyzed. On average, the birds had titers less
than 20, meaning that 1.5 years after vaccination, we cannot
detect HI titers in serum samples.
Antibody titers against HPAI H5N1 showed a similar trend
as those against the homologous strain, with 34.1% of birds
developing a titer of ?32 (animals vaccinated with successive
vaccines, H5N9 and H5N3) and 20.3% of the animals receiving
only the H5N3 vaccine showing seroprotective titers. However,
both groups showed lower titers than the results reported by
Philippa et al. (16), where 61.2% of the birds had a titer of ?40
against the HPAI strain tested, and more than 80% had a
seroprotective titer against the homologous strain.
Taking into account that inactivated H5N3 vaccine induces
strong immune responses and, more importantly, limits viral
shedding (sterile immunity), a prime (H5N9)-boost (H5N3)
vaccine strategy in future vaccination programs within exotic
valuable zoo birds and in particular in the Psittaciformes, Gal-
liformes, and Struthioniformes orders would be more adequate
and advisable. Together with increased biosecurity measures
and monitoring, vaccination may represent the best alternative
to protect valuable and/or endangered bird species against
HPAI virus infection. However, variations in elicited antibody
responses among different bird orders and species must be
carefully scrutinized in designing future vaccination programs.
This will not only protect vaccinated birds from infection but
also restrict further dissemination of otherwise devastating
HPAI virus.
ACKNOWLEDGMENTS
This work was partially supported by the AGL2007-60434/GAN
project funded by the Spanish Government and by the EUROFLU
project (SP5B-CT-2007-044098) funded by the European Union.
FIG. 5. Comparison of serum hemagglutination inhibition (HI) antibody titers against the H5N3 vaccine and H5N1 field virus following
vaccination with either a single vaccine (H5N3) or two successive heterologous vaccines (H5N9 and H5N3). HI titers against the vaccine
component (A/ck/VN/C58/04; H5N3) and the field strain (A/Mallard/It/3401/05; H5N1) were determined in 338 birds 12 weeks after starting VP2.
VOL. 18, 2011 SUCCESSIVE HETEROLOGOUS H5 VACCINES IN ZOOS 705

Page 10

We are grateful to staff at participating zoos for their collaboration
and kind help in data compilation, including Rocío Canales Merino
(Safari Park Vergel), Loles Carbonell (Jardín Zoolo ´gico de Valencia),
Sergio Ferna ´ndez Herna ´ndez (Selwo Marina and Selwo Aventura),
Daniel García Pa ´rraga (L’Oceanogra `fic), Candelaria Gonza ´lez Villavi-
cencio (A ´guilas Jungle Park), Ayose Melia ´n Melia ´n (Palmitos Park),
Tania Monreal Pawlowsky (Marineland Mallorca), Miguel Angel
Quevedo Mun ˜oz (Zoo Bota ´nico Jerez), Jose ´ María Rodríguez Linde
(Oasys Parque del Desierto de Tabernas), and Fernanda Valde ´s
García (Senda del Retiro), as well as staff at Faunia, Zoo Aquarium de
Madrid, Zoo de Fuengirola, and Parc Zoolo `gic de Barcelona.
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