15

Outbreaks of Highly Pathogenic Avian Influenza in Japan
Toshihiro ITO
Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University
Tottori 680-8553, Japane-mail: toshiito@muses.tottori-u.ac.jp

Abstract
In January 2004, an outbreak of highly pathogenic avian influenza, caused by the virus subtype H5N1,
occurred at a farm in Yamaguchi Prefecture, Japan. This was Japan’s first outbreak since 1925, a span of
79 years. By March, there were additional outbreaks in Oita and Kyoto Prefectures at a total of four
premises, resulting in the sacrifice of about 275,000 domestic fowl. The disease was successfully eradicatedin Japan, and infection damage was kept to a minimum thanks to an intense eradication campaign. On theother hand, extensive outbreaks of the disease were confirmed at around the same time, mainly in Southeast
Asian countries.
Three years later, in January 2007, there was another outbreak of highly pathogenic avian influenza
caused by the H5N1 virus in Miyazaki Prefecture, Japan. Within less than two weeks, there were two morecases in Miyazaki Prefecture and another in Okayama Prefecture, for a total of four confirmed cases. At
each of the four farms where cases occurred, the outbreak was quickly discovered and reported, and rapid
epidemic prevention measures, such as culling, burial and incineration, were successful. The outbreaks were
contained and no reoccurrences were reported.
However, the risk of H5N1 virus entering Japan again is not decreasing, because the virus is endemic
in several parts of Asia. Therefore, it is still top priority for poultry producers to maintain biosecurity practices to prevent introduction of the virus into their flocks.
Key words: chicken, highly pathogenic avian influenza virus, Japan, wild waterfowl

1. Introduction
Highly pathogenic avian influenza is a peracutecontagious disease of poultry that causes systemic infection (Easterday & Tumova, 1978). Since the mortality ofthe disease in poultry is as high as 100%, and it canspread rapidly, an outbreak can devastate the poultry
industry. The main symptoms of highly pathogenic
avian influenza in poultry are depression; loss of appetite; decreased or absent egg production; nervous behavioral signs; facial, comb or leg edema; blood spots or
cyanosis; respiratory symptoms; and diarrhea (Fig. 1).
In addition, symptoms and virus excretion vary depending on the bird species, their age and virus strain. Spreadof this disease is mainly by contact with infected birds
or with feces, saliva, nasal secretions, drinking water,
feed, dust, insects, wild birds, human beings, small animals, equipment or vehicles contaminated with thevirus.
In Japan, in cases where highly pathogenic avian
influenza has been confirmed, appropriate measures
such as eradication and movement restrictions on theaffected holdings are implemented based on the Domestic Animal Infectious Diseases Control Law. In addition,
Global Environmental Research
12: 15-20 (2008)
printed in Japan

although some countries use vaccination as a preventive
measure, Japan in principle does not. Vaccination may
suppress symptom onset and allow birds to shed thevirus while remaining asymptomatic. Vaccination,
therefore, may induce the habitual presence of the virus
in the field. As special testing is required to distinguishthe antibodies produced by vaccination from those produced by natural infection, Japan engages in prevention

Fig. 1 Dead layers infected with highly pathogenic avian
influenza virus. (provided by Kyoto Prefecture)©2008 AIRIES

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T. ITOby using testing to detect infected poultry and cull them.
In this paper, an overview of highly pathogenic avian
influenza outbreaks in Japan is presented and the possible sources of introduction of the virus are discussed.

2. Outbreaks in 2004
Japan had been free from highly pathogenic avian
influenza (formerly known as “fowl plague”) until January and March 2004, when an outbreak of highly pathogenic avian influenza caused by the virus subtype H5N1
occurred on four premises in three prefectures (Fig. 2).
This was the first report of highly pathogenic avian
influenza in Japan for 79 years since the 1925 outbreak,which was caused by the virus subtype H7N7.
Case 1
The first case of highly pathogenic avian influenza
occurred on a layer farm (35,000 birds) in Ato Town,
Abu County, Yamaguchi Prefecture, in January 2004,
after dead chickens were confirmed on December 28,
2003. Since the number of chicken deaths subsequently
increased and spread to other houses, the egg farm requested the local Livestock Hygiene Service Center to
conduct a disease diagnosis. On January 11, the suspected outbreak of avian influenza was reported to the
Ministry of Agriculture, Forestry and Fisheries (MAFF).
Case 2In February 2004, the second case of highly pathogenic avian influenza occurred at the home of a hobbyist
bantam breeder (13 bantams and one domestic duck) in
Kokonoe Town, Kusu County, Oita Prefecture. The sudden deaths of three bantams were discovered on February 14, 2004. Therefore, the raiser reported the cases tothe local Livestock Hygiene Service Center. Receiving

the report, the Service Center conducted an on-site
inspection the same day and, taking two of the deadbirds, started virus isolation tests. Furthermore, since
four more bantams raised in the same shed died twodays later (February 16), an on-site inspection was again
conducted. In the evening of the same day, the virus
suspected of being avian influenza was isolated from thespecimens removed on the 14th, and the Service Center
reported the fact to Oita Prefecture. At the same time,
the six bantams and one duck still remaining at thehome of the raiser were culled with the raiser’s agreement.
Case 3
The third case in Tanba Town, Kyoto Prefecture, on
February 17, 2004, was particularly aggravated. The
outbreak occurred in one poultry house (No. 8, capacity:
approx. 30,000 fowl) of an egg production farm
(225,000 fowl), and despite the deaths of many chickens,no report was made. Furthermore, infected chickens
were shipped to meat processing plants in Hyogo andAichi Prefectures. The infection spread to virtually all of
the houses within a few days. The Service Center, which
had received an anonymous telephone call reporting
large-scale deaths at the farm, conducted an on-site
inspection before dawn on February 27. Infection by thehighly pathogenic avian influenza virus subtype H5N1
was confirmed on February 29.
Furthermore, avian influenza virus subtype H5N1
was also isolated from a total of nine crows between
May 7 and April 9 in the restricted movement zone. No
other infection of wild birds was confirmed.
Case 4Since there was a rapid increase in the number ofdead chickens on March 3 at a meat chicken farm

SEOUL

KOREA

In 2003-2004
In 2006-2007
Hawk-eagle

Fig. 2 Location of outbreaks of highly pathogenic avian influenza in Japan and Korea
from 2003 to 2007.

Outbreaks of Highly Pathogenic Avian Influenza in Japan(15,000 birds) in Kyoto Prefecture, located about 4 km
northeast of the infected farm of the third outbreak, thefarm manager notified the Livestock Hygiene Service
Center. The Service Center promptly conducted an
on-site inspection of the farm and a simple test of thedead fowl at the farm. The virus was confirmed to be
subtype H5N1 on March 8.
As an epidemic prevention response, necessary
measures in accordance with the Domestic Animal
Infectious Diseases Control Law were performed. All
chickens on outbreak farms were slaughtered, disinfection was carried out, movement at nearby farms was
restricted and epidemiological surveys were performed.
This procedure prevented the spread of the virus tonearby farms and halted the outbreaks in all four cases.
Though all virus strains isolated in these outbreaks
were from the same source, they were different virus
strains that had mutated in a relatively short period.
There is the possibility that the outbreaks in the threeprefectures occurred independently due to different
infection sources. The isolated virus strains indicate extremely strong pathogenicity to chickens. As ducks cansurvive infection (Kishida et al., 2005), they may be
carriers of the virus. In experiments on infecting mice,the virus was not detected in the feces, and it is thought

Fig. 3 Epidemiological investigation of an infected farm by
the expert “Investigation Team on the Infection Routeof Highly Pathogenic Avian Influenza.”

17that proliferation through the feces does not readily
occur (Isoda et al., 2004). Infection has not been established in hogs, suggesting that they also have resistance.
With respect to infection routes, an expert “Investigation Team on the Infection Route of Highly Pathogenic Avian Influenza” was formed (Fig. 3). The teamconducted analysis and evaluation based on epidemiological surveys originating at each outbreak farm, as
well as analysis of the characteristics of isolated viruses.
An investigation of wild birds, including migratorywaterfowl, was also conducted within a 10-km radius ofeach infected farm (Fig. 4). Although no highly pathogenic avian influenza viruses were isolated fromfree-living birds, the team reported that it was probablethat migratory birds, such as ducks, brought the pathogenic virus to Japan from the Korean Peninsula, and thatthe feces of these ducks and other migratory birds werea source of infection that was carried by resident birds,
rats, other animals and humans into poultry houses(http://www.maff.go.jp/tori/20040630e_report.pdf).

3. Outbreaks in 2005
On June 26, 2005, avian influenza virus subtype
H5N2 was isolated in Mitsukaido, Ibaraki Prefecture.
By December 25, there were 41 confirmed cases ofinfection (40 in Ibaraki Prefecture and 1 in Saitama
Prefecture), including farms that tested positive for antibodies but had no mortalities and no virus detection.
The virus was isolated in nine cases and each virus was
closely related. No clinical abnormalities were found on
the outbreak farms. Pathogenicity testing revealed thatthe virus belonged to the attenuated type, producing
minimal symptoms and mortality. Although investigation of infection routes by the Investigation Team on the
Infection Route of Highly Pathogenic Avian Influenza
was unable to specify the virus origins or entry routes,
in light of the virus characteristics and the localized outbreak area, it was unable to rule out illegal importation
and use of an unauthorized vaccine derived from a
Central and South American virus strain or of the virus
itself. In addition, an epidemiological survey found that
there was a high probability that the virus was transmitted among some of the farms by the movement of chickens between farms. In addition, the survey suggested
that neighborhood transmission and the comings andgoings of people and goods were major causes oftransmission.

4. Outbreaks in 2007

Fig. 4 Investigation of free-living birds, including migratorywaterfowl, within a 10-km radius of the infected
premises.

In January and February 2007, there were four outbreaks: on a meat-chicken breeding farm, a meatchicken farm and an egg farm in Miyazaki Prefecture, as
well as an egg farm in Okayama Prefecture. In each case,the virus was an H5N1 subtype related to viruses
isolated in China, Mongolia and South Korea.

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T. ITO

Case 1
In early January, an increased number of deaths andfacial puffiness were confirmed in a poultry house on ameat-chicken breeding farm (12,000 birds) in Kiyotake
Town, Miyazaki Prefecture. An attending veterinarian
was consulted, but simple testing results were negative.
Since the number of dead birds subsequently increased,
simple testing was repeated, and the result was positive.
Therefore, the Livestock Hygiene Service Center for thearea carried out virus isolation. Since it resulted in theisolation of a virus suspected of being a type A influenza virus on January 12, the National Institute ofAnimal Health carried out virus identification testing.
On January 13, it confirmed that the virus in question
was an H5 subtype. The dead chickens all came fromone poultry house complex without separations, so it
was not possible to specify the source of the outbreak.
Initially, there were more dead males than females.
Case 2On January 22, when a rapid increase in dead chickens in the central part of a poultry house on ameat-chicken farm (53,000 birds) in Hyuga City wasconfirmed, the attending veterinarian carried out simple
testing. The results were all negative, but the attending
veterinarian reported an overview to the Livestock
Hygiene Service Center. On January 23, the Livestock
Hygiene Service Center and the attending veterinarian
visited the farm and assessed the illness. On January 23,
the number of dead chickens increased by 326, andsimple testing results were positive. That day, the farm
was quarantined, and the farm and the area around thepoultry house were disinfected and limed. On January
25, the National Institute of Animal Health confirmed
infection with an H5 subtype, type A influenza virus.
Case 3On January 22, two dead birds in one cage in a poultry house were discovered. On January 27, a further
15 birds were confirmed dead in locations near the firsttwo deaths, so the Livestock Hygiene Service Center
carried out a clinical examination and sampling.
Symptomatic chickens were limited to the left rearcorner of the poultry house as viewed from the entrance.
Lethargy, lassitude and ruffled feathers were observedin several birds. Since the results of simple testing werepositive, quarantine of the farm and voluntary restrictions on movement were requested. On the following
day, January 28, disinfection and liming of the farm, thearea around the poultry house, and the interior of thepoultry house affected by the outbreak were performed.
Furthermore, even before confirmation, poultry and egg
shipping inspections were implemented for farms within
a 10-km radius based on the recommendation of theDomestic Fowl Disease Committee. On January 29, theNational Institute of Animal Health confirmed infection
with an H5 subtype type A influenza virus. Samples
were taken on January 30 as culling began. Antibody
testing and virus isolation testing isolated the virus only

from specimens taken from the left rear corner, which
confirmed that the spread of the infection was limited.
Case 4On an egg farm (93,000 birds) in Shintomi Town,
Miyazaki Prefecture, no abnormalities were observed
until January 30, when a number of dead chickens were
found in the southwest corner of a poultry house.
Extremely mild cyanosis in the combs of some deadchickens and lethargy in a number of live ones wereconfirmed. Simple testing by the Livestock Hygiene
Service Center was positive. Virus isolation began thatsame day, and on February 1, the National Institute ofAnimal Health confirmed infection with an H5 subtype
type A influenza virus.
During these outbreaks the farms reported promptly,
and quick and effective disease-control response
measures, such as culling, incineration, burial, anddisinfection, were implemented. Furthermore, movement-restriction areas were set up in a 10-km radiusfrom each outbreak farm. The movement restrictions
were lifted on March 1, after all tests on poultry farms
and pet chickens were negative, and no secondary cases
were found.
The viruses isolated from each of the four farms
were all H5N1 subtype, highly pathogenic, avian influenza viruses. Genetic analysis found at least 99%
homology for each of eight gene segments. In addition,
there was at least 99% homology with H5N1 subtype
viruses isolated during previous outbreaks in Mongolia
and South Korea in 2006. Phylogenetic tree analysis
showed that all these viruses belonged to the same lineage (Fig. 5), which was the same group as the virus isolated from wild birds in Qinghai Province, China, in2005. They differed from viruses isolated in 2004 in
Yamaguchi, Oita and Kyoto Prefectures, and in
Thailand, Vietnam and Indonesia.
On intravenous inoculation testing using an isolated
virus, every inoculated chicken died within 26 hours of
inoculation. Therefore, it was clear that the virus has
high pathogenicity in chickens. Clinical presentation
included comb cyanosis in some chickens, with grossly
visible pathology, such as cardiac edema and hepatic
petechiae. This pathology differed from that of the virus
isolated in 2004, which caused sudden death without
other notable symptoms.
In nasal inoculation testing of mice using an isolated
virus, all 18 mice died within ten days after inoculation.
Although no virus was recovered from the animals’
digestive tracts, the virus was recovered from theirbrains, lungs, spleens, livers and kidneys, indicatingstrong pathogenicity in mice. On the other hand, in rats
tested under the same conditions, although antibodies
were produced, no clinical symptoms were evident, andno virus growth within the rats’ bodies was found.
These results indicate that even though they are bothrodents, susceptibility differs between rats and mice.
On January 4, 2007, an emaciated mountain hawkeagle (adult female) was captured in Sagaramura,Outbreaks of Highly Pathogenic Avian Influenza in Japan

Kumagun, Kumamoto Prefecture, and it died immediately thereafter. Since the bird was emaciated despite
having no external injuries, the Kyushu Regional
Environment Office of Ministry of the Environment
suspected lead poisoning and sent the carcass to the
Ministry’s Kushiro-shitsugen Wildlife Center, which
was engaged in a study of lead poisoning in raptors.
Along with testing for lead poisoning, the Center used a
simple kit to test for avian influenza. Since the simple
kit test was positive, on February 10, the Center asked
the Avian Zoonoses Research Center, Faculty ofAgriculture, Tottori University, to carry out further testing. On March 18, isolation of the H5N1 subtype avian
influenza virus was reported, and on March 23, it wasdetermined to be a highly virulent form. The directcause of death of the mountain hawk-eagle was
unknown.
Mountain hawk-eagles are large raptors that inhabit
mountainous areas from Hokkaido to Kyushu. They
feed on a variety of birds, small mammals, reptiles, etc.
They will feed on poultry or pets such as cats if they areleft outside. They will also feed on carrion. Those thatbreed in Japan do not travel long distances, but remainin the area around their nests year round. Their range issaid to be 5 to 10 km. Young birds may travel longer
distances. In southwestern Japan, there may be individuals who have crossed over from the continent. In addi-

19tion to Japan, they inhabit Sri Lanka, southern India, theHimalayas, southeastern China south of the Yangtze
River, the Indochinese Peninsula, Taiwan, Hainan
Island and the Korean Peninsula, and they have also
been reported in northeastern China.
Analysis of the HA gene of the H5N1 highly pathogenic avian influenza virus isolated from the mountain hawk-eagle found the virus to have the
G-E-R-R-R-K-K-R amino acid sequence in the HA
cleavage region, which is typical of virulent types. As
for homology with other H5 viruses, it is most homologous (at least 99.7%) with the virus isolated from wild
birds in Mongolia in 2006. A phylogenetic tree for the
HA gene was created in order to analyze the details of
the relationships with other viruses (Fig. 5). It is related
most closely to the A/chicken/Miyazaki/K11/07 strain
isolated in Miyazaki, demonstrating that this virus isalso a Qinghai Lake-type.
These results indicate that the Qinghai Lake-type
H5N1 HPAI virus was newly introduced from the Asian
continent into Japan and already existed in the naturalenvironment of the Kyushu district at the beginning ofJanuary 2007. However, the mountain hawk-eagle is a
resident bird in Japan and its range of inhabitation iswithin 5 to 20 km. Therefore, it would be unlikely that it
carried the virus from the Asian continent to Japan. Thus,
the mountain hawk-eagle was likely infected secondar-

Ck/Korea/IS/06
Ck/Miyazaki/S749/07
Ck/Miyazaki/H358/07
Ck/Miyazaki/K11/07
Dk/Korea/ASN5/2006

Mountain hawk-eagle/Kumamoto/1/2007
Ck/Okayama/T6/07

Common goldeneye/Mongolia/12/2006
Whooper swan/Mongolia/2/06
Grebe/Tyva/Tyv06-1/2006
Dk/Tuva/01/2006
Ck/Afghanistan/1207/2006
Swan/Iran/754/2006
Cygnus cygnus/Iran/754/2006
Cygnus olor/Italy/742/2006
Ck/Adygea/203/06
Ck/Krasnodar/01/2006
Swan/Germany/R65/2006
Cygnus olor/Croatia/1/2005
Dk/Niger/914/2006
Cygnus olor/Astrakhan/Ast05-2-1/2005
Gs/Crimea/615/05
Ck/Tambov/570-2/05
Dk/Novosibirsk/56/2005
Dk/Cote d Ivoire/1787-18/2006
Ck/Nigeria/641/2006
Ck/Ivory Coast/1787/2006
Ck/Sudan/2115-9/2006
Ck/Volgograd/236/06
Iraq/207-NAMRU3/2006
Domestic goose/Iraq/812/2006
Turkey/651242/06
Ck/Kurgan/05/2005
Bh goose/Mongolia/1/05
Whooper swan/Mongolia/6/05
Whooper swan/Mongolia/3/05
Goosander/Bavaria/18/2006
Egypt/14725-NAMRU3/2006
Ck/Egypt/2253-1/2006
Mal/Italy/332/2006
Cygnus olor/Czech Republic/5170/2006
Bh Goose/Qinghai/5/05
Bh goose/Qinghai/0510/05
migratory duck/Jiangxi/2300/2005
Ck/Yamaguchi/7/04
0.002

Fig. 5 Evolutionary tree of the H5 HA gene for the Qinghai Lake-type virus group.

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T. ITO

ily by preying on other infected wildlife.
The initial outbreaks in Cases 2 through 4 were inparts of the poultry houses that were away from the entrances and areas where people usually work. Each farm
had curtains, bird nets, and wire mesh installed, butthere were tears in the wire, gaps in exterior walls andareas where bird nets were not in place; thus, anti-bird
measures were insufficient. Many wild birds andanimals were confirmed to be living around the outbreakfarms. Evidence of incursions of such animals into poultry houses included the presence of rat feces inside theCase 1 poultry house and the presence of carcasses ofwild birds inside the Case 4 poultry house. In addition,at the Case 1 farm where males and females were kept
together, the dead chickens were predominantly male,suggesting that males, which are aggressive during thebreeding season, may have attacked infected wild animals that invaded the poultry house.
So, it appears that the viruses in these outbreaks
entered the farms and poultry houses not through artificial means, such as the movement of people, feed, ormaterials, but through incursions by wild birds and/or
animals (http://www.maff.go.jp/tori/ 20070906e_report.
pdf).

5. Conclusion
Extensive epidemiological investigations were conducted on the affected farms to discover the source of
the introduction of the virus into the flocks. However,except for the outbreaks in 2005, there was no evidence
to indicate that the virus had been introduced throughdomestic or international movement/transportation ofpeople, animals, vehicles or any other commodities.
Therefore, though there is no direct evidence, such as
isolation of virus from migratory birds, it can be surmised that such birds brought the viruses into Japan, andwild birds and/or animals brought them onto the farms.
In order to carry out better investigation of infection
routes from now on, ongoing monitoring of wild birds

and on-site investigation before epidemic-control measures begin will be necessary.
Furthermore, the risk of H5N1 virus entering Japanagain is not decreasing, because the virus is endemic inseveral parts of Asia. Therefore, it is still top priority forpoultry producers to maintain biosecurity practices toprevent introduction of the virus into their flocks.
References
Easterday, B. C. and B. Tumova (1978) Avian influenza. In: M.S.
Hofstad et al., eds., Biester and Schwarte: Disease of poultry,
7th edn., Iowa State University Press, Ames, pp. 549-573.
Isoda, N., Y. Sakoda, N. Kishida, A.S. Mweene, G. Bai, K.
Matsuda, T. Umemura and H. Kida (2004) Pathogenicities ofhighly pathogenic avian influenza virus Japanese isolate
A/chicken/Yamaguchi/7/04 (H5N1) in chickens, quails, starlings, budgerigars, mice and miniature pigs. In: Proc. 52nd
Conference of the Japanese Society for Virology, Yokohama,
21-23 November. Japanese Society for Virology, Yokohama,110. (in Japanese)
Kishida, N., Y. Sakoda, N. Isoda, K. Matsuda, M. Eto, Y. Sunaga,
T. Umemura and H. Kida (2005) Pathogenicity of H5 influenza
viruses for ducks. Archives of Virology, 150 (7): 1383-1392.

Toshihiro ITO
Toshihiro ITO is a Professor of Veterinary
Public Health at the Faculty of Agriculture, Tottori University. He has also
been the designated Director of the Avian
Zoonosis Research Center at Tottori University since 2006. His academic interests
cover host range restriction of influenza
viruses, the evolution of the myxovirus
genome and mutational changes in viralpathogenicity. He has served as Chairman of the Investigation Team on
the Infection Route of Highly Pathogenic Avian Influenza (Ministry ofAgriculture, Forestry and Fisheries of Japan).

(Received 7 May 2008, Accepted 30 July 2008)