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Avian Pathology (August 2004) 33(4), 405 /412
Vaccination of chickens against H5N1 avian
influenza in the face of an outbreak interrupts virus
transmission
Trevor M. Ellis
1,+
, Connie Y. H. C. Leung
2
, Mary K. W. Chow
3
, Lucy A.
Bissett
1
, William Wong
1
, Yi Guan
2
and J. S. Malik Peiris
2
1
Tai Lung Veterinary Laboratory, Agriculture Fisheries and Conservation Department, Lin Tong Mei, Sheung
Shui, Hong Kong SAR, China,
2
Department of Microbiology, The University of Hong Kong, SAR, China, and
3
Livestock Farm Division, Agriculture Fisheries and Conservation Department, Lin Tong Mei, Sheung Shui,
Hong Kong SAR, China
Vaccination of chickens with a commercially available killed H5N2 vaccine was being evaluated as an additional
tool to enhanced biosecurity measures and intensive surveillance for control of highly pathogenic avian influenza
subtype H5N1 disease in Hong Kong in 2002. In December 2002 to January 2003, there were outbreaks of
H5N1 disease in waterfowl in two recreational parks, wild water birds, several poultry markets and five chicken
farms. In addition to quarantine, depopulation of the affected sheds and increased biosecurity, vaccination of the
unaffected sheds and surrounding unvaccinated farms was undertaken on three farms. In at least two farms,
infection spread to the recently vaccinated sheds with low rates of H5N1 mortality in sheds when the chickens
were between 9 and 18 days post-vaccination. However, after 18 days post-vaccination no more deaths from
H5N1 avian influenza occurred and intensive monitoring by virus culture on these farms showed no evidence of
asymptomatic shedding of the virus. This provides evidence that H5 vaccine can interrupt virus transmission in a
field setting.
Introduction
Outbreaks of H5N1 highly pathogenic avian influ-
enza (HPAI) have occurred in Hong Kong in
chickens and other gallinaceous poultry in 1997,
2001, 2002 and 2003 (Sims et al ., 2003; Ellis et al .,
2004a). High mortality rates were seen in gallinac-
eous birds on farms (1997, 2002 and 2003) and/or
in poultry markets (1997, 2001, 2002, 2003) in all
outbreaks and in wild or captive waterfowl (geese,
ducks and swans) in outbreaks in two bird parks
during December 2002 to January 2003. Deaths
also occurred in other wild or captive water birds
(Little Egrets, Egretta garzetta ; Greater Flamingo,
Phoenicopterus ruber; Grey Heron, Ardea cinerea ;
Black-headed Gull, Larus ridibundus ) during these
outbreaks (Ellis et al ., 2004a). Outbreaks of H5N1
HPAI were also detected in five chicken farms in
Hong Kong in late December 2002 and January
2003. These were detected after the outbreaks were
detected in water birds in the two bird parks and
the detection of H5N1 HPAI in the two wild Grey
Herons.
The 1997, 2001 and early 2002 H5N1 outbreaks
had substantial economic impacts in Hong Kong
due to costs of partial or total depopulation of
poultry, closure of live poultry markets, reductions
in tourism and the costs of a comprehensive H5N1
testing and surveillance system for local and
imported poultry. After the outbreak in 2001, the
poultry farm and market biosecurity measures and
monitoring systems in place since 1998 were
enhanced. Following a detailed epidemiological
study of the February to April 2002 H5N1 HPAI
*To whom correspondence should be addressed. Tel: /852 2455 2156. Fax: /852 2461 6412. E-mail: ellis_trevor@afcd.gov.hk
Received 26 February 2004. Accepted 5 April 2004
ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/04/04405-08 # 2004 Houghton Trust Ltd
DOI: 10.1080/03079450410001724012
406 T. M. Ellis et al.
outbreak, further measures were introduced to
improve farm and market biosecurity. However,
due to the large daily movement of poultry into
retail live poultry markets of Hong Kong from
farms in Hong Kong and elsewhere in southern
China, together with the possibility of H5N1 virus
infections occurring in the wider region, the Hong
Kong SAR Government investigated the use of H5
avian influenza vaccination as an additional control
measure for H5N1 avian influenza.
A field evaluation trial using Nobilis
†
Influenza
H5, an inactivated avian influenza Type A H5N2
virus (A/Chicken/Mexico/232-CPA/94) water-in-oil
emulsion vaccine (Intervet International, Boxmeer,
The Netherlands), on chicken farms was com
-
menced in April 2002 in the district where the last
four farm outbreaks of H5N1 had occurred, and
evaluation continued until March 2003. The eva
-
luation trial showed that acceptable flock H5
antibody responses were generated by the vaccine
and vaccinated chickens were protected from high-
dose laboratory challenge with H5N1 HPAI viruses
from the February to April 2002 outbreaks (Ellis et
al ., 2004b). In December 2002, the H5N1 out
-
breaks in waterfowl and wild birds and the detec-
tion of viruses in several retail markets, together
with the encouraging vaccine trial results, led to the
decision to broaden the chicken farm vaccination
area to cover those areas most exposed to wild bird
movements. H5N1 outbreaks occurred in five
unvaccinated chicken farms in late December
2002 and January 2003. Strict quarantine and
movement controls were initiated immediately,
followed on two farms by total depopulation with
ring vaccination of surrounding farms. In three
other affected farms only sheds showing high or
rising mortality rates were depopulated together
with vaccination of unaffected sheds and surround
-
ing farms. The effect of killed H5N2 vaccination in
the face of these outbreaks was monitored in terms
of protection from disease and ability to interrupt
transmission of H5N1 virus.
Materials and Methods
Outbreak history. The three chicken farms referred to in this report rear
chickens in semi-enclosed sheds in A-frame cage banks, as do most of
the 154 chicken farms in Hong Kong. Farm sizes in Hong Kong vary
between approximately 20 000 and 100 000 chickens and they rear slow-
growing yellow
/brown broiler chickens, which are imported as one-
day-old chickens from Mainland China and are usually marketed at
between 80 and 100 days of age.
Vaccinations on the affected farms were given using a single standard
0.5 ml dose of the Nobilis
†
Influenza H5 vaccine using the subcuta-
neous route at the base of the neck. Separate farm staff members were
used to vaccinate the different sheds and all staff wore their standard
working uniform. Disinfectant footbaths containing Virkon
TM
were
located at the entrance of each shed and hand washing facilities were
available and were used by staff. Experienced teams of Agriculture,
Fisheries and Conservation Department (AFCD) staff were involved in
depopulation of chicken sheds and they wore impervious protective
overalls with built-in hoods, boots, gloves and masks and were not
permitted into other sheds on the farms.
Farm 1. A moderate sized farm (51 000 chickens) in Tai Kong Po (see
farm plan in Figure 1) reported increased mortalities (about 35
chickens) on 6 January 2003, and follow-up inspection by AFCD staff
detected further deaths in three sheds (shed 7 had 107 dead/6000 in 98-
day-old birds, shed 8 had 44 dead/1500 in 65-day-old birds and shed 11
had eight dead/2600 in 77-day-old birds) by that afternoon. Rapid
testing (real-time reverse transcriptase polymerase chain reaction [RRT
-
PCR] for H5) detected HPAI H5 virus that evening. Movement control
and strict attention to biosecurity was initiated immediately and the
farm was officially quarantined on 7 January 2003. However, un
-
resolved issues with compensation resulted in culling of chickens in the
affected sheds being delayed. Vaccination with the killed H5N2 vaccine
was started in the six unaffected sheds on the farm on 7 January 2003.
Mortalities commenced in sheds 3, 6 and 10, which were adjacent to the
three initially affected sheds, on 8 January 2003. Subsequently
mortalities started to rise rapidly in sheds 7, 8 and 11, then in sheds
3, 6 and 10 and by 15 January 2003 in sheds 4 and 5 (Table 1). The 8000
remaining chickens in sheds 7, 8 and 11 were killed on 10 January 2003
and the 12 000 remaining chickens in sheds 3, 4, 5, 6 and 10 were killed
on 15 January 2003.
On 16 January 2003 (day 9 post-vaccination) deaths, confirmed as
H5N1 HPAI after postmortem examination, H5 RRT-PCR and virus
culture in chicken embryos, were detected in the remaining large shed
on the farm (shed 2 containing 19 000 chickens). This shed was
physically well isolated from the other sheds on the farm and staffed
by a separate group of workers. Mortalities confirmed as H5-positive
continued at a low level until 25 January (day 18 post-vaccination)
(Table 1). Sequential measurements of haemagglutination inhibition
(HI) test antibody levels to H5 avian influenza were conducted for
chickens from this shed (days 15, 22, 28, 34, 38 and 42 post
-
vaccination). Cloacal swabs were collected from 60 randomly selected
chickens in this shed for virus culture on days 15, 22 and 28 post
-
vaccination by AFCD. A larger cross-sectional sampling of chickens in
the shed for virus culture (300 throat and 300 cloacal swabs each time)
was conducted by the Department of Microbiology, University of Hong
Kong on days 28, 33 and 38 post-vaccination.
Farm 2 . A second farm (35 000 chickens) in Tai Kong Po reported
increased mortalities (about 150 chickens in two batches of chickens) in
two adjacent sheds (shed 1 with 46-day-old chickens and shed 3 with 39-
day-old chickens) on 20 January 2003. Rapid testing (postmortem
examination and H5 RRT-PCR) diagnosed H5 HPAI. Movement
controls and strict adherence to biosecurity procedures were instigated
immediately. The farm was placed in quarantine and the 5300 chickens
in those sheds were killed on 21 January 2003. This farm was near the
first outbreak farm at Tai Kong Po and had been included in the ring
vaccination programme around that farm. Shed 3 was vaccinated on 8
January 2003 and Shed 1 on the 14 January 2003. Other sheds were
vaccinated on 9 to 15 January 2003.
The remaining chickens on the farm were checked daily for
mortalities that were investigated to determine the cause of death. On
22 to 23 January 2003 AFCD staff collected 180 chicken cloacal swabs
and 120 fresh faecal droppings the trays under the cages from sheds 2, 4
and 5 for virus culture. On 11 February 2003 60 cloacal swabs were
randomly collected from the market-aged chickens (about 100 days old)
and on 20 February 2003 100 swabs of fresh faecal droppings from the
cage trays were also collected and tested by virus culture from the
market aged birds. Subsequently, the seven batches of market-aged
chickens between February and April 2003 all had 60 cloacal swabs
randomly collected and tested by H5 RRT-PCR and virus culture.
Serum antibody levels were measured by H5 HI tests on 14 birds from
shed 4 at 10 days post-vaccination and 14 birds from shed 2 at 13 days
post-vaccination on 22 January 2003, then 30 birds each from shed 2
and 4 (30 to 33 days post-vaccination) on 11 February 2003.
Farm 3 . A third farm (20 700 chickens) in Shek Kong Tsuen reported
increased mortalities (about 100 chickens of 38 days old) in a single shed
on 20 January 2003. Rapid testing (post-mortem examination and H5
RRT-PCR) detected HPAI H5 virus that day. This farm was in a
separate valley 1.5 km away from the Tai Kong Po farms. The farm was
immediately placed in quarantine and the 5600 chickens in the affected
shed were killed on 21 January 2003. All remaining chickens on the
farm between 8 and 55 days old were vaccinated and ring vaccination
Vaccination interrupts H5N1 virus transmission 407
Figure 1. Location plan of chicken sheds on Farm 1.
was conducted on the nine farms nearby (involving 212 000 chickens)
on 23 January 2003.
The remaining chickens on the farm were checked daily for
mortalities, which were investigated immediately to determine the cause
of death. Blood samples were collected from 14 chickens in all batches
of market age chickens to measure H5 antibody responses. From each
batch of market-aged chickens between February and April 2003, 60
cloacal swabs were randomly collected for H5 RRT-PCR testing and
virus culture.
Laboratory test procedures. Dead birds were subjected to postmortem
examination as described for previous H5N1 outbreak investigations
(Ellis et al ., 2004a). Pooled cloacal and tracheal swabs from each dead
bird were suspended in antibiotic containing viral transport media and
subsequently inoculated into the allantoic cavity of 9-day-old to 11-day-
old specific pathogen free chicken embryos following standard proce
-
dures (Alexander, 2000). Cloacal swabs, throat swabs and swabs of
faecal droppings from clinically healthy chickens that were collected for
avian influenza virus surveillance in the chicken sheds were similarly
tested. Allantoic fluid from eggs with dead embryos and all eggs at 4
days post-inoculation were tested for presence of haemagglutinins (HA)
of chicken red blood cells, and HA-positive allantoic fluid was routinely
subjected to HI tests using reference antisera (Veterinary Laboratory
Agency, Weybridge, UK and USDA, Ames, IA, USA) to avian
influenza subtypes H5 and H9 and Newcastle disease virus by standard
procedures (Alexander, 2000).
Cloacal and faecal dropping swabs for surveillance testing of chicken
sheds were also tested for presence of the H5 HA gene by the RRT-PCR
test described by Spackman et al . (2002). HA-positive allantoic fluids
from the virus cultures were also tested for the H5 gene by the
RRT-PCR. Any isolated H5 virus was further characterized by
sequencing of virus gene segments at the Department of Microbiology,
University of Hong Kong using procedures described previously (Guan
et al ., 2002).
Antibody levels to H5 avian influenza virus in chicken sera were
tested by the standard HI test procedures described by Alexander
(2000) using avian influenza A/chicken/Hong Kong/97 (H5N1) virus
antigen.
Results
Affected dead chickens from all three farms had
gross and microscopic pathology changes consis
-
tent with previous H5N1 HPAI cases in chickens in
Hong Kong. The chickens shown as H5N1 positive
in Tables 1 and 2 gave positive results by H5 RRT
-
PCR tests on cloacal swabs and H5N1 virus
was isolated by chick embryo allantoic cavity
inoculation. Partial gene sequencing of eight gene
c
408 T. M. Ellis et al.
Table 1. Record of the number of dead chickens in respective sheds on Farm 1 (Tai Kong Po)
Date Sheds 7, 8, 11 Shed 10 Sheds 3, 6 Sheds 4, 5 Shed 2
Number of chickens (age)
3/1/03
4/1/03
5/1/03
6/1/03
7/1/03
8/1/03
9/1/03
10/1/03
a
11/1/03
12/1/03
13/1/03
14/1/03
15/1/03
c
16/1/03
17/1/03
18/1/03
19/1/03
20/1/03
21/1/03
22/1/03
23/1/03
241/03
25/1/03
26/1/03
27/1/03
28/1/03
29/1/03
30/1/03
31/1/03
1/2/03 to 7/2/03
10 300 (65 to 98 days old)
7 to 8
5 to 6
11 to 12
194
403
318
493
819
Depopulated
8000 (26 days old)
Vaccinated
36
0
700
210
220
250
600
2500
Depopulated
3000 (62 days old)
Vaccinated
7
4
15
0
12
0
18
26
Depopulated
10 300 (28 days old)
Vaccinated
0
0
0
0
0
0
0
6000
Depopulated
19 000 (62 to 74 days old)
Vaccinated
0
0
0
7 (negative)
b
0
4 (negative)
0
2 (negative)
10 (H5-positive)
d
4 (H5-positive)
4 (H5-positive)
3 (H5-positive)
6 (negative)
8 (H5-positive)
10 (H5-positive)
11 (H5-positive)
12 (H5-positive)
6 (H5-positive)
6 (negative)
4 (negative)
5 (negative)
5 (negative)
4 (negative)
6 (negative)
All 0
a
A total of 8000 chickens were culled from sheds 7, 8 and 11 on 10 January 2003.
b
(negative), negative for H5 virus isolation.
A total of 12 000 chickens were culled from sheds 3, 4, 5, 6 and 10 on 15 January 2003.
d
(H5-positive), H5N1 avian influenza virus was isolated from dead chickens.
segments from representative viruses from these
farms showed that all viruses were similar and they
were also similar to H5N1 viruses that had been
detected from the outbreak in waterfowl at one
water bird park and in wild Grey Herons in
December 2002.
Farm 1 investigation. H5N1 infection was detected
in shed 2 on 16 January, 9 days post-vaccination,
and dead chicken continued to be detected until 25
January (day 18 post-vaccination). However, sub
-
sequently no sick or dead birds were diagnosed as
H5 avian influenza in shed 2 (Table 1). No
subclinical infection with H5N1 virus was detected
by virus culture of random samples of cloacal and
throat swabs from 60 clinically normal chickens
from this shed by AFCD on days 15, 22 and 28
post-vaccination or from cloacal and throat swabs
collected from 300 clinically normal chickens on
days 28, 33 and 37 post-vaccination in the cross-
sectional sampling in this shed by University of
Hong Kong.
The sequential antibody responses to H5 avian
influenza in vaccinated chickens in shed 2 are
indicated in Table 3. By day 22 post-vaccination,
81.7% chickens had H5 antibody titre ]
/16 and the
overall GMT for these birds was 33.9. By this time
there had been no mortalities due to H5 avian
influenza or H5 virus isolations for 4 days.
Farm 2 investigation. The dead bird monitoring in
sheds 2, 4 and 5 showed that a small number of
chickens in each of these sheds died of H5N1 HPAI
(diagnosed by postmortem examination, RRT
-
PCR and virus culture) on 25 to 26 January 2003,
indicating that these sheds had also been exposed
to H5N1 virus. Two other more isolated sheds of
younger chickens on the farm showed no H5N1
HPAI (Table 2).
By the time the affected chickens were detected
in sheds 2, 4 and 5, these batches were already 13 to
17 days post-vaccination and there was only limited
disease in these sheds over a 2-day period.
Prior to marketing of the first batch of chickens
from this farm, virus culture testing of cloacal
swabs collected from 60 randomly sampled chick
-
ens and swabs of 100 randomly collected fresh
faecal droppings from the cage trays were shown to
be negative for H5N1 viruses. Subsequently, no
H5N1 virus was detected by H5 RRT-PCR or by
virus culture from 60 randomly sampled cloacal
Vaccination interrupts H5N1 virus transmission 409
Table 2. Record of the number of dead chickens in respective sheds on Farm 2 (Tai Kong Po)
Date Shed 1 Shed 3 Shed 2 Shed 4 Shed 5 Sheds A, B
Number of 2800 2 500 5000 5800 10 200 8700
chickens (age) (46 days old) (39 days old) (95 to 102 days old) (52 to 88 days old) (59 to 81 days old) (16 to 32 days old)
Date vaccinated 14/1/03 8/1/03 9/1/03 12-13/1/03 10-11/1/03 15/1/03
20/1/03 150 (14 H5-positive pools)
a
0 0
21/1/03 Depopulated 0 0
22/1/03 0 0
23/1/03 0 0
24/1/03 0 0
25/1/03 2 (1 H5
/ve pool) 0
26/1/03 8 (2 H5
/ve pools) 2 ( /ve)
b
27/1/03 0 5 ( /ve) (1 NDV /ve)
c
28/1/03 /1/2/03 0 0
2/2/03 0 20 (
/ve) (1 H9 /ve)
d
3/2/03 0 3 ( /ve)
4/2/03 0 3 (
/ve)
5/2/03 to 6/2/03 0 0
7/2/03 0 2 ( /ve)
a
(H5-positive pool), H5N1 avian influenza virus was isolated from pooled cloacal and throat swabs from the dead chickens.
b
(negative), negative for H5 virus isolation.
c
NDV, Newcastle disease virus.
d
H9, avian influenza H9N2 virus.
swabs from the next seven batches of market-aged
chickens from this farm.
H5 HI antibody titre ]
/16 was detected in six of
14 (42.9%) of chickens in shed 4 at 10 days post
-
vaccination, four of 14 (28.6%) of chickens in shed
2 at 13 days post-vaccination and in all chickens
from both shed 2 and 4 at 30 to 33 days post
-
vaccination (57 of 60 had H5 HI titre ]
/32 and
three chickens had titre
/16).
Farm 3 investigation. No H5N1 HPAI occurred in
other sheds on the farm or in the nearby nine farms
subsequent to this outbreak. H5N1 virus was not
detected by H5 RRT-PCR or by virus culture from
60 randomly sampled cloacal swabs from all
batches of market-aged chickens from this farm
between February and April 2003.
The first batch of vaccinated chickens was
marketed on day 37 post-vaccination, and blood
tests on these chickens showed that all (14/14) had
H5 HI antibody titres ]
/32.
Discussion
One of the concerns in the use of vaccine to control
HPAI in poultry farms is the possibility that while
vaccine may protect from disease, asymptomatic
virus circulation may continue, resulting in spread
of infection to other farms. The monitoring and
surveillance conducted on these three chicken
farms showed that use of this killed H5N2 vaccine
in the face of HPAI H5N1 virus challenge was able
to protect chickens from disease and interrupt virus
transmission. The protective effect of vaccine
became apparent after day 18 post-vaccination.
On farms 1 and 2, clear evidence of H5N1 infection
was demonstrated in sheds of vaccinated chickens,
and subsequently extensive surveillance by clinical
inspection and virus detection tests, both H5 RRT
-
PCR and virus culture, showed that the virus
transmission had been interrupted. For farm 3,
the rapid depopulation of the affected shed and
strict biosecurity measures applied combined to
minimize the level of challenge to other sheds. No
Table 3. Antibody responses to H5 virus
a
in vaccinated chickens on Farm 1
Number of days Number of Titre 4 Titre 8 Titre 16 Titre 32 Titre 64 Titre Titre % birds Geometric
post-vaccination (date) chickens 128 256 positive mean titre
15 (22/1/03) 60 28 11 15 4 2 53.3 11.7
22 (29/1/03) 60 11 6 16 15 7 5 81.7 33.9
28 (4/2/03) 60 6 5 49
b
90 n/a
34 (10/2/03) 60 4 56
b
93.3 n/a
38 (14/2/03) 14 14
b
100 n/a
42 (18/2/03) 14 14
b
100 n/a
a
Antibody responses were detected by HI tests using A/chicken/Hong Kong/97 (H5N1) antigen.
b
Tested in a three-dilution test (8, 16, 32) only.
n/a indicates geometric mean titre was not calculated because end-point titres were not available.
410 T. M. Ellis et al.
evidence of clinical disease or H5N1 infection was
demonstrated in the sheds of vaccinated chickens
so it is possible that the other sheds on this farm
may not have received significant exposure to the
H5N1 virus from the initial infected shed.
Vaccines have been used in other countries to
assist in the control of avian influenza. Countries
that have used vaccines for avian influenza control
include Italy (Capua et al. , 2002), the US (Halvor
-
son, 2002), Mexico (Villarreal & Flores, 1998) and
Pakistan (Naeem, 1998). Mostly vaccination has
been directed against low pathogenic strains of
avian influenza virus but Mexico and Pakistan have
successfully used vaccine against highly pathogenic
H5 or H7 avian influenza viruses. Experimental
studies have shown that commercially available H5
avian influenza vaccines could protect poultry from
1997 Hong Kong strains of H5N1 HPAI virus
(Swayne et al ., 2001).
On Farm 2, avian influenza H9N2 virus was
detected in the sheds containing 16-day-old to 32-
day-old chickens. Recent experimental studies have
suggested that infection with H9N2 virus may
stimulate cell-mediated immune responses that
could cross-protect chickens from intranasal
H5N1 virus challenge that was lethal in uninocu
-
lated controls (Seo & Webster, 2001). This cross-
protectivity was effective at 15 days after intranasal
inoculation with H9N2 virus given in a low
challenge dose (10 50% lethal chicken doses), but
its effectiveness was diminished by 30 days post
-
inoculation. Infection of chickens with H9N2 avian
influenza viruses is quite common in chickens in
Hong Kong based on monthly serological surveil
-
lance conducted by our laboratories on local and
imported chickens between 1999 and 2001. The
H9N2 viruses isolated from chickens in Hong
Kong belong to a lineage of viruses related to A/
Duck/Hong Kong/Y280/97 (H9N2) (Guan et al .,
2000), which generally causes mild or inapparent
infections of the upper respiratory tract in chickens.
On local farms where H9N2 infection has been
monitored, it generally occurs in chickens under 30
days that are reared on litter. By the time they are
moved to the A-frame cages infection is less
common and chickens of multiple ages on affected
farms are H9N2 antibody positive. On farm 2 with
H9N2 infection circulating in the 16-day-old to 32-
day-old birds it would be highly probable that the
older birds (39 to 46 days old) in sheds 1 and 3
would have been exposed to this virus, but this did
not prevent the H5N1 outbreak in these sheds.
During the 2002 H5N1 outbreak on chicken farms
in Hong Kong there appeared to be no correlation
between exposure to H9N2 virus, measured by
serology, and the severity of the outbreak. The
H9N2 AI virus exposure and resulting immunity
had no protective effect against the field challenge
by H5N1 AI virus possibly because of either short-
lived cross-protective cellular immunity or a high
environmental challenge dose of H5N1 AI virus.
Avian influenza vaccination has generally been
used in uninfected flocks in control areas around
but not including infected flocks. From this in
-
vestigation we are definitely not suggesting that the
use of vaccination to assist in the control of an
avian influenza outbreak could be delayed in the
control area until evidence of spread from infected
farm(s) occurs. Nor do we recommend the use of
partial depopulation plus vaccination on an in
-
fected farm as a normal practice. In the first Tai
Kong Po farm, five sheds with 22 000 chickens had
to be killed before vaccination had a chance to
work in the final shed, and in the meantime
outbreaks occurred on two nearby farms that
were ring vaccinated at the same time as the initial
farm. Generally, when ring vaccination is used for
avian influenza control, the infected farm and high-
risk contact farms within an epidemiologically
sustainable perimeter (usually several kilometres)
are quarantined, monitored and possibly depopu
-
lated. Ring vaccination is used outside this zone
where there is a good chance for immunity to
develop to the virus before exposure occurs. The
close proximity of farms and limited land avail
-
ability makes this approach difficult in Hong Kong.
For the three farms involved in this investigation
the individual circumstances at the time, together
with expanding use of preventative vaccination
throughout Hong Kong, led to an unusual control
strategy involving quarantine, partial depopulation
and vaccination of unaffected sheds and surround
-
ing farms. As part of this strategy very strict
attention had to be paid to movement control of
birds, people and materials onto and from the farm
and strict biosecurity practices had to be main
-
tained. This was combined with an intensive
monitoring programme on the vaccinated sheds
and the surrounding farms to rapidly detect any
spread of the infection. This strategy was very
resource intensive and would have been very
difficult to sustain in a more widespread outbreak.
Another factor that should be considered with
vaccinating in the face of an outbreak is the
possibility of selection of variant viruses when the
virus is replicating rapidly in the presence of partial
or incomplete flock immunity. The chance of this
occurring will clearly be lower if virus is introduced
to a fully vaccinated flock that has had time to
develop its immunity. However, concerns expressed
about the risk of enhanced H5N1 virus evolution in
the presence of a vaccinated antibody-positive
chicken population needs to be kept in perspective.
If you do not vaccinate, all exposed chickens have
the potential to become infected with H5N1 viruses
that will replicate to high titres and shed large
quantities of virus in faeces and respiratory secre
-
tions that will infect further chickens. Each replica-
tion cycle increases the number of mutations and
Vaccination interrupts H5N1 virus transmission 411
the potential for antigenic variation. There are also
many examples of emergence of HPAI avian
influenza viruses from low or medium pathogenic
avian influenza viruses without any influence from
vaccination (Alexander et al ., 2000). Inactivated oil
emulsion avian influenza vaccines have given good
protection despite variation of up to 10.9% in
haemagglutinin-deduced amino acid sequence
(Swayne et al ., 1999, 2000). Avian influenza
vaccination has been most widely practiced in
Mexico, beginning in January 1995, and it con
-
tinues to be used. Over 1.4 billion doses of
inactivated vaccine and 500 million doses of
fowlpox-AI-H5 recombinant vaccine have been
used and the vaccines are still considered protective
(Villarreal-Chavez & Rivera-Cruz, 2003).
The ultimate goal of any control programme for
avian influenza should be to eradicate HPAI. This
was also the goal in Hong Kong during this
outbreak, and this goal was achieved. With the
presence of these viruses in wild water birds in the
region and the large daily cross-border movement
of poultry the risk of H5N1 virus incursions
infection in Hong Kong is very high. A compre
-
hensive package of measures including enhanced
biosecurity programmes for farms, wholesale and
retail poultry markets, the use of rest days in
markets to break cycles of infection and a compre
-
hensive monitoring and surveillance programme
for early detection of any H5 avian influenza virus
incursions have been in place since 2001 and were
enhanced after the February to April 2002 out
-
break. As stressed by international animal health
authorities (Alexander et al ., 2000), avian influenza
vaccination in Hong Kong is used to complement
the strict biosecurity measures and a comprehen
-
sive monitoring and surveillance programme al-
ready in place. Comprehensive vaccination of all
chicken farms supplying the local retail markets
was introduced as an additional layer of protection
after a one year long vaccination evaluation trial
(Ellis et al ., 2004b). This investigation showed that
the use of killed H5N2 vaccine on three farms
undergoing H5N1 HPAI outbreaks was able to
protect chickens against disease and also to inter
-
rupt asymptomatic virus shedding. This is particu-
larly relevant when dealing with viruses such as
H5N1 where the virus also poses a significant risk
to human health.
Acknowledgements
The authors thank the staff of the Avian Influenza
Serology, Avian Virology, Molecular Biology, His
-
tology and Bacteriology laboratories at Tai Lung
Veterinary Laboratory, the staff of the Department
of Microbiology, University of Hong Kong and the
field staff of Livestock Farm Division for their
excellent technical support. The studies at The
University of Hong Kong were supported by The
Wellcome Trust Grant 067072/D/02/Z and a Public
Health Research Grant AI95357 from the National
Institutes of Allergy and Infectious Diseases.
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Translations of the abstract in French, German and Spanish are available on the Avian Pathology website.
